Methods for Diagnosis, Prognosis and Monitoring of Breast Cancer and Reagents Therefor

TECHNICAL FIELD

The present disclosure generally relates to methods and reagents for the diagnosis, prognosis or the monitoring of estrogen receptor 1 (ESR1) positive breast cancer, for example, ESR1 positive breast cancer which is responsive to endocrine therapy and/or ESR1 positive breast cancer which is refractory to endocrine therapy. The present disclosure also relates generally to treatment management of ESR1 positive breast cancer.

BACKGROUND

Cancer is a leading cause of disease worldwide. Breast cancer is one of the most common forms of cancer, affecting both females and males globally. Various subtypes of breast cancer have been distinguished based on a number of factors including the histopathological type of tumor, the grade of the tumor, the stage of the tumor, and the expression of genes which are characteristic of particular subtypes of breast cancer. Determination of the particular subtype of cancer in a patient is often of critical importance in determining the most appropriate course of treatment for the patient.

Estrogen receptor (ER) negative (ER-ve) breast cancer and ER positive (ER+ve) breast cancer are two recognised subtypes of breast cancer, defined by the presence or absence of expression of the estrogen receptor gene. The steroid hormone estrogen activates the estrogen receptor (ESR1) to mediate a variety of functions that are central to the normal development and maintenance of multiple tissues, including breast tissue. Inappropriate activation of the ESR1-signalling network in mammary epithelial cells initiates neoplastic transformation and drives ESR1-positive breast cancer. Patients with this disease commonly receive adjuvant endocrine therapy, which serves to inhibit ESR1-signalling. Although endocrine therapy reduces the risk of disease recurrence and breast cancer-related mortality, a third of patients with ESR1-positive breast cancer acquire drug resistance and experience disease relapse. Currently, no tests exist which can predict resistance to endocrine therapy with certainty. Thus, there is a need to identify a robust method by which ESR1-positive breast cancer patients can be stratified according to their responsiveness or resistance to endocrine therapy to enable more informed disease management.

Previous efforts to stratify early breast cancer prognosis have primarily focused on multi-gene expression signatures. In addition to multi-gene expression assays, DNA methylation signatures are being assessed as potential molecular biomarkers of cancer. Despite growing interest in the prognostic significance of DNA methylation in breast cancer, there have been no studies specifically investigating the DNA methylation profile of human ESR1-positive breast cancer and its association with disease outcome in response to treatment.

There is a need in the art for improved methods for the diagnosis of breast cancer, as well as for the diagnosis of specific subtypes of breast cancer, including ESR1-positive breast cancer which is responsive to endocrine therapy and ESR1-positive breast cancer which is resistant to endocrine therapy. There is also a need for methods of prognosis, and predicting responsiveness to treatment, in patients diagnosed with breast cancer and undergoing treatment.

SUMMARY

The present inventors performed a genome-wide DNA methylation profiling analysis from ESR1-positive endocrine therapy sensitive breast cancer cells and ESR1-positive endocrine resistant cells. In doing so, the inventors identified significant enrichment of hypermethylated probes in enhancer regions of the genome for ESR1-positive endocrine resistant cells in comparison to ESR1-positive endocrine therapy sensitive breast cancer cells. The inventors also identified a subset of 856 ESR1 binding sites that overlap enhancer regions that contain hypermethylated loci in the ESR1-positive endocrine resistant cells, 617 of which were identified as being intragenic. Furthermore, using RNA-seq and HM450 methylation data derived from a TCGA breast cancer cohort, the inventors identified that out of the 856 ESR1 binding sites which overlap enhancer regions identified, hypermethylation of 328 of those sites correlated with reduced expression of the genes with which they were most closely associated, representing 291 unique genes. These markers have been demonstrated to have significant value in the diagnosis and prognosis of ESR1-positive breast cancer which is resistant or responsive to endocrine therapy (i.e., whether the ESR1-positive cancer is in an endocrine responsive state), including determining whether a subject has acquired resistance to endocrine therapy during treatment of ESR1-positive breast cancer. The inventors have also shown that the methylation profile at the 856 ESR1 binding sites is indicative of the particular subtypes of ESR1-positive breast cancer e.g., luminal A breast cancer subtype or a luminal B breast cancer subtype.

Particular examples of enhancer regions of the disclosure which harbour CpG dinucleotide sequences identified as having significant value in the diagnosis and/or prognosis of ESR1-positive breast cancer which is, or is likely to be, resistant or responsive to endocrine therapy include those located within DAXX, MSI2, NCOR2, RXRA, C8orf46, GATA3, ITPK1, ESR1 and GET4.

Accordingly, the present disclosure provides a method for predicting response to endocrine therapy in a subject suffering from estrogen receptor 1 (ESR1) positive breast cancer, said method comprising:

(i) determining the methylation status of one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in the subject; and

(ii) identifying differential methylation of said one or more CpG dinucleotide sequences in the subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences;

wherein differential methylation of said one or more CpG dinucleotide sequences in the subject relative to the reference level is indicative of the subject's likely response to endocrine therapy.

For example, increased methylation at the one or more CpG dinucleotide sequences within the one or more estrogen responsive enhancers relative to the reference level is indicative of the ESR1-positive breast cancer being refractory to endocrine therapy.

The present disclosure also provides a method for diagnosing estrogen receptor 1 (ESR1) positive breast cancer which is refractory to endocrine therapy in a subject suffering from ESR1 positive breast cancer, said method comprising:

(i) determining the methylation status of one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in the subject; and

wherein differential methylation of said one or more CpG dinucleotide sequences in the subject relative to the reference level is indicative of the subject having breast cancer which is refractory to endocrine therapy.

The present disclosure also provides a method for predicting the therapeutic outcome of and/or monitoring the progression of estrogen receptor 1 (ESR1) positive breast cancer in a subject receiving or about to receive endocrine therapy, said method comprising:

(i) determining the methylation status of one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in the subject; and

wherein differential methylation identified at (ii) is indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer.

In one example, the method comprises determining whether the ESR1-positive breast cancer is a luminal A breast cancer subtype or a luminal B breast cancer subtype.

The present disclosure also provides a method for detecting differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in a subject suffering from estrogen receptor 1 (ESR1) positive breast cancer, said method comprising:

(i) performing an assay on a sample from the subject configured to determine methylation status at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in the subject; and

(ii) detecting differential methylation at the one or more CpG dinucleotide sequences within the one or more estrogen responsive enhancers in the subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences.

In one example, detecting differential methylation at the one or more CpG dinucleotide sequences at (ii) comprises comparing a level of methylation at the one or more CpG dinucleotide sequences as determined at (i) to the reference level of methylation for the corresponding one or more CpG dinucleotide sequences, and determining whether methylation at the one or more CpG dinucleotide sequences in the subject differs to the corresponding reference level(s) of methylation.

In any of the methods disclosed herein, methylation status may be determined for one or more CpG dinucleotide sequences within one or more ESR1 binding sites. In one example, the one or more CpG dinucleotide sequences are within one or more ESR1-binding sites as defined in Table 1. In another example, the one or more CpG dinucleotide sequences are within one or more ESR1-binding sites as defined in Table 2. In another example, the one or more CpG dinucleotide sequences are within one or more ESR1-binding sites as defined in Table 3.

In one example of the methods disclosed herein, methylation status is determined at one or more CpG dinucleotide sequences within an estrogen responsive enhancer of a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1. For example, the methylation status may be determined at one or more CpG dinucleotide sequences within an estrogen responsive enhancer within a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1. Exemplary CpG dinucleotide sequences for which methylation status may be determined in accordance with the methods of the disclosure are selected from those defined in rows 57, 111-113, 256-258, 288-289, 469-470, 805, 821-822 and 824-826 of Table 1.

In another example of the methods disclosed herein, methylation status is determined at one or more CpG dinucleotide sequences within an estrogen responsive enhancer of a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46. For example, the methylation status may be determined at one or more CpG dinucleotide sequences within an estrogen responsive enhancer within a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46.

In yet another example of the methods disclosed herein, methylation status is determined at one or more CpG dinucleotide sequences within an estrogen responsive enhancer of a gene selected from FOXA1, ESR1 and/or GATA3.

In any of the methods disclosed herein, methylation status of one or more CpG dinucleotide sequences may be determined according to any suitable method known in the art. For example, methylation status of one or more CpG dinucleotide sequences within the one or more estrogen responsive enhancers may be determined by one or more techniques selected from the group consisting of a nucleic acid amplification, polymerase chain reaction (PCR), methylation specific PCR, bisulfite pyrosequencing, single-strand conformation polymorphism (SSCP) analysis, restriction analysis, microarray technology, and proteomics. For example, methylation status of one or more CpG dinucleotide sequences within the one or more estrogen responsive enhancers in the subject is determined by one or more of the following:

(i) performing methylation-sensitive endonuclease digestion of DNA from the subject;

(ii) treating nucleic acid from the subject with an amount of a compound that selectively mutates non-methylated cytosine residues in nucleic acid under conditions sufficient to induce mutagenesis thereof and produce a mutant nucleic acid and amplifying the mutant nucleic acid using at least one primer that selectively hybridizes to the mutant nucleic acid;

(iii) treating nucleic acid from the subject with an amount of a compound that selectively mutates non-methylated cytosine residues in nucleic acid under conditions sufficient to induce mutagenesis thereof and produce a mutant nucleic acid, hybridizing a nucleic acid probe or primer capable of specifically hybridizing to the mutant nucleic acid and detecting the hybridized probe or primer;

(iv) treating nucleic acid from the subject with an amount of a compound that selectively mutates non-methylated cytosine residues in nucleic acid under conditions sufficient to induce mutagenesis thereof and produce a mutant nucleic acid, amplifying the mutant nucleic acid with promoter-tagged primers, transcribing the mutant nucleic acid in vitro to produce a transcript, subjecting the transcript to an enzymatic base-specific cleavage, and determining differences in mass and/or size of any cleaved fragments resulting from mutated cysteine residues, such as by MALDI-TOF mass spectrometry;

(v) treating nucleic acid from the subject with an amount of a compound that selectively mutates non-methylated cytosine residues in nucleic acid under conditions sufficient to induce mutagenesis thereof, thereby producing a mutant nucleic acid, and determining the nucleotide sequence of the mutant nucleic acid; and

(vi) performing methylation DNA capture or immunoprecipitation on DNA from the subject to detect and/or capture methylated DNA from the subject, and optionally determining the nucleotide sequence of the DNA fragments detected and/or captured.

The method used for methylation DNA capture or immunoprecipitation may be methylated DNA immunoprecipitation (MeDIP) or capture of methylated DNA by methyl-CpG binding domain-based (MBD) proteins (MBDCap).

The compound that selectively mutates non-methylated cytosine residues may be any compound suitable for that purpose, including, for example, a salt of bisulphite.

The methods disclosed herein may be performed on any test sample taken from a subject. For example, the methylation status of one or more CpG dinucleotide sequences within the one or more estrogen responsive enhancers may be determined in a test sample from the subject comprising tissue and/or a body fluid comprising, or suspected of comprising, a breast cancer cell or components of a breast cancer cell. The sample may comprise tissue, a cell and/or an extract thereof taken from a breast or lymph node. When the sample comprises a body fluid, the body fluid may be selected from the group consisting of whole blood, a fraction of blood such as blood serum or plasma, urine, saliva, breast milk, pleural fluid, sweat, tears and mixtures thereof.

In any of the methods disclosed herein, the reference level of methylation is a level of methylation determined for one or more CpG dinucleotide sequences within a corresponding estrogen responsive enhancer of a sample selected from the group consisting of:

(i) a sample from a normal or healthy tissue;

(ii) a sample comprising a non-cancerous cell;

(iii) a sample comprising a cancerous cell, other than a breast cancer cell characterized as being ESR1-negative subtype;

(iv) a sample comprising a cancerous cell, other than a breast cancer cell characterized as being a ESR1-positive subtype which is refractory to endocrine therapy;

(v) a sample comprising a breast cancer cell characterized as being a ESR1-positive subtype which is refractory to endocrine therapy;

(vi) a sample comprising a breast cancer cell characterized as being a ESR1-positive subtype which is responsive to endocrine therapy;

(vii) an extract of any one of (i) to (vi);

(viii) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in a normal or healthy individual or a population of normal or healthy individuals;

(ix) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in an individual or a population of individuals having cancer other than ESR1-negative breast cancer subtype;

(x) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in an individual or a population of individuals having cancer other than a ESR1-positive breast cancer subtype which is refractory to endocrine therapy;

(xi) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in an individual or a population of individuals having breast cancer characterized as being a ESR1-positive breast cancer subtype which is refractory to endocrine therapy;

(xii) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in an individual or a population of individuals having breast cancer characterized as being a ESR1-positive breast cancer subtype which is responsive to endocrine therapy; and

(xiii) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in the subject being tested wherein the levels of methylation are determined for a matched sample having normal cells.

The cancerous cell in (iii) and/or (iv) above may be, for example, a breast cancer cell. Alternatively or in addition, the cancer in (ix) and/or (x) above may be, for example, breast cancer.

In any of the methods disclosed herein, the method may additionally provide a step of treating ESR1-positive breast cancer e.g., following performance of a diagnostic or prognostic method disclosed herein. In this way, the methods of the disclosure may comprise, for example, diagnosing ESR1-positive breast cancer using a method of the disclosure described in any one or more examples described herein and, based on whether the subject is determined as being responsive or resistant to endocrine therapy, administering a suitable therapeutic compound or performing surgery or recommending treatment with a suitable therapeutic compound or recommending performance of surgery. For example, if, after performing the method of diagnosis or prognosis of the disclosure, the subject is determined as being responsive to endocrine therapy, the method may comprise commencing endocrine therapy e.g., by administering a therapeutic compound suitable for endocrine therapy, or recommending that the subject commence endocrine therapy. In another example, if, after performing the method of diagnosis or prognosis of the disclosure, the subject is determined as being resistance/refractory to endocrine therapy, the method may comprise commencing treatment other than endocrine therapy e.g., chemotherapy or radiotherapy, and/or performing surgery, or recommending that the subject commences treatment other than endocrine therapy e.g., chemotherapy or radiotherapy, and/or recommending surgery.

Drugs suitable for use in endocrine therapy are well known in the art, and include, for example, anastrozole, exemestane, fulvestrant, goserelin, letrozole, leuprorelin, leuprolide acetate, megestrol, palbociclib, tamoxifen and toremifene.

Chemotherapeutic drugs suitable for treatment of breast cancer are known in the art, but may include, for example, docetaxel, paclitaxel, platinum agents (cisplatin, carboplatin), vinorelbine, capecitabine, liposomal doxorubicin, gemcitabine, mitoxantrone, ixabepilone, albumin-bound paclitaxel and eribulin.

The present disclosure also provides a method of treating a subject suffering from estrogen receptor 1 (ESR1) positive breast cancer, wherein the subject has been diagnosed as being refractory to endocrine therapy based on differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in the subject as determined relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequence(s), said method comprising administering chemotherapy and/or radiotherapy to the subject, and/or performing surgery on the subject to remove the cancer or a portion thereof.

In one example, the subject has been diagnosed as being refractory to endocrine therapy based on increased methylation at the one or more CpG dinucleotide sequences within the one or more estrogen responsive enhancers relative to the reference level of methylation for the corresponding one or more CpG dinucleotide sequence(s).

In accordance with an example in which the subject has been diagnosed as being refractory to endocrine therapy, the reference level of methylation is a level of methylation determined for one or more CpG dinucleotide sequences within a corresponding estrogen responsive enhancer of a sample selected from the group consisting of:

(i) a sample from a normal or healthy tissue;

(ii) a sample comprising a non-cancerous cell;

(iii) a sample comprising a cancerous cell, other than a breast cancer cell characterized as being ESR1-negative subtype;

(iv) a sample comprising a cancerous cell, other than a breast cancer cell characterized as being a ESR1-positive subtype which is refractory to endocrine therapy;

(v) a sample comprising a breast cancer cell characterized as being a ESR1-positive subtype which is responsive to endocrine therapy

(vi) an extract of any one of (i) to (v);

(vii) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in a normal or healthy individual or a population of normal or healthy individuals;

(viii) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in an individual or a population of individuals having cancer other than ESR1-negative breast cancer subtype;

(x) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in an individual or a population of individuals having ESR1-positive breast cancer subtype which is responsive to endocrine therapy; and

(xi) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in the subject being tested wherein the levels of methylation are determined for a matched sample having normal cells.

The cancerous cell in (iii) and/or (iv) above may be, for example, a breast cancer cell. Alternatively or in addition, the cancer in (viii) and/or (ix) above may be, for example, breast cancer.

In one example, the method comprises administering chemotherapy to the subject who has been diagnosed as being refractory to endocrine therapy. Alternatively, or in addition, the method comprises administering radiotherapy to the subject who has been diagnosed as being refractory to endocrine therapy. Alternatively, or in addition, the method comprises performing surgery to remove the cancer or a portion thereof from the subject who has been diagnosed as being refractory to endocrine therapy. For example, the subject may receive chemotherapy and radiotherapy, or chemotherapy and surgery, or radiotherapy and surgery, or chemotherapy, radiotherapy and surgery. According to an example in which more than one of chemotherapy, radiotherapy and surgery are performed on the subject who has been diagnosed as being refractory to endocrine therapy, the respective treatments may be performed in any particular order.

Chemotherapeutic drugs suitable for treatment of breast cancer are known in the art and described herein.

The present disclosure also provides a method of treating a subject suffering from estrogen receptor 1 (ESR1) positive breast cancer, wherein the subject has been diagnosed as being responsive to endocrine therapy based on a differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in the subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequence(s), said method comprising administering endocrine therapy to the subject.

In one example, the subject has been diagnosed as being responsive to endocrine therapy based on a decreased level methylation at the one or more CpG dinucleotide sequences within the one or more estrogen responsive enhancers relative to the reference level of methylation for the corresponding one or more CpG dinucleotide sequence(s), wherein the reference level of methylation is a level of methylation determined for one or more CpG dinucleotide sequences within a corresponding estrogen responsive enhancer of a sample selected from the group consisting of:

(i) a sample comprising a breast cancer cell characterized as being a ESR1-positive subtype which is refractive to endocrine therapy;

(ii) an extract of (i); and

(iii) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in an individual or a population of individuals having ESR1-positive breast cancer subtype which is refractive to endocrine therapy.

In one example, the subject has been diagnosed as being responsive to endocrine therapy based on a level methylation at the one or more CpG dinucleotide sequences within the one or more estrogen responsive enhancers which corresponds or is equivalent to the reference level of methylation for the corresponding one or more CpG dinucleotide sequence(s), wherein the reference level of methylation is a level of methylation determined for one or more CpG dinucleotide sequences within a corresponding estrogen responsive enhancer of a sample selected from the group consisting of:

(i) a sample comprising a breast cancer cell characterized as being a ESR1-positive subtype which is responsive to endocrine therapy;

(ii) an extract of (i); and

Drugs suitable for use in endocrine therapy are well known in the art and are described herein.

In any of the methods of treatment disclosed herein, the one or more CpG dinucleotide sequences are within one or more ESR1-binding sites as defined in Table 1. In another example, the one or more CpG dinucleotide sequences are within one or more ESR1-binding sites as defined in Table 2. In another example, the one or more CpG dinucleotide sequences are within one or more ESR1-binding sites as defined in Table 3.

In one example of the methods disclosed herein, the subject has been diagnosed as being refractory to endocrine therapy or responsive to endocrine therapy based on differential methylation at one or more CpG dinucleotide sequences within an estrogen responsive enhancer of a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1. For example, the subject has been diagnosed as being refractory to endocrine therapy or responsive to endocrine therapy based on differential methylation at one or more CpG dinucleotide sequences within an estrogen responsive enhancer within a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1. In one particular example, the subject has been diagnosed as being refractory to endocrine therapy or responsive to endocrine therapy based on differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 57, 111-113, 256-258, 288-289, 469-470, 805, 821-822 and 824-826 of Table 1.

In another example of the methods disclosed herein, the subject has been diagnosed as being refractory to endocrine therapy or responsive to endocrine therapy based on differential methylation at one or more CpG dinucleotide sequences within an estrogen responsive enhancer of a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46. For example, the subject has been diagnosed as being refractory to endocrine therapy or responsive to endocrine therapy based on differential methylation at one or more CpG dinucleotide sequences within an estrogen responsive enhancer within a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46.

In yet another example of the methods disclosed herein, the subject has been diagnosed as being refractory to endocrine therapy or responsive to endocrine therapy based on differential methylation at one or more CpG dinucleotide sequences within an estrogen responsive enhancer within a gene selected from FOXA1, ESR1 and/or GATA3.

The present disclosure also provides a kit for predicting response to endocrine therapy in a subject suffering from estrogen receptor 1 (ESR1) positive breast cancer, said kit comprising:

(i) one or more reagents configured to determine the methylation status of one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in the subject; and

(ii) a reference material which provides a reference level of methylation of the corresponding one or more CpG dinucleotide sequences.

The present disclosure also provides a kit for predicting the therapeutic outcome of and/or monitoring the progression of estrogen receptor 1 (ESR1) positive breast cancer in a subject receiving or about to receive endocrine therapy, said kit comprising:

(i) one or more reagents configured to determine methylation status of one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in the subject; and

(ii) a reference material which provides a reference level of methylation of the corresponding one or more CpG dinucleotide sequences.

In any of the kits disclosed herein, the one or more reagents is/are configured to determine methylation status of one or more CpG dinucleotide sequences within one or more ESR1 binding sites as defined in Table 1. In another example, the one or more reagents is/are configured to determine methylation status of one or more CpG dinucleotide sequences within one or more ESR1 binding sites as defined in Table 2. In yet another example, the one or more reagents is/are configured to determine methylation status of one or more CpG dinucleotide sequences within one or more ESR1 binding sites as defined in Table 3.

Reagents which may be particularly useful in kits of the disclosure may be those that are configured to determine methylation status of one or more CpG dinucleotide sequences within an estrogen responsive enhancer of a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1. For example, the kit may comprise one or more reagents configured to determine methylation status of one or more CpG dinucleotide sequences within an estrogen responsive enhancer within a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1. Exemplary reagents for inclusion in a kit of the disclosure include those configured to determine methylation status of one or more CpG dinucleotide sequences within one or more genomic regions selected from those defined in rows 57, 111-113, 256-258, 288-289, 469-470, 805, 821-822 and 824-826 of Table 1.

In another example, kits of the disclosure may comprise one or more reagents configured to determine methylation status of one or more CpG dinucleotide sequences within an estrogen responsive enhancer within a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46 e.g., such as a reagent configured to determine methylation status at one or more CpG dinucleotide sequences within an estrogen responsive enhancer within a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46.

In yet another example, kits of the disclosure may comprise one or more reagents configured to determine methylation status of one or more CpG dinucleotide sequences within an estrogen responsive enhancer within a gene selected from FOXA1, ESR1 and/or GATA3.

In any of the kits disclosed herein, the reference level of methylation is a level of methylation determined for one or more CpG dinucleotide sequences within a corresponding genomic region of a sample selected from the group consisting of:

(i) a sample from a normal or healthy tissue;

(ii) a sample comprising a non-cancerous cell;

(iii) a sample comprising a cancerous cell, other than a breast cancer cell characterized as being ESR1-negative subtype;

(iv) a sample comprising a cancerous cell, other than a breast cancer cell characterized as being a ESR1-positive subtype which is refractory to endocrine therapy;

(v) an extract of any one of (i) to (iv);

(vi) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in a normal or healthy individual or a population of normal or healthy individuals;

(vii) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in an individual or a population of individuals having cancer other than ESR1-negative breast cancer subtype;

(ix) a data set comprising levels of methylation for the one or more CpG dinucleotide sequences within the corresponding estrogen responsive enhancer in the subject being tested wherein the levels of methylation are determined for a matched sample having normal cells.

The cancerous cell in (iii) and/or (iv) above may be, for example, a breast cancer cell. Alternatively or in addition, the cancer in (vii) and/or (viii) above may be, for example, breast cancer.

The present disclosure also provides any one of the kits disclosed herein when used in any one or more of the methods disclosed herein.

In addition, the present disclosure provides use of one or more reagents in the preparation of a medicament for predicting response to endocrine therapy in a subject suffering from estrogen receptor 1 (ESR1) positive breast cancer, wherein the one or more reagents is/are configured to determine methylation status of one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in the subject.

The present disclosure also provides the use of one or more reagents in the preparation of a medicament for predicting the therapeutic outcome of and/or monitoring the progression of estrogen receptor 1 (ESR1) positive breast cancer in a subject receiving or about to receive endocrine therapy, wherein the one or more reagents is/are configured to determine methylation status of one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in the subject.

In one example, the one or more reagents is/are configured to determine methylation status of one or more CpG dinucleotide sequences within one or more ESR1 binding sites as defined in Table 1. In another example, the one or more reagents is/are configured to determine methylation status of one or more CpG dinucleotide sequences within one or more ESR1 binding sites as defined in Table 2. In yet another example, the one or more reagents is/are configured to determine methylation status of one or more CpG dinucleotide sequences within one or more ESR1 binding sites as defined in Table 3.

Reagents which may be particularly useful for in the preparation of medicaments as disclosed herein may be those that are configured to determine methylation status of one or more CpG dinucleotide sequences within an estrogen responsive enhancer of a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1. For example, the medicament may comprise one or more reagents configured to determine methylation status of one or more CpG dinucleotide sequences within an estrogen responsive enhancer within a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1. Exemplary reagents for inclusion in a medicament as described herein include those configured to determine methylation status of one or more CpG dinucleotide sequences within one or more genomic regions selected from those defined in rows 57, 111-113, 256-258, 288-289, 469-470, 805, 821-822 and 824-826 of Table 1.

In another example, reagents which are particularly useful for in the preparation of medicaments as disclosed herein include those that are configured to determine methylation status of one or more CpG dinucleotide sequences within an estrogen responsive enhancer of a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46 e.g., such as a reagent configured to determine methylation status at one or more CpG dinucleotide sequences within an estrogen responsive enhancer within a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46.

In yet another example, reagents which are particularly useful for in the preparation of medicaments as disclosed herein include those that are configured to determine methylation status of one or more CpG dinucleotide sequences within an estrogen responsive enhancer within a gene selected from FOXA1, ESR1 and/or GATA3.

In addition, any of the methods disclosed herein may further comprise a step of administering a therapeutic treatment to a subject. For example, the determination of the presence of a particular subtype of ESR1 positive breast cancer e.g., ESR1 positive breast cancer which is responsive to endocrine therapy or ESR1 positive breast cancer which is resistant to endocrine therapy, in a subject may lead to the administration of a particular therapeutic treatment to that subject, which therapeutic treatment is particularly tailored to that particular subtype of breast cancer.

Each feature of any particular aspect or embodiment or example of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment or example of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.

FIG. 1. Genome-wide DNA methylation profiling of endocrine resistant MCF7 cell models. (a-c) A colorimetric density plot showing correlation between the HM450 methylation profile of the endocrine resistant MCF7X (a), TAMR (b) and FASR (c) cells and the parent (endocrine sensitive) MCF7 cells. The plots show that while the methylation profile of the endocrine resistant cell lines is strongly correlated with the parent MCF7 cells (MCF7X, r²=0.895; TAMR, r²=0.91; FASR, r²=0.848; Pearson's Coefficient), both the MCF7X and TAMR cells predominantly gain DNA methylation, whereas the FASR cells exhibit both hyper and hypomethylation events relative to parent MCF7 cells. (d) A Venn diagram showing the overlap of HM450 methylation probes that are more heavily methylated in multiple endocrine resistant cells compared to the parent MCF7 cells (FDR<0.01). (e) A bar plot showing the association of differentially methylated HM450 probes that were common to all endocrine resistant cell lines (compared to the parent MCF7 cells) across functional/regulatory regions of the genome as determined by MCF7 ChromHMM annotation¹³. The height of the bars represents the level of enrichment measured as a ratio between the frequency of hypermethylated (dark blue) or hypomethylated (light blue) probes overlapping a functional element over the expected frequency if such overlaps were to occur at random in the genome. Statistically significant enrichments (p-value <<0.0001; hyper-geometric test) are marked with an asterisk. The numbers of commonly hyper/hypomethylated probes located within each specific region are presented in the respective column.

FIG. 2. ESR1 regulation of enhancer sites commonly hypermethylated in endocrine resistant cell models. (a) A bar plot showing the association of HM450 probes that were more heavily methylated in endocrine resistant cell models (compared to MCF7 cells) and also specifically located in enhancer regions, across ESR1, FOXA1 and GATA3 binding sites in MCF7 cells. The height of the bars represents enrichment measured as a ratio between the frequency of hypermethylated probes in enhancers overlapping a transcription factor binding site over the expected frequency if such overlaps were to occur at random across the genome (*p-value <<0.0001; hyper-geometric test). The numbers of commonly hyper/hypomethylated probes located within each specific region are presented in the columns. (b) A Venn diagram showing the overlap of enhancer-specific HM450 methylation probes that are more heavily methylated in multiple endocrine resistant cell models (compared to MCF7 cells) across ESR1, FOXA1 and GATA3 binding sites. (c) A box plot showing the log fold change (log FC) in ESR1 binding signal at ESR1-enhancer sites that contain at least one commonly hypermethylated probe (yellow box) and all other ESR1-enhancer sites that overlap a HM450 probe (grey box) in TAMR cells compared to the parent MCF7 cells. The mean log FC in ESR1 binding at hypermethylated ER-enhancer sites is −2.29 and the mean log FC of all other ESR1-enhancer sites is −0.52 (* p<<0.0001; t-test). (The whiskers of the boxplot extend to the most extreme data point, which is no more than 1.5×IQR from the box). (d) IGV screen shots to illustrate the loss of ESR1 binding in TAMR cells compared to the parent MCF7 cells in enhancer regions that overlap methylation probes that are more heavily methylated in the endocrine resistant cell models. The MCF7 ChromHMM regions are colour coded as follows; blue=enhancer, yellow=transcribed, green=promoter, light blue=CTCF, burgundy=transcribed. The HM450 beta values are shown for the MCF7 (green), MCF7X (burgundy), TAMR (orange) and FASR cells (red) and are representative of biological duplicates. ESR1 ChIP data (blue) is presented in duplicate for both MCF7 and TAMR cells. The ESR1-enhancers that overlap regions of endocrine-resistant specific hypermethylation are highlighted by the blue boxes.

FIG. 3. Characterisation of genes whose expression is negatively affected by ESR1-enhancer hypermethylation in human breast cancer. (a) Hypergeometric testing of genes whose expression is negatively affected by ESR1-enhancer hypermethylation in human breast cancer (n=291) in the MSigDB C2 database. The height of the bars represents the level of enrichment measured as a ratio between the number of genes overlapping an MSigDB C2 gene set over the expected frequency if such overlaps were to occur at random in the genome (p-value <<0.0001; hyper-geometric test). (b) Unsupervised clustering of the gene set whose expression is negatively correlated with ESR1-enhancer methylation (n=291) in ESR1 positive (red) (n=174) and ESR1 negative (n=588) breast cancer patients (obtained from TGCA breast cohort RNA-seq data).

FIG. 4. Graphical representation of the correlation between the technical replicates presented in FIG. 5. Scatter plots showing the correlation between the technical replicates of multiplex bisulphite-PCR resequencing data presented in FIG. 5 (R=Pearson correlation).

FIG. 5. Association between ESR1 enhancer methylation and breast cancer subtype. (a) A boxplot showing the median methylation of all HM450 probes that overlap an enhancer region, an ESR1 binding site and demonstrate hypermethylation in endocrine resistant vs parental MCF7 cells (n=801 probes), in normal breast tissue (green) (n=97), luminal A (light blue) (n=301), luminal B (dark blue) (n=52) and ESR1-negative (red) (n=105) breast cancer (data obtained from TCGA breast cancer cohort) (* p<0.05, ** p<<0.0001; Mann-Whitney U test). (The whiskers of the boxplot extend to the most extreme data point, which is no more than 1.5×IQR from the box). (b) A heatmap showing the methylation profile of 801 ESR1-enhancer specific HM450 probes that are more heavily methylated in endocrine resistant vs parent MCF7 cells in normal breast tissue (green) (n=97), luminal A (light blue) (n=301), luminal B (dark blue) (n=52) and ESR1-negative (red) (n=105) breast cancer. Columns are patient samples and rows are HM450 probes. The level of methylation is represented by a colour scale—blue for low levels and red for high levels of methylation. (c) Boxplots showing distribution of methylation beta values in normal n=97 (green), luminal A (light blue) (n=301), luminal B (dark blue) (n=52) and ESR1-negative (red) (n=105) breast cancer samples across HM450 probes overlapping the ESR1-binding site located within the DAXX enhancer (Chr6: 33288112-33288670) (left panel) and the DAXX promoter region (1000 bp upstream and 100 bp downstream of the transcription start site) (Chr6: 33290693-33291793) (right panel). (The whiskers of the boxplots extend to the most extreme data point, which is no more than 1.5×IQR from the box).

FIG. 6. ESR1-Enhancer DNA hypermethylation in acquired endocrine resistance in human breast cancer. (a-d) (Left panel) A scatter plot showing the methylation of individual CpG sites across the ESR1-enhancer region of interest (a—GATA3—Chr10: 8103616-8103673; b—ITPK1—Chr14: 93412603-93412703; c—ESR1—Chr6: 152124782-152125008; d—GET4—Chr7: 922042-922114) in 3 primary luminal A breast cancers from patients that received adjuvant endocrine therapy and experienced relapse free survival (RFS) (green), 3 primary luminal A breast cancers from patients that relapsed following adjuvant endocrine therapy (n/RFS) (blue) and their matched local relapse (red). Each dot represents the % methylation at an individual CpG site for a single patient and the lines represent the average methylation for the region in primary RFS (green), primary n/RFS (blue) and matched recurrent tumours (red). (Right Panel) Box plots showing the distribution of methylation values across the ESR1-enhancer region depicted in the left panel for RFS (green), prognosis/RFS (blue) and matched recurrent tumours (red); p-values correspond to t-test comparison between RFS vs n/RFS, and n/RFS vs relapse tumours. (The whiskers of the boxplot extend to the most extreme data point, which is no more than 1.5×IQR from the box).

FIG. 7. ESR1-Enhancer DNA hypermethylation in cell models of acquired endocrine resistance. (a-I) (Left panel) A scatter plot showing the methylation of individual CpG sites across the ESR1-enhancer region of interest (a—DAXX—Chr6: 33288296-33288372; b—GET4—Chr7: 922042-922114; c—ESR1—Chr6: 152124782-152125008; d-NCOR2—Chr12: 124844786-124844883; e—GATA3—Chr10: 8103616-8103673; f—ITPK1—Chr14: 93412603-93412703; g—RXRA—Chr9: 137252867-137252967; h—MSI2—Chr17: 55371693-55371786; i—C8orf46—Chr8: 67425069-67425134) in the parental MCF7 cells (green), and the endocrine resistant derivatives, TAMR (orange), MCF7X (purple) and FASR (red). Each dot represents the % methylation at an individual CpG site and the lines represent the average methylation for the region. (Right Panel) Box plots showing the distribution of methylation values across the ESR1-enhancer region depicted in the left panel for the parental MCF7 cells (green), and the endocrine resistant derivatives, TAMR (orange), MCF7X (purple) and FASR (red) (mean±SD) (*p<0.05, **p<0.01, ***p<0.001; t-test). (The whiskers of the boxplot extend to the most extreme data point, which is no more than 1.5×IQR from the box.)

FIG. 8. Graphical representation of the correlation between the technical replicates presented in FIG. 7. Scatter plots showing the correlation between the technical replicates of multiplex bisulphite-PCR resequencing data presented in FIG. 7 (R=Pearson correlation).

FIG. 9. ESR1 enhancer DNA hypermethylation in acquired endocrine resistance in human breast cancer. (a-e) (Left panel) A scatter plot showing the methylation of individual CpG sites across the ESR1-enhancer region of interest (a—DAXX—Chr6: 33288296-33288372; b—MSI2—Chr17: 55371693-55371786; c—NCOR2—Chr12: 124844786-124844883; d—RXRA—Chr9: 137252867-137252967; e—C8orf46—Chr8: 67425069-67425134) in 3 primary luminal A breast cancers from patients that received adjuvant endocrine therapy and exhibited relapse free survival (RFS) (green), 3 primary luminal A breast cancers from patients that relapsed following adjuvant endocrine therapy, defined as no relapse free survival (n/RFS) (blue) and their matched local relapse (red). Each dot represents the % methylation at an individual CpG site for a single patient and the lines represent the average methylation for the region in primary RFS (green), primary n/RFS (blue) and matched recurrent tumours (red). (Right Panel) Box plots showing the distribution of methylation values across the ESR1-enhancer region depicted in the left panel for RFS (green), prognosis/RFS (blue) and matched recurrent tumours (red); p-values correspond to t-test comparison between RFS vs n/RFS, and n/RFS vs relapse tumours. (The whiskers of the boxplots extend to the most extreme data point, which is no more than 1.5×IQR from the box).

FIG. 10. This figure provides a flow-chart illustrating a computer system of the disclosure which may be used for predicting response to endocrine therapy in a subject suffering from ESR1 positive breast cancer.

KEY TO THE SEQUENCE LISTING

SEQ ID NO: 1: DNA sequence for bisulphite-PCR primer designated GATA3_ct_f2

SEQ ID NO: 2: DNA sequence for bisulphite-PCR fusion primer designated GATA3_ct_f2

SEQ ID NO: 3: DNA sequence for bisulphite-PCR primer designated GATA3_ct_r2

SEQ ID NO: 4: DNA sequence for bisulphite-PCR fusion primer designated GATA3_ct_r2

SEQ ID NO: 5: DNA sequence for bisulphite-PCR primer designated ESR1_ct_f1

SEQ ID NO: 6: DNA sequence for bisulphite-PCR fusion primer designated ESR1_ct_f1

SEQ ID NO: 7: DNA sequence for bisulphite-PCR primer designated ESR1_ct_r1

SEQ ID NO: 8: DNA sequence for bisulphite-PCR fusion primer designated ESR1_ct_r1

SEQ ID NO: 9: DNA sequence for bisulphite-PCR primer designated ESR1_ct_f2

SEQ ID NO: 10: DNA sequence for bisulphite-PCR fusion primer designated ESR1_ct_f2

SEQ ID NO: 11: DNA sequence for bisulphite-PCR primer designated ESR1_ct_r2

SEQ ID NO: 12: DNA sequence for bisulphite-PCR fusion primer designated ESR1_ct_r2

SEQ ID NO: 13: DNA sequence for bisulphite-PCR primer designated GET4_ct_f1

SEQ ID NO: 14: DNA sequence for bisulphite-PCR fusion primer designated GET4_ct_f1

SEQ ID NO: 15: DNA sequence for bisulphite-PCR primer designated GET4_ct_r1

SEQ ID NO: 16: DNA sequence for bisulphite-PCR fusion primer designated GET4_ct_r1

SEQ ID NO: 17: DNA sequence for bisulphite-PCR primer designated ITPK1_ct_f1

SEQ ID NO: 18: DNA sequence for bisulphite-PCR fusion primer designated ITPK1_ct_f1

SEQ ID NO: 19: DNA sequence for bisulphite-PCR primer designated ITPK1_ct_r2

SEQ ID NO: 20: DNA sequence for bisulphite-PCR fusion primer designated ITPK1_ct_r2

SEQ ID NO: 21: DNA sequence for bisulphite-PCR primer designated MSI2_ct_f2

SEQ ID NO: 22: DNA sequence for bisulphite-PCR fusion primer designated MSI2_ct_f2

SEQ ID NO: 23: DNA sequence for bisulphite-PCR primer designated MSI2_ct_r2

SEQ ID NO: 24: DNA sequence for bisulphite-PCR fusion primer designated MSI2_ct_r2

SEQ ID NO: 25: DNA sequence for bisulphite-PCR primer designated C8orf46_ga_f1

SEQ ID NO: 26: DNA sequence for bisulphite-PCR fusion primer designated C8orf46_ga_f1

SEQ ID NO: 27: DNA sequence for bisulphite-PCR primer designated C8orf46_ga_r1

SEQ ID NO: 28: DNA sequence for bisulphite-PCR fusion primer designated C8orf46_ga_r1

SEQ ID NO: 29: DNA sequence for bisulphite-PCR primer designated DAXX_ga_f2

SEQ ID NO: 30: DNA sequence for bisulphite-PCR fusion primer designated DAXX_ga_f2

SEQ ID NO: 31: DNA sequence for bisulphite-PCR primer designated DAXX_ga_r2

SEQ ID NO: 32: DNA sequence for bisulphite-PCR fusion primer designated DAXX_ga_r2

SEQ ID NO: 33: DNA sequence for bisulphite-PCR primer designated NCOR2_ga_f1

SEQ ID NO: 34: DNA sequence for bisulphite-PCR fusion primer designated NCOR2_ga_f1

SEQ ID NO: 35: DNA sequence for bisulphite-PCR primer designated NCOR2ga_r1

SEQ ID NO: 36: DNA sequence for bisulphite-PCR fusion primer designated NCOR2_ga_r1

SEQ ID NO: 37: DNA sequence for bisulphite-PCR primer designated RXRA_ga_f1

SEQ ID NO: 38: DNA sequence for bisulphite-PCR fusion primer designated RXRAga_f1

SEQ ID NO: 39: DNA sequence for bisulphite-PCR primer designated RXRA_ga_r1

SEQ ID NO: 40: DNA sequence for bisulphite-PCR fusion primer designated RXRAga_r1

DETAILED DESCRIPTION

General

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or

“X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Selected Definitions

As used herein, the term “diagnosis”, and variants thereof, such as, but not limited to “diagnose” or “diagnosing” shall include, but not be limited to, a primary diagnosis of a clinical state or any primary diagnosis of a clinical state. A diagnostic method described herein is also useful for assessing responsiveness of a subject to a particular form of therapy, such as determining whether a subject having cancer will be responsive to endocrine therapy. A diagnostic method described herein is also useful for assessing the remission of a subject, or monitoring disease recurrence, or tumor recurrence, such as following surgery, radiation therapy, adjuvant therapy or chemotherapy, or determining the appearance of metastases of a primary tumor. All such uses of the assays described herein are encompassed by the present disclosure.

As used herein, the term “prognosis”, and variants thereof, such as, but not limited to “prognosing” shall refer to the prediction of the likelihood that a cancer patient e.g., a breast cancer patient, will have a cancer-attributable death, or that the cancer will progress to a worsening stage in the subject, such as recurrence or metastatic spread, or that the cancer will have or develop drug resistance, such as resistance to endocrine therapy.

As used herein, the term “cancer” shall be taken to include a disease that is characterized by uncontrolled growth of cells within a subject. The term “cancer” shall not be limited to cancer of a specific tissue or cell type. Those skilled in the art will be aware that as a cancer progresses, metastases occur in organs and tissues outside the site of the primary cancer. Accordingly, the term “cancer” as used herein shall be taken to include a metastasis of a cancer in addition to a primary tumor. A particularly preferred cancer in the context of the present disclosure is breast cancer.

As used herein, the term “breast cancer” shall be understood to include a disease that is characterized by uncontrolled growth of cells from breast tissue of a subject.

As used herein, the term “estrogen receptor 1 (ESR1) positive breast cancer” shall be understood to refer to a breast cancer which is characterised by increased expression of the ESR1 gene when compared to a non-cancerous sample or an ESR1 negative cancerous sample, or which is characterised by a level of expression of the ESR1 gene which is different from the level of expression of a housekeeping gene.

As used herein, the term “estrogen receptor 1 (ESR1) negative breast cancer” shall be understood to refer to a breast cancer which is characterised by reduced expression of the ESR1 gene when compared to a non-cancerous sample, or an ESR1 positive cancerous sample, or which is characterised by a level of expression of the ESR1 gene which is not significantly different from the level of expression of a housekeeping gene, or which is characterised by the absence of a detectable level of expression of the ESR1 gene, or which is characterised by the absence of expression of the ESR1 gene.

As used herein, the term “estrogen responsive enhancer”, “estrogen responsive enhancers”, or similar, refers to a region or regions of the genome to which estrogen-bound estrogen receptor protein, including estrogen receptor 1 (ESR1) protein bound to estrogen, binds to activate transcription of a gene. It will be appreciated that an “estrogen responsive enhancer” may be located within the gene it activates or may be cis-acting and located away from the gene it activates e.g., upstream or downstream from the gene's start site or in an unrelated part of the genome. For example, an “estrogen responsive enhancer” may be defined according to the means described in Example 2 herein, and in particular, using the ChromHMM segmentation program as described in Taberlay et al., (2014).

The term “estrogen receptor 1 binding site”, “ESR1 binding site”, or similar, as used herein refers to a region of the genome to which the ESR1 protein binds e.g., including free ESR1 protein or ESR1 protein bound to estrogen. An “estrogen responsive enhancer” may comprise one or more “estrogen receptor 1 binding sites”.

The term “endocrine therapy” is given to those treatments which target the estrogen receptor e.g., ESR1, by blocking receptor binding with an antagonist or by depriving a cancer e.g., breast cancer, of estrogen. In the context of the present disclosure, the term “endocrine therapy” shall include therapy or treatment with an agent or compound which inhibits estrogen e.g., from acting on breast cancer cells. Such therapy is routine in treatment of breast cancer which is determined to be estrogen receptor positive i.e., expresses estrogen receptor protein, such as breast cancer which is ESR1 positive. Drugs suitable for use in endocrine therapy are well known in the art, and include, for example, anastrozole, exemestane, fulvestrant, goserelin, letrozole, leuprorelin, leuprolide acetate, megestrol, palbociclib, tamoxifen and/or toremifene.

As used herein, breast cancer which is characterised as being “resistant” or “refractory” or as having “resistance” to endocrine therapy, refers to a breast cancer which does not or will not respond to treatment with endocrine therapy.

The term “tumor” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. It will also be understood that the term “tumor sample” or similar in the context of a patient having cancer refers to a sample comprising tumor material obtained from a cancer patient. The term encompasses tumor tissue samples, for example, tissue obtained by surgical resection and tissue obtained by biopsy, such as for example, a core biopsy or a fine needle biopsy. In a particular embodiment, the tumor sample is a fixed, wax-embedded tissue sample, such as a formalin-fixed, paraffin-embedded tissue sample. Additionally, the term “tumor sample” encompasses a sample comprising tumor cells obtained from sites other than the primary tumor, e.g., circulating tumor cells.

The term “test sample” as used herein is taken to mean any tissue or body fluid sample taken from a subject having or suspected of having breast cancer. The presence of breast cancer in the subject may therefore already have been determined. Thus, the methods of the present disclosure may be used to determine a particular subtype of breast cancer (such as ESR1-positive breast cancer which is responsive to endocrine therapy or ESR1-positive breast cancer which is resistance to endocrine therapy) in a subject known to have ESR1-positive breast cancer. Thus, the “test sample” may be a “tumor sample” as defined herein. Alternatively, the methods of the present disclosure may be used to determine the presence of breast cancer e.g., such as ESR1 positive breast cancer, in a subject in whom the presence of breast cancer has not previously been determined.

As used herein, the term “methylation” will be understood to mean the presence of a methyl group added by the action of a DNA methyl transferase enzyme to a cytosine base or bases in a region of nucleic acid e.g. genomic DNA. Accordingly, the term, “methylation status” as used herein refers to the presence or absence of methylation in a specific nucleic acid region e.g., genomic region. In particular, the present disclosure relates to detection of methylated cytosine (5-methylcytosine). A nucleic acid sequence may comprise one or more CpG methylation sites.

As used herein, the term “differential methylation” shall be taken to mean a change in the relative amount of methylation of a nucleic acid e.g., genomic DNA, in a biological sample e.g., such as a cell or a cell extract, or a body fluid (such as blood), obtained from a subject. In one example, the term “differential methylation” is an increased level of methylation of a nucleic acid. In another example, the term “differential methylation” is a decreased level of methylation of a nucleic acid. In the present disclosure, “differential methylation” is generally determined with reference to a baseline level of methylation for a given genomic region, such as a non-cancerous sample, including a non-cancerous matched sample from a subject known to have cancer e.g., breast cancer. For example, the level of differential methylation may be at least 2% greater or less than a baseline level of methylation, for example at least 5% greater or less than a baseline level of methylation, or at least 10% greater or less than a baseline level of methylation, or at least 15% greater or less than a baseline level of methylation, or at least 20% greater or less than a baseline level of methylation, or at least 25% greater or less than a baseline level of methylation, or at least 30% greater or less than a baseline level of methylation, or at least 40% greater or less than a baseline level of methylation, or at least 50% greater or less than a baseline level of methylation, or at least 60% greater or less than a baseline level of methylation, or at least 70% greater or less than a baseline level of methylation, or at least 80% greater or less than a baseline level of methylation, or at least 90% greater or less than a baseline level of methylation. Thus, the level of differential methylation may be at least 10%, at least 15%, at least 20%, or at least 25% greater than or less than a baseline level of methylation. For example, the level of differential methylation may be at least 10%, at least 15%, at least 20%, or at least 25% greater than a baseline level of methylation.

As used herein, a “CpG dinucleotide”, “CpG methylation site” or equivalent, shall be taken to denote a cytosine linked to a guanine by a phosphodiester bond. CpG dinucleotides are targets for methylation of the cytosine residue and may reside within coding or non-coding nucleic acids. Non-coding nucleic acids are understood in the art to include introns, 5′-untranslated regions, 3′ untranslated regions, promoter regions of a genomic gene, or intergenic regions.

As used herein, a “reference level of methylation” shall be understood to include a level of methylation detected in a corresponding nucleic acid from a normal or healthy cell or tissue or body fluid, or a data set produced using information from a normal or healthy cell or tissue or body fluid. A “reference level of methylation” can also include a level of methylation detected in a corresponding nucleic acid from a cell or tissue or body fluid from a subject suffering from ESR1-positive breast cancer who is refractory to endocrine therapy, or a data set produced using information from a cell or tissue or body fluid from a subject suffering from ESR1-positive breast cancer who is refractory to endocrine therapy i.e., to provide a baseline level of methylation in a subject who is refractive to endocrine therapy. A “reference level of methylation” can also include a level of methylation detected in a corresponding nucleic acid from a cell or tissue or body fluid from a subject suffering from ESR1-positive breast cancer who is responsive to endocrine therapy, or a data set produced using information from a cell or tissue or body fluid from a subject suffering from ESR1-positive breast cancer who is responsive to endocrine therapy i.e., to provide a baseline level of methylation in a subject who is responsive to endocrine therapy. For example, a “reference level of methylation” may be a level of methylation in a corresponding nucleic acid from:

(i) a sample from a normal or healthy tissue;

(ii) a sample comprising a non-cancerous cell;

(iii) a sample comprising a cancerous cell, other than a breast cancer cell characterized as being ESR1-negative subtype;

(iv) a sample comprising a cancerous cell, other than a breast cancer cell characterized as being a ESR1-positive subtype which is refractory to endocrine therapy;

(v) a sample comprising a breast cancer cell characterized as being a ESR1-positive subtype which is refractory to endocrine therapy;

(vi) a sample comprising a breast cancer cell characterized as being a ESR1-positive subtype which is responsive to endocrine therapy;

(vii) an extract of any one of (i) to (vi);

Preferably, the non-cancerous sample is (i) or (ii) or (viii) or (xi).

In one example, the reference level of methylation may be a level of methylation determined for one or more CpG dinucleotide sequences within a corresponding genomic region of a healthy breast epithelial cell. Thus, the normal or healthy cell or tissue may comprise a breast epithelial cell. In addition, the “non-cancerous cell” may be a breast epithelial cell. The extract of the normal or healthy cell or tissue, or of the non-cancerous cell may be an extract from a breast epithelial cell.

As used herein, the term “subject” or “patient” shall be taken to mean any animal including a human, preferably a mammal. Exemplary subjects include but are not limited to humans, primates, livestock (e.g. sheep, cows, horses, donkeys, pigs), companion animals (e.g. dogs, cats), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs, hamsters), captive wild animals (e.g. fox, deer). Preferably the mammal is a human or primate. More preferably the mammal is a human.

DNA Methylation Biomarkers

The present disclosure provides a method for detecting differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in a subject suffering from ESR1 positive breast cancer, said method comprising performing an assay on a sample from the subject configured to determine methylation status at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in the subject, and detecting differential methylation at the one or more CpG dinucleotide sequences within the one or more estrogen responsive enhancers in the subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences. For example, detecting differential methylation at the one or more CpG dinucleotide sequences may comprise comparing a level of methylation at the one or more CpG dinucleotide sequences in the subject to the reference level of methylation for the corresponding one or more CpG dinucleotide sequences, and determining whether methylation at the one or more CpG dinucleotide sequences in the subject differs to the corresponding reference level(s) of methylation.

The present disclosure also provides a method for predicting response to endocrine therapy in a subject suffering from ESR1 positive breast cancer, comprising detecting the methylation status of one or more CpG dinucleotides within one or more estrogen responsive enhancers in the subject, and determining differential methylation at said one or more CpG dinucleotides in the subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences, wherein differential methylation at said one or more CpG dinucleotides in the subject relative to the reference level is indicative of the subject's likely response to endocrine therapy. For example, determining increased methylation at the one or more CpG dinucleotide sequences within the one or more estrogen responsive enhancers relative to the reference level may be indicative of the ESR1-positive breast cancer being refractory to endocrine therapy.

The present disclosure also provides a method for diagnosing ESR1 positive breast cancer which is refractory to endocrine therapy in a subject suffering from ESR1 positive breast cancer, comprising detecting the methylation status of one or more CpG dinucleotides within one or more estrogen responsive enhancers in the subject, and determining differential methylation at said one or more CpG dinucleotides in the subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences, wherein differential methylation at said one or more CpG dinucleotides in the subject relative to the reference level is indicative of the subject having breast cancer which is refractory to endocrine therapy.

The present disclosure also provides a method for predicting the therapeutic outcome of and/or monitoring the progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy, comprising detecting the methylation status of one or more CpG dinucleotides within one or more estrogen responsive enhancers in the subject, and determining differential methylation at said one or more CpG dinucleotides in the subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences, wherein differential methylation at said one or more CpG dinucleotides in the subject relative to the reference level is indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. For example, increased methylation at the one or more CpG dinucleotide sequences within the one or more estrogen responsive enhancers relative to the reference level may be indicative of the ESR1-positive breast cancer being refractory to endocrine therapy and/or that the subject is not responding to the endocrine therapy.

In any one of the foregoing methods, identifying differential methylation at the one or more CpG dinucleotides in the subject relative to the reference level may be used to determine whether the ESR1-positive breast cancer is a luminal A breast cancer subtype or a luminal B breast cancer subtype. Accordingly, methods of the disclosure may also comprise determining whether the ESR1-positive breast cancer is a luminal A breast cancer subtype or a luminal B breast cancer subtype.

The one or more CpG dinucleotide sequences may be within one or more ESR1 binding sites which are within one or more estrogen responsive enhancers. For example, the one or more CpG dinucleotide sequences may be within one or more of the ESR1 binding sites set forth in Table 1. The ESR1 binding sites set forth in Table 1 are defined with reference to human genome assembly version 19 (“hg19”). As used herein, “hg19” refers to the February 2009 human reference sequence (Genome Reference Consortium GRCh37), which was produced by the International Human Genome Sequencing Consortium. Further information about this assembly is provided under the reference Genome Reference Consortium GRCh37 in the NCBI Assembly database. Thus, the nucleotide sequences of each of the regions identified in Table 1 (or in any of the Tables disclosed herein) can be identified by reference to hg19, using the “start” and “end” positions described in Table 1 (or in any of the Tables disclosed herein).

The 856 genomic regions listed in Table 1 encompass ESR1 binding sites that overlap estrogen responsive enhancer regions containing hypermethylated CpG dinucleotides in multiple models of endocrine resistance (i.e., MCF7-derived cell lines, tamoxifen-resistant (TAMR)10, fulvestrant-resistant (FASR)11 and estrogen deprivation resistant (MCF7X)12 cells) relative to ESR1-positive hormone sensitive MCF7 cells. Increased methylation at the 856 genomic regions listed in Table 1 was found to be associated with a reduction in ESR1 binding. For each of the ESR1 binding sites set forth in Table 1, the following information is provided:

(i) chromosome ID (Column 2);

(ii) genomic coordinates of ESR1-binding site with respect to hg19 (Columns 3-4);

(iii) ESR1-binding site name (Column 5);

(iv) name of gene within which ESR1-binding site is located (Column 6); and

(v) whether hypermethylation resulted in loss of ESR1 binding in TAMR cells

TABLE 1

Hypermethylated ESR1 enhancer binding sites

Loss of ESR1

binding in TAMR

Row No.
Chromosome
Start
End
ESR1 site name
Gene
Cells

1
7
27210940
27211286
ER_100307
HOXA-AS4
No

2
7
43288514
43288831
ER_100892
HECW1
Yes

3
7
44224085
44224425
ER_100934
GCK
No

4
7
47611478
47611980
ER_101074
TNS3
No

5
7
55620324
55620676
ER_101291
VOPP1
No

6
7
73866691
73867043
ER_101653
GTF2IRD1
Yes

7
7
74020804
74021327
ER_101674
GTF2IRD1
No

8
7
74024605
74024980
ER_101675
GTF2IRD1
No

9
7
75279353
75279713
ER_101692
HIP1
Yes

10
7
75362309
75362707
ER_101699
HIP1
Yes

11
1
223854199
223854500
ER_10205
CAPN8
Yes

12
7
87855579
87855879
ER_102057
SRI
No

13
1
224400118
224400678
ER_10231
NVL
Yes

14
7
98989800
98990147
ER_102479
ARPC1B
Yes

15
7
99719664
99719969
ER_102522
CNPY4
Yes

16
7
100463589
100463889
ER_102585
SLC12A9
No

17
7
100464090
100464390
ER_102586
SLC12A9
Yes

18
7
100769916
100770235
ER_102602
SERPINE1
Yes

19
7
100800324
100800624
ER_102605
AP1S1
Yes

20
7
101017180
101017480
ER_102622
EMID2
No

21
7
101180514
101180818
ER_102635
EMID2
Yes

22
7
101273902
101274202
ER_102645
MYL10
No

23
7
101768479
101768882
ER_102709
CUX1
Yes

24
7
101959130
101959535
ER_102722
SH2B2
Yes

25
7
102080614
102081003
ER_102733
ORAI2
No

26
7
105313870
105314183
ER_102843
ATXN7L1
Yes

27
1
27240488
27241066
ER_1029
NR0B2
No

28
7
107886705
107887046
ER_103011
NRCAM
No

29
1
228598287
228598587
ER_10389
TRIM17
No

30
7
138560635
138560935
ER_104150
KIAA1549
Yes

31
7
139345059
139345423
ER_104192
HIPK2
Yes

32
7
140206668
140207069
ER_104247
DENND2A
No

33
7
143106427
143106737
ER_104387
EPHA1-AS1
Yes

34
7
144104415
144104715
ER_104415
NOBOX
No

35
7
148901363
148901733
ER_104483
ZNF282
No

36
7
149569864
149570164
ER_104518
ATP6V0E2
Yes

37
7
151418719
151419146
ER_104616
PRKAG2
Yes

38
7
151424787
151425151
ER_104617
PRKAG2
No

39
7
151442252
151442633
ER_104619
PRKAG2
No

40
7
151493516
151493829
ER_104624
PRKAG2
Yes

41
7
151553656
151553979
ER_104631
PRKAG2
No

42
7
155600847
155601325
ER_104760
SHH
Yes

43
1
230882258
230882814
ER_10488
CAPN9
Yes

44
7
157069948
157070302
ER_104899
UBE3C
Yes

45
7
157916425
157916765
ER_104936
PTPRN2
Yes

46
1
230895945
230896482
ER_10494
CAPN9
Yes

47
1
231113407
231113970
ER_10502
TTC13
Yes

48
8
1946769
1947125
ER_105037
KBTBD11
No

49
8
17394868
17395391
ER_105282
SLC7A2
Yes

50
8
21948482
21948782
ER_105469
FAM160B2
No

51
8
28223166
28223486
ER_105673
ZNF395
Yes

52
1
28012971
28013583
ER_1057
IFI6
Yes

53
8
37700545
37700845
ER_105921
GPR124
No

54
8
48739114
48739414
ER_106145
PRKDC
Yes

55
8
56791360
56791707
ER_106342
LYN
Yes

56
8
62567898
62568253
ER_106497
ASPH
Yes

57
8
67425037
67425360
ER_106600
C8orf46
Yes

58
8
74002442
74002818
ER_106800
SBSPON
No

59
8
90772144
90772507
ER_107685
RIPK2
No

60
8
98881668
98882017
ER_108508
MATN2
No

61
8
101017572
101017872
ER_108722
RGS22
Yes

62
1
246868690
246869132
ER_10884
SCCPDH
Yes

63
1
249106283
249106588
ER_10898
SH3BP5L
Yes

64
8
102526632
102526954
ER_109025
GRHL2
Yes

65
8
103588959
103589377
ER_109184
ODF1
Yes

66
10
416280
416842
ER_10930
DIP2C
Yes

67
10
579019
579393
ER_10949
DIP2C
Yes

68
8
124172628
124172928
ER_110725
WDR67
No

69
8
124179711
124180074
ER_110727
FAM83A
No

70
8
124193498
124193984
ER_110732
FAM83A
No

71
8
124194626
124194965
ER_110733
FAM83A
No

72
8
126082161
126082738
ER_111167
KIAA0196
Yes

73
8
126398383
126398799
ER_111257
NSMCE2
No

74
10
5538429
5538782
ER_11143
CALML5
Yes

75
8
129103245
129103607
ER_111781
PVT1
No

76
8
134224471
134224833
ER_112110
WISP1
No

77
8
134249814
134250165
ER_112114
NDRG1
No

78
8
135490722
135491122
ER_112226
ZFAT
Yes

79
8
142139549
142139849
ER_112470
DENND3
Yes

80
8
142247703
142248043
ER_112484
SLC45A4
Yes

81
8
143395413
143395760
ER_112563
TSNARE1
Yes

82
8
143625782
143626082
ER_112589
BAI1
Yes

83
8
143690473
143690892
ER_112597
ARC
No

84
8
143763120
143763420
ER_112608
PSCA
No

85
8
143823681
143823981
ER_112619
SLURP1
No

86
8
143851193
143851559
ER_112624
LYNX1
Yes

87
8
143867136
143867477
ER_112628
LY6D
Yes

88
8
144129484
144129962
ER_112655
C8orf31
No

89
10
7984711
7985286
ER_11272
TAF3
No

90
8
145048592
145048911
ER_112777
PLEC
Yes

91
8
145086414
145086732
ER_112784
SPATC1
Yes

92
8
145721323
145721690
ER_112829
PPP1R16A
Yes

93
8
145728596
145728968
ER_112830
GPT
Yes

94
9
33443026
33443378
ER_113615
AQP3
No

95
9
34372740
34373087
ER_113641
KIAA1161
No

96
9
71660820
71661159
ER_113876
FXN
Yes

97
9
79629665
79630009
ER_114134
FOXB2
No

98
9
95834504
95834841
ER_115179
SUSD3
Yes

99
1
3388133
3388514
ER_116
ARHGEF16
Yes

100
9
124979962
124980333
ER_116729
LHX6
No

101
1
32039497
32039797
ER_1168
TINAGL1
No

102
1
3398747
3399296
ER_117
ARHGEF16
Yes

103
9
129433798
129434130
ER_117042
LMX1B
Yes

104
9
130484264
130484588
ER_117145
TTC16
Yes

105
9
130487106
130487456
ER_117146
TTC16
No

106
9
130524258
130524602
ER_117150
SH2D3C
Yes

107
9
130727796
130728104
ER_117181
FAM102A
Yes

108
9
131934253
131934574
ER_117307
IER5L
Yes

109
9
133974376
133974700
ER_117471
AIF1L
Yes

110
9
136728378
136728709
ER_117634
VAV2
Yes

111
9
137252760
137253089
ER_117681
RXRA
Yes

112
9
137258516
137258927
ER_117685
RXRA
Yes

113
9
137302122
137302482
ER_117702
RXRA
Yes

114
9
138997255
138997607
ER_117799
C9orf69
Yes

115
9
139069147
139069469
ER_117809
LHX3
Yes

116
9
139734802
139735102
ER_117864
RABL6
Yes

117
9
139949038
139949338
ER_117897
ENTPD2
Yes

118
9
140188799
140189099
ER_117923
NRARP
No

119
9
140374065
140374394
ER_117940
PNPLA7
Yes

120
9
140408341
140408692
ER_117943
PNPLA7
Yes

121
10
24496562
24497177
ER_11835
KIAA1217
Yes

122
X
16889511
16889834
ER_118379
RBBP7
Yes

123
X
18708777
18709167
ER_118438
PPEF1
No

124
X
40007163
40007510
ER_118823
BCOR
Yes

125
1
32404117
32404574
ER_1191
PTP4A2
No

126
X
133792539
133792870
ER_120294
PLAC1
Yes

127
10
30316774
30317298
ER_12062
KIAA1462
No

128
X
153586498
153586870
ER_120632
FLNA
Yes

129
1
3471648
3472248
ER_122
MEGF6
Yes

130
1
37321565
37322055
ER_1299
GRIK3
Yes

131
1
931045
931617
ER_13
HES4
Yes

132
1
37503121
37503497
ER_1301
GRIK3
Yes

133
10
71213510
71214074
ER_13175
TSPAN15
Yes

134
10
72202041
72202482
ER_13208
NODAL
Yes

135
10
73828553
73829113
ER_13269
SPOCK2
Yes

136
10
74020315
74020712
ER_13281
DDIT4
Yes

137
10
74021102
74021625
ER_13282
DDIT4
Yes

138
1
3614361
3614966
ER_133
TP73
Yes

139
10
76858883
76859398
ER_13395
DUSP13
Yes

140
10
77872231
77872922
ER_13427
C10orf11
No

141
10
82189362
82189690
ER_13657
FAM213A
Yes

142
10
88475369
88475919
ER_13815
LDB3
No

143
10
88703091
88703639
ER_13821
MMRN2
Yes

144
10
95228052
95228774
ER_14079
MYOF
Yes

145
10
99331269
99331862
ER_14202
UBTD1
No

146
10
102792785
102793085
ER_14340
SFXN3
No

147
10
105238778
105239360
ER_14449
CALHM3
No

148
10
112259180
112259480
ER_14629
DUSP5
No

149
1
42420202
42420763
ER_1481
HIVEP3
Yes

150
10
119102102
119102657
ER_14914
PDZD8
No

151
10
121079102
121079402
ER_15001
GRK5
No

152
10
121415148
121415773
ER_15018
BAG3
Yes

153
10
123900770
123901156
ER_15113
TACC2
No

154
10
124888975
124889540
ER_15133
HMX3
No

155
10
126211691
126211991
ER_15194
LHPP
Yes

156
10
126315723
126316102
ER_15199
FAM53B
Yes

157
10
126700346
126700820
ER_15221
CTBP2
Yes

158
1
44300618
44300993
ER_1532
ST3GAL3
Yes

159
10
128606764
128607329
ER_15340
DOCK1
Yes

160
10
134079116
134079490
ER_15434
STK32C
Yes

161
10
134224980
134225280
ER_15443
PWWP2B
Yes

162
10
134261296
134261809
ER_15450
C10orf91
No

163
10
134332284
134332680
ER_15463
INPP5A
Yes

164
10
134346912
134347484
ER_15465
INPP5A
Yes

165
10
134420347
134420647
ER_15478
INPP5A
No

166
10
134498671
134499284
ER_15480
INPP5A
Yes

167
10
134728825
134729477
ER_15487
TTC40
Yes

168
10
134729867
134730225
ER_15488
TTC40
Yes

169
10
135178534
135178842
ER_15502
ECHS1
Yes

170
10
135337035
135337690
ER_15505
CYP2E1
No

171
11
569384
569784
ER_15531
MIR210HG
No

172
11
849066
849366
ER_15551
TSPAN4
No

173
11
1224796
1225357
ER_15568
MUC5B
Yes

174
11
1275364
1275883
ER_15574
MUC5B
No

175
11
1460055
1460355
ER_15581
BRSK2
Yes

176
11
1507075
1507659
ER_15583
MOB2
Yes

177
11
1777371
1777938
ER_15595
CTSD
Yes

178
11
1990361
1990661
ER_15612
MRPL23
No

179
11
2004135
2004733
ER_15613
MRPL23-AS1
No

180
11
2011314
2011614
ER_15617
MRPL23-AS1
No

181
11
2181411
2181715
ER_15620
INS
Yes

182
11
2181411
2181715
ER_15620
INS-IGF2
Yes

183
11
2212159
2212517
ER_15626
MIR4686
Yes

184
11
2431263
2431688
ER_15638
TRPM5
No

185
11
10764241
10764827
ER_15808
CTR9
No

186
11
12309323
12309637
ER_15849
MICALCL
Yes

187
11
18616740
18617317
ER_16065
SPTY2D1-AS1
Yes

188
11
20063465
20064029
ER_16131
NAV2
No

189
11
33744676
33745230
ER_16397
CD59
Yes

190
11
45928675
45929091
ER_16775
C11orf94
Yes

191
11
46373841
46374290
ER_16803
DGKZ
No

192
11
61466759
61467374
ER_17096
DAGLA
Yes

193
11
62437396
62437722
ER_17173
C11orf48
Yes

194
11
62647962
62648262
ER_17194
SLC3A2
No

195
11
62690015
62690315
ER_17200
CHRM1
No

196
11
64007699
64008267
ER_17271
FKBP2
No

197
11
64034904
64035204
ER_17276
PLCB3
No

198
11
64053773
64054355
ER_17285
GPR137
Yes

199
11
64322980
64323660
ER_17306
SLC22A11
Yes

200
11
64618305
64618873
ER_17326
EHD1
Yes

201
11
65121625
65122198
ER_17368
TIGD3
Yes

202
11
65548866
65549271
ER_17431
AP5B1
No

203
11
65582282
65582857
ER_17439
OVOL1
Yes

204
11
66659547
66660139
ER_17525
PC
No

205
11
67165633
67165963
ER_17593
PPP1CA
No

206
11
67186225
67186704
ER_17595
CARNS1
Yes

207
11
67810737
67811051
ER_17643
TCIRG1
No

208
11
67914354
67914874
ER_17656
SUV420H1
Yes

209
11
69061731
69062031
ER_17743
MYEOV
No

210
11
69515571
69515871
ER_17789
FGF19
Yes

211
11
70917115
70917490
ER_17848
SHANK2
No

212
11
71276995
71277590
ER_17878
KRTAP5-10
Yes

213
11
73000242
73000542
ER_17968
P2RY6
Yes

214
11
76371803
76372137
ER_18134
LRRC32
Yes

215
1
59058083
59058687
ER_1844
TACSTD2
No

216
11
85468982
85469571
ER_18506
SYTL2
No

217
1
6330275
6330725
ER_187
ACOT7
Yes

218
11
117300693
117300993
ER_19145
DSCAML1
No

219
11
118013408
118013932
ER_19187
SCN4B
Yes

220
1
6445555
6445855
ER_192
ACOT7
No

221
11
118758401
118758701
ER_19225
CXCR5
No

222
11
119994594
119994894
ER_19291
TRIM29
No

223
11
119995602
119996221
ER_19292
TRIM29
No

224
11
120106109
120106716
ER_19293
POU2F3
No

225
12
2021757
2022057
ER_19684
CACNA2D4
Yes

226
12
48340415
48340727
ER_21318
TMEM106C
Yes

227
12
48396469
48396769
ER_21327
COL2A1
No

228
12
49759304
49759746
ER_21394
SPATS2
Yes

229
12
51236349
51236665
ER_21514
TMPRSS12
No

230
12
52542519
52542858
ER_21611
KRT80
Yes

231
12
52579425
52579733
ER_21620
KRT80
Yes

232
12
53300210
53300676
ER_21729
KRT8
Yes

233
12
53552932
53553480
ER_21786
CSAD
Yes

234
12
56123942
56124242
ER_21950
CD63
Yes

235
12
56652692
56653098
ER_22003
ANKRD52
No

236
12
57559601
57559901
ER_22058
LRP1
No

237
12
58160686
58160986
ER_22088
CYP27B1
No

238
12
58209624
58209992
ER_22094
AVIL
No

239
12
63193501
63193816
ER_22367
PPM1H
Yes

240
12
65043934
65044256
ER_22510
RASSF3
Yes

241
12
65066737
65067037
ER_22517
RASSF3
Yes

242
12
68633903
68634289
ER_22744
IL22
No

243
1
7705733
7706033
ER_240
CAMTA1
No

244
12
103344027
103344491
ER_24357
ASCL1
No

245
12
109162594
109162897
ER_24689
SSH1
Yes

246
12
111029308
111029650
ER_24787
PPTC7
No

247
12
111840744
111841077
ER_24815
SH2B3
Yes

248
12
112207002
112207577
ER_24835
ALDH2
No

249
12
113547314
113547643
ER_24903
RASAL1
No

250
12
117471560
117471860
ER_25133
FBXW8
No

251
12
120703585
120704001
ER_25278
PXN
Yes

252
12
122019200
122019628
ER_25371
KDM2B
No

253
12
122458756
122459117
ER_25409
BCL7A
No

254
12
123446240
123446640
ER_25491
ABCB9
Yes

255
12
123449086
123449400
ER_25493
ABCB9
No

256
12
124844847
124845161
ER_25578
NCOR2
Yes

257
12
124879925
124880326
ER_25592
NCOR2
Yes

258
12
125004808
125005149
ER_25616
NCOR2
Yes

259
12
131438531
131438856
ER_25787
GPR133
Yes

260
12
131622396
131622696
ER_25800
GPR133
No

261
12
132285353
132285666
ER_25837
SFSWAP
Yes

262
12
132348290
132348625
ER_25849
MMP17
Yes

263
12
132671184
132671527
ER_25874
GALNT9
Yes

264
12
132694663
132695050
ER_25875
GALNT9
No

265
13
20965643
20965996
ER_25987
CRYL1
No

266
13
25691436
25692026
ER_26197
PABPC3
Yes

267
13
34208323
34209020
ER_26636
STARD13
Yes

268
13
41590103
41590403
ER_26934
ELF1
Yes

269
13
51854732
51855052
ER_27435
FAM124A
No

270
13
113651709
113652104
ER_28798
MCF2L
Yes

271
13
113677094
113677394
ER_28802
MCF2L
Yes

272
13
113979939
113980263
ER_28813
GRTP1
Yes

273
14
21494973
21495345
ER_28907
NDRG2
No

274
1
8823899
8824414
ER_290
RERE
No

275
14
23623250
23623562
ER_29019
SLC7A8
Yes

276
14
23623676
23623976
ER_29020
SLC7A8
Yes

277
14
38054124
38054508
ER_29794
FOXA1
Yes

278
14
64932503
64932803
ER_31140
AKAP5
No

279
14
68871364
68871773
ER_31488
RAD51B
Yes

280
14
69263206
69263506
ER_31542
ZFP36L1
Yes

281
14
74214079
74214466
ER_31871
C14orf43
Yes

282
14
74238204
74238510
ER_31884
C14orf43
Yes

283
1
1141942
1142242
ER_32
TNFRSF18
Yes

284
14
76447292
76447654
ER_32084
TGFB3
No

285
14
88942632
88943092
ER_32519
PTPN21
No

286
14
91723710
91724098
ER_32707
GPR68
Yes

287
14
91730894
91731408
ER_32708
CCDC88C
No

288
14
93412574
93412953
ER_32818
ITPK1
Yes

289
14
93473727
93474077
ER_32832
ITPK1
Yes

290
14
94392599
94392913
ER_32902
FAM181A-AS1
Yes

291
14
95047627
95047927
ER_32949
SERPINA5
No

292
14
95078366
95078753
ER_32959
SERPINA3
Yes

293
14
95615683
95616180
ER_32995
DICER1
No

294
1
1143577
1143920
ER_33
TNFRSF18
Yes

295
14
96691960
96692332
ER_33117
BDKRB2
No

296
14
100055296
100055707
ER_33296
CCDC85C
No

297
14
100196895
100197241
ER_33310
CYP46A1
No

298
14
100618272
100618577
ER_33370
DEGS2
No

299
14
103541236
103541566
ER_33566
CDC42BPB
Yes

300
14
103566340
103566640
ER_33567
EXOC3L4
Yes

301
14
103576070
103576413
ER_33570
EXOC3L4
No

302
14
104094520
104094858
ER_33611
KLC1
Yes

303
14
104165149
104165544
ER_33615
XRCC3
Yes

304
14
104637698
104638043
ER_33656
KIF26A
Yes

305
14
105131984
105132284
ER_33687
MIR4710
Yes

306
14
105181841
105182228
ER_33695
INF2
Yes

307
14
105215618
105215967
ER_33701
ADSSL1
Yes

308
14
105285893
105286336
ER_33720
LINC00638
Yes

309
14
105434204
105434522
ER_33730
AHNAK2
Yes

310
14
105446288
105446760
ER_33737
AHNAK2
Yes

311
14
105823215
105823515
ER_33780
PACS2
Yes

312
14
105992102
105992449
ER_33807
TMEM121
No

313
15
28352967
28353510
ER_33941
HERC2
Yes

314
15
32951599
32952135
ER_33988
SCG5
Yes

315
15
41258295
41258881
ER_34198
CHAC1
Yes

316
15
50519199
50519766
ER_34615
SLC27A2
Yes

317
1
1173574
1173890
ER_35
B3GALT6
Yes

318
15
53095569
53095869
ER_35206
ONECUT1
No

319
1
10075638
10076128
ER_353
RBP7
No

320
15
63074443
63074894
ER_35498
TLN2
No

321
15
63345507
63345850
ER_35518
TPM1
Yes

322
15
63671577
63672181
ER_35532
CA12
Yes

323
15
63672612
63673165
ER_35533
CA12
Yes

324
15
69588677
69589124
ER_35794
PAQR5
Yes

325
15
70384192
70384714
ER_35866
TLE3
Yes

326
15
73667419
73667968
ER_36062
HCN4
Yes

327
15
74232754
74233252
ER_36102
LOXL1
Yes

328
15
74495260
74495560
ER_36118
STRA6
Yes

329
15
74537511
74538135
ER_36123
CCDC33
Yes

330
15
75974558
75974909
ER_36216
CSPG4
Yes

331
15
79223130
79223938
ER_36351
CTSH
Yes

332
15
80878455
80879031
ER_36421
ARNT2
No

333
15
81596013
81596551
ER_36480
IL16
No

334
15
83781576
83782122
ER_36550
TM6SF1
Yes

335
15
86219506
86219806
ER_36659
AKAP13
Yes

336
15
89027778
89028410
ER_36781
MRPS11
No

337
15
89630906
89631554
ER_36798
ABHD2
Yes

338
15
90328102
90328625
ER_36856
ANPEP
Yes

339
15
90349924
90350224
ER_36858
ANPEP
Yes

340
15
90649755
90650347
ER_36890
IDH2
Yes

341
15
93573071
93573371
ER_37056
CHD2
No

342
15
96866722
96867218
ER_37215
NR2F2
Yes

343
15
99236506
99236926
ER_37297
IGF1R
No

344
15
99271960
99272504
ER_37301
IGF1R
Yes

345
15
101548985
101549285
ER_37458
LRRK1
No

346
16
374320
374831
ER_37511
AXIN1
Yes

347
16
585341
586026
ER_37534
MIR5587
Yes

348
16
615200
615500
ER_37536
C16orf11
Yes

349
16
633459
633759
ER_37539
PIGQ
Yes

350
16
635443
635747
ER_37540
PIGQ
Yes

351
16
701631
702182
ER_37550
WDR90
Yes

352
16
757105
757566
ER_37561
FBXL16
Yes

353
16
760308
760829
ER_37562
METRN
Yes

354
16
832843
833202
ER_37573
RPUSD1
Yes

355
16
854207
854703
ER_37576
PRR25
Yes

356
16
863331
863631
ER_37577
PRR25
No

357
16
1098718
1099027
ER_37589
SSTR5-AS1
Yes

358
16
1132936
1133498
ER_37595
SSTR5
No

359
16
1138312
1138612
ER_37596
C1QTNF8
No

360
16
1209882
1210346
ER_37604
CACNA1H
Yes

361
16
1232244
1232599
ER_37605
CACNA1H
Yes

362
16
1240629
1241133
ER_37606
CACNA1H
No

363
16
1275504
1275804
ER_37610
TPSG1
No

364
16
1305610
1305910
ER_37611
TPSD1
No

365
16
1350736
1351036
ER_37623
UBE2I
No

366
16
1353340
1353767
ER_37624
UBE2I
Yes

367
16
1430147
1430478
ER_37632
UNKL
Yes

368
16
1479242
1479542
ER_37638
CCDC154
No

369
16
1827853
1828153
ER_37665
SPSB3
No

370
16
2004491
2004791
ER_37679
RPL3L
No

371
16
2047651
2048147
ER_37687
ZNF598
No

372
16
2285522
2286213
ER_37716
E4F1
Yes

373
16
2294337
2294666
ER_37717
ECl1
Yes

374
16
2879820
2880369
ER_37757
ZG16B
Yes

375
16
3009266
3009645
ER_37769
KREMEN2
Yes

376
16
3199417
3199873
ER_37797
ZNF213
Yes

377
16
3704538
3704838
ER_37830
DNASE1
No

378
16
3707018
3707318
ER_37831
DNASE1
No

379
16
4367762
4368278
ER_37883
GLIS2
Yes

380
16
4421377
4421694
ER_37887
CORO7
Yes

381
16
4425919
4426536
ER_37892
VASN
Yes

382
16
4478392
4478714
ER_37899
DNAJA3
Yes

383
16
4741141
4741534
ER_37916
MGRN1
Yes

384
16
4746859
4747392
ER_37919
ANKS3
Yes

385
16
4838430
4839026
ER_37923
Sep-12
Yes

386
16
11422282
11422834
ER_38071
RMI2
Yes

387
16
14030414
14030714
ER_38174
ERCC4
No

388
16
14580747
14581084
ER_38229
PARN
No

389
16
15602986
15603303
ER_38266
C16orf45
Yes

390
16
16090418
16090901
ER_38297
ABCC1
Yes

391
16
16108606
16109211
ER_38301
ABCC1
No

392
16
22103014
22103632
ER_38518
VWA3A
No

393
16
24747980
24748466
ER_38621
TNRC6A
Yes

394
16
24855743
24856361
ER_38625
SLC5A11
No

395
16
28511293
28511661
ER_38732
IL27
Yes

396
16
28608240
28608616
ER_38739
SULT1A2
Yes

397
16
30906082
30906783
ER_38861
BCL7C
No

398
16
68321665
68322263
ER_39511
SLC7A6
No

399
16
69358300
69358626
ER_39598
VPS4A
Yes

400
16
70472947
70473558
ER_39656
ST3GAL2
No

401
16
70687883
70688183
ER_39667
IL34
No

402
16
72994910
72995462
ER_39877
ZFHX3
Yes

403
16
78991185
78991807
ER_40213
WWOX
Yes

404
16
81248258
81248721
ER_40354
PKD1L2
Yes

405
16
83847710
83848298
ER_40473
HSBP1
No

406
16
84400643
84401271
ER_40523
ATP2C2
Yes

407
16
84552526
84553023
ER_40542
KIAA1609
Yes

408
16
84628881
84629206
ER_40553
COTL1
Yes

409
16
84840044
84840619
ER_40577
CRISPLD2
No

410
16
84871395
84871718
ER_40585
CRISPLD2
No

411
16
85669299
85669599
ER_40724
KIAA0182
No

412
16
85723351
85723651
ER_40737
GINS2
No

413
16
85786954
85787520
ER_40750
C16orf74
Yes

414
1
109373005
109373612
ER_4085
AKNAD1
No

415
16
87739399
87739859
ER_40900
KLHDC4
No

416
16
87778638
87778938
ER_40904
KLHDC4
No

417
16
87812661
87813207
ER_40908
KLHDC4
No

418
16
87868043
87868626
ER_40912
SLC7A5
No

419
16
87890385
87890763
ER_40918
SLC7A5
Yes

420
16
87914990
87915446
ER_40927
CA5A
Yes

421
16
88110906
88111273
ER_40942
BANP
Yes

422
16
88111279
88111579
ER_40943
BANP
No

423
16
88548864
88549438
ER_40969
ZFPM1
Yes

424
16
88704888
88705257
ER_40987
IL17C
Yes

425
16
88988838
88989268
ER_41018
CBFA2T3
Yes

426
16
88991561
88992120
ER_41020
CBFA2T3
No

427
16
89004344
89004862
ER_41024
CBFA2T3
No

428
16
89043365
89043665
ER_41029
CBFA2T3
Yes

429
16
89638485
89638811
ER_41092
CPNE7
Yes

430
16
89648350
89648952
ER_41093
CPNE7
Yes

431
16
89665706
89666006
ER_41094
CPNE7
Yes

432
1
11020446
11021019
ER_411
C1orf127
No

433
16
89900078
89900645
ER_41112
SPIRE2
Yes

434
16
89927268
89927731
ER_41118
SPIRE2
Yes

435
16
90012005
90012305
ER_41125
DEF8
No

436
17
151960
152279
ER_41149
RPH3AL
Yes

437
17
1634282
1634616
ER_41197
WDR81
Yes

438
17
1901327
1901683
ER_41216
RTN4RL1
No

439
17
1987743
1988066
ER_41224
SMG6
Yes

440
17
3635535
3635874
ER_41267
ITGAE
Yes

441
1
109826384
109826898
ER_4128
PSRC1
No

442
17
3870398
3870698
ER_41284
ATP2A3
No

443
17
4400749
4401049
ER_41310
SPNS2
Yes

444
17
4436972
4437323
ER_41315
SPNS2
No

445
17
4455826
4456126
ER_41316
MYBBP1A
No

446
17
7283657
7283982
ER_41423
TNK1
No

447
17
7959803
7960158
ER_41460
ALOX15B
No

448
17
14109320
14109680
ER_41695
COX10
No

449
17
16322433
16322733
ER_41788
TRPV2
No

450
17
16954969
16955288
ER_41817
MPRIP
Yes

451
17
17628505
17628831
ER_41872
RAI1
No

452
17
17718324
17718694
ER_41889
SREBF1
Yes

453
17
18139202
18139554
ER_41939
LLGL1
Yes

454
17
18280720
18281075
ER_41950
EVPLL
No

455
17
19627818
19628193
ER_42003
SLC47A2
No

456
17
26578307
26578607
ER_42148
PPY2
Yes

457
17
27295913
27296324
ER_42201
SEZ6
Yes

458
17
29649808
29650164
ER_42279
NF1
Yes

459
1
111006535
111007108
ER_4230
PROK1
No

460
17
39577357
39577695
ER_42572
KRT37
Yes

461
17
39662933
39663289
ER_42574
KRT13
No

462
17
39677996
39678528
ER_42579
KRT19
No

463
17
39685761
39686063
ER_42583
KRT19
No

464
17
39694022
39694351
ER_42588
KRT19
No

465
17
40931971
40932360
ER_42648
WNK4
Yes

466
17
44896572
44896908
ER_42790
WNT3
Yes

467
17
48048352
48048668
ER_42976
DLX4
Yes

468
17
48261929
48262229
ER_43003
COL1A1
Yes

469
17
55371434
55371787
ER_43290
MSI2
Yes

470
17
55673703
55674091
ER_43343
MSI2
Yes

471
1
12192916
12193471
ER_444
TNFRSF8
No

472
17
58498280
58498903
ER_44815
C17orf64
No

473
1
1609934
1610467
ER_45
SLC35E2B
No

474
17
59484187
59484499
ER_45541
TBX2
Yes

475
1
114218048
114218488
ER_4610
MAGI3
No

476
17
64940732
64941081
ER_46841
CACNG4
Yes

477
17
64954251
64954635
ER_46847
CACNG4
Yes

478
17
65487266
65487622
ER_46920
PITPNC1
No

479
17
66291281
66291581
ER_46984
ARSG
Yes

480
17
70636643
70636952
ER_47135
LINC00511
Yes

481
17
71612589
71612889
ER_47199
SDK2
No

482
17
72439108
72439488
ER_47235
GPRC5C
Yes

483
17
72732636
72732936
ER_47245
RAB37
No

484
17
72740901
72741201
ER_47248
RAB37
No

485
17
73500571
73500871
ER_47351
CASKIN2
Yes

486
17
73641528
73641923
ER_47388
RECQL5
Yes

487
17
73696463
73696795
ER_47396
SAP30BP
Yes

488
17
73761501
73761882
ER_47406
GALK1
Yes

489
17
73805905
73806251
ER_47422
UNK
Yes

490
17
73872405
73872705
ER_47428
TRIM47
No

491
17
74494140
74494511
ER_47474
RHBDF2
Yes

492
17
74684239
74684546
ER_47498
MXRA7
No

493
17
75181779
75182110
ER_47526
SEC14L1
No

494
17
75473604
75473943
ER_47548
Sep-09
Yes

495
17
76498871
76499229
ER_47612
DNAH17
No

496
17
76522848
76523165
ER_47614
DNAH17
Yes

497
17
76588325
76588770
ER_47620
DNAH17
No

498
17
76858208
76858508
ER_47631
TIMP2
No

499
17
76973124
76973427
ER_47643
LGALS3BP
Yes

500
17
77782827
77783366
ER_47666
CBX8
No

501
17
77785183
77785533
ER_47667
CBX8
No

502
17
77818303
77818633
ER_47681
CBX4
Yes

503
17
78522351
78522735
ER_47743
RPTOR
Yes

504
17
78667839
78668195
ER_47754
RPTOR
Yes

505
17
78791604
78791917
ER_47760
RPTOR
Yes

506
17
78793449
78793890
ER_47761
RPTOR
Yes

507
17
78796720
78797088
ER_47762
RPTOR
Yes

508
17
79018691
79019077
ER_47773
BAIAP2
No

509
17
79251153
79251492
ER_47785
SLC38A10
No

510
17
79447600
79447963
ER_47803
BAHCC1
Yes

511
17
79961758
79962058
ER_47823
ASPSCR1
No

512
17
80162846
80163196
ER_47840
CCDC57
No

513
17
80174646
80174968
ER_47845
CCDC57
Yes

514
17
80419732
80420082
ER_47870
NARF
No

515
17
80662180
80662480
ER_47885
RAB40B
Yes

516
17
81025415
81025759
ER_47909
METRNL
Yes

517
17
81031364
81031696
ER_47911
METRNL
Yes

518
18
3446546
3446906
ER_48111
TGIF1
Yes

519
1
114521332
114522214
ER_4835
OLFML3
No

520
18
74536197
74536522
ER_49880
ZNF236
Yes

521
19
930678
930978
ER_49971
ARID3A
Yes

522
19
1169031
1169402
ER_49996
SBNO2
No

523
19
1496339
1496639
ER_50033
REEP6
No

524
19
1907761
1908143
ER_50049
SCAMP4
Yes

525
19
2167382
2167682
ER_50060
DOT1L
No

526
19
2624590
2624934
ER_50099
GNG7
Yes

527
19
2723050
2723398
ER_50102
DIRAS1
Yes

528
19
2727038
2727338
ER_50103
SLC39A3
Yes

529
19
3374719
3375065
ER_50124
NFIC
Yes

530
19
6276448
6276748
ER_50272
MLLT1
Yes

531
19
7684955
7685267
ER_50326
XAB2
Yes

532
19
7714289
7714687
ER_50328
STXBP2
No

533
19
11617772
11618120
ER_50514
ECSIT
Yes

534
19
14066552
14066906
ER_50640
DCAF15
Yes

535
19
14544916
14545316
ER_50670
PKN1
Yes

536
19
15590207
15590541
ER_50728
PGLYRP2
No

537
19
15618423
15618788
ER_50733
CYP4F22
Yes

538
19
15622532
15622947
ER_50734
CYP4F22
Yes

539
19
16045737
16046329
ER_50746
CYP4F11
No

540
19
16603746
16604192
ER_50800
CALR3
Yes

541
19
17407033
17407362
ER_50855
ABHD8
Yes

542
19
18385725
18386025
ER_50903
KIAA1683
Yes

543
19
33167485
33167785
ER_51224
RGS9BP
No

544
19
33624481
33624781
ER_51258
WDR88
Yes

545
19
33726540
33726840
ER_51263
SLC7A10
Yes

546
19
35531859
35532247
ER_51354
HPN
No

547
19
35800367
35800739
ER_51381
MAG
Yes

548
19
35801186
35801551
ER_51382
MAG
No

549
19
35940219
35940897
ER_51391
FFAR2
Yes

550
19
38793369
38793685
ER_51493
YIF1B
Yes

551
19
39222375
39222739
ER_51519
CAPN12
Yes

552
19
41633774
41634080
ER_51655
CYP2F1
Yes

553
19
45843797
45844097
ER_51894
KLC3
No

554
19
45848214
45848514
ER_51895
KLC3
Yes

555
19
49059555
49059855
ER_52131
SULT2B1
No

556
19
50458158
50458475
ER_52226
SIGLEC11
Yes

557
19
50969943
50970265
ER_52248
FAM71E1
Yes

558
19
51568129
51568429
ER_52287
KLK13
No

559
19
54600186
54600532
ER_52363
OSCAR
No

560
19
55874846
55875146
ER_52410
FAM71E2
Yes

561
19
55880474
55880774
ER_52411
IL11
No

562
19
56047757
56048080
ER_52419
SBK2
No

563
2
3452679
3453034
ER_52628
TRAPPC12
Yes

564
2
8833486
8833786
ER_52806
ID2
No

565
2
19555335
19555655
ER_53364
OSR1
Yes

566
2
25094778
25095111
ER_53566
ADCY3
Yes

567
2
25562754
25563086
ER_53585
DNMT3A
Yes

568
2
26199821
26200281
ER_53612
KIF3C
No

569
2
26947077
26947447
ER_53662
KCNK3
No

570
2
27319112
27319450
ER_53686
KHK
No

571
2
28549119
28549437
ER_53751
BRE
Yes

572
2
28569483
28570005
ER_53759
BRE
Yes

573
2
45998277
45998784
ER_54551
PRKCE
Yes

574
2
46361686
46362029
ER_54598
PRKCE
Yes

575
2
47236009
47236309
ER_54678
TTC7A
Yes

576
2
54760101
54760460
ER_54825
SPTBN1
Yes

577
1
16061307
16061646
ER_549
PLEKHM2
Yes

578
1
16074063
16074363
ER_550
TMEM82
No

579
1
115211543
115212151
ER_5516
DENND2C
Yes

580
2
74152981
74153296
ER_55613
DGUOK
Yes

581
2
85280957
85281340
ER_55852
KCMF1
Yes

582
2
85621930
85622230
ER_55867
CAPG
No

583
1
16251091
16251574
ER_559
SPEN
No

584
2
95719289
95719611
ER_56072
MAL
Yes

585
2
97508396
97508742
ER_56156
ANKRD23
No

586
2
102012642
102013116
ER_56390
RFX8
No

587
1
16403340
16403801
ER_564
FAM131C
Yes

588
2
106007690
106008035
ER_56517
FHL2
No

589
2
113875147
113875494
ER_56808
IL1RN
No

590
2
121036360
121036660
ER_56956
RALB
Yes

591
2
121036666
121036969
ER_56957
RALB
Yes

592
2
121071534
121071901
ER_56964
RALB
No

593
2
128458070
128458447
ER_57147
SFT2D3
No

594
2
129066951
129067320
ER_57178
HS6ST1
No

595
1
116219030
116219646
ER_5750
VANGL1
No

596
2
175499224
175499524
ER_59117
WIPF1
No

597
1
16950505
16950951
ER_599
CROCCP2
Yes

598
2
197158771
197159125
ER_59918
HECW2
No

599
2
197466173
197466509
ER_59929
HECW2
Yes

600
1
17035334
17035751
ER_603
ESPNP
No

601
2
208494280
208494580
ER_60350
METTL21A
No

602
2
216478034
216478350
ER_60685
LINC00607
No

603
2
220007041
220007402
ER_60937
NHEJ1
No

604
1
17047514
17047814
ER_610
ESPNP
No

605
2
224624781
224625214
ER_61179
AP1S3
No

606
2
236447665
236448023
ER_61661
AGAP1
Yes

607
2
239169600
239169933
ER_61869
PER2
No

608
2
240186821
240187208
ER_61904
HDAC4
Yes

609
2
240241009
240241336
ER_61909
HDAC4
Yes

610
2
241807610
241808275
ER_61956
AGXT
Yes

611
2
241832846
241833163
ER_61959
C2orf54
No

612
2
241936626
241937048
ER_61974
SNED1
Yes

613
2
241975925
241976252
ER_61978
SNED1
Yes

614
2
242138256
242138571
ER_61990
ANO7
Yes

615
2
242500199
242500499
ER_62006
BOK
Yes

616
20
17595352
17595711
ER_62469
RRBP1
Yes

617
20
18035798
18036353
ER_62490
OVOL2
No

618
20
30432800
30433405
ER_62916
FOXS1
Yes

619
20
32446738
32447321
ER_63011
CHMP4B
No

620
20
32888452
32889022
ER_63021
AHCY
Yes

621
20
34205164
34205464
ER_63067
SPAG4
No

622
20
35093680
35094313
ER_63095
DLGAP4
No

623
20
35493191
35493491
ER_63113
SOGA1
No

624
20
36767493
36768090
ER_63162
TGM2
No

625
1
118727703
118728260
ER_6319
SPAG17
Yes

626
1
17634485
17634785
ER_634
PADI4
Yes

627
20
43343181
43343741
ER_63515
WISP2
No

628
20
44048102
44048440
ER_63559
PIGT
No

629
20
44330526
44330841
ER_63571
WFDC13
Yes

630
20
47278241
47278894
ER_65136
PREX1
No

631
1
144989293
144989659
ER_6529
PDE4DIP
No

632
20
47448122
47448615
ER_65321
PREX1
No

633
20
49345259
49346053
ER_65723
PARD6B
No

634
20
49346308
49346885
ER_65724
PARD6B
No

635
20
49411019
49411779
ER_65780
BCAS4
No

636
20
52205856
52206621
ER_66116
ZNF217
Yes

637
1
17887921
17888496
ER_662
ARHGEF10L
Yes

638
1
18006751
18007051
ER_669
ARHGEF10L
No

639
20
55200110
55200410
ER_67539
TFAP2C
No

640
20
58325746
58326345
ER_68282
PHACTR3
No

641
20
60510103
60510565
ER_68459
CDH4
Yes

642
20
60924954
60925254
ER_68486
LAMA5
No

643
20
60932217
60932572
ER_68489
LAMA5
Yes

644
20
61451571
61451871
ER_68537
COL9A3
Yes

645
20
62184027
62184327
ER_68582
C20orf195
No

646
20
62282532
62282832
ER_68587
STMN3
No

647
1
150333685
150334193
ER_6892
RPRD2
Yes

648
1
2036450
2036863
ER_69
PRKCZ
Yes

649
21
37802129
37802579
ER_69231
CHAF1B
No

650
21
42212292
42212834
ER_69514
DSCAM
No

651
21
43107677
43108088
ER_69583
LINC00111
No

652
21
43135959
43136350
ER_69588
LINC00479
No

653
21
43735388
43735910
ER_69642
TFF3
Yes

654
21
44816541
44817155
ER_69713
SIK1
No

655
21
44897853
44898245
ER_69724
LINC00313
Yes

656
21
46172674
46173357
ER_69814
UBE2G2
Yes

657
21
46321203
46321769
ER_69832
ITGB2
No

658
21
46325777
46326117
ER_69834
ITGB2
Yes

659
21
46331748
46332340
ER_69835
ITGB2
No

660
21
46409729
46410331
ER_69844
LINC00163
Yes

661
21
46953747
46954295
ER_69890
SLC19A1
Yes

662
22
18919539
18919839
ER_69990
PRODH
Yes

663
22
19718480
19718835
ER_70025
GP1BB
No

664
22
19755289
19755589
ER_70033
TBX1
Yes

665
22
19879093
19879435
ER_70044
TXNRD2
No

666
22
24384589
24384947
ER_70184
GSTT1
No

667
1
151554445
151555180
ER_7042
TUFT1
Yes

668
22
35695235
35695568
ER_70647
TOM1
Yes

669
22
35931960
35932283
ER_70657
RASD2
Yes

670
22
38092597
38092897
ER_70759
TRIOBP
No

671
1
151818574
151819202
ER_7079
THEM5
No

672
22
38610003
38610303
ER_70793
MAFF
No

673
22
39759998
39760357
ER_70842
SYNGR1
No

674
22
40404687
40405045
ER_70880
FAM83F
Yes

675
22
43525157
43525457
ER_70998
BIK
Yes

676
22
46921821
46922121
ER_71163
CELSR1
Yes

677
22
50450954
50451412
ER_71287
IL17REL
Yes

678
22
50720235
50720566
ER_71300
PLXNB2
No

679
22
50738759
50739090
ER_71305
PLXNB2
Yes

680
22
50918140
50918446
ER_71311
ADM2
No

681
3
8693628
8694000
ER_71572
C3orf32
Yes

682
3
9757534
9757834
ER_71643
CPNE9
No

683
3
9996819
9997181
ER_71662
PRRT3-AS1
No

684
3
11550571
11550871
ER_71746
ATG7
No

685
3
11643071
11643504
ER_71757
VGLL4
No

686
3
11763408
11763780
ER_71771
VGLL4
Yes

687
3
12985852
12986194
ER_71863
IQSEC1
No

688
3
12994633
12995020
ER_71864
IQSEC1
No

689
3
13517660
13517983
ER_71911
HDAC11
No

690
3
14920746
14921046
ER_72049
FGD5
No

691
3
15310870
15311256
ER_72070
SH3BP5
Yes

692
3
15687178
15687551
ER_72120
BTD
No

693
1
19663685
19664277
ER_722
CAPZB
Yes

694
1
19664905
19665205
ER_723
CAPZB
Yes

695
1
154166420
154166720
ER_7253
MIR190B
Yes

696
1
154298772
154299072
ER_7259
ATP8B2
No

697
1
154377532
154377832
ER_7267
IL6R
No

698
3
38067281
38067610
ER_73005
PLCD1
No

699
3
46734109
46734463
ER_73287
ALS2CL
Yes

700
3
48589861
48590161
ER_73356
PFKFB4
No

701
1
155161639
155162099
ER_7336
MUC1
No

702
3
50638992
50639632
ER_73426
CISH
Yes

703
3
52280070
52280428
ER_73463
PPM1M
No

704
3
58028409
58028709
ER_73666
FLNB
No

705
1
155912401
155912737
ER_7372
RXFP4
Yes

706
1
156095999
156096299
ER_7395
LMNA
No

707
3
61793488
61793816
ER_73972
PTPRG
No

708
1
156679461
156679823
ER_7437
CRABP2
Yes

709
1
156822285
156822644
ER_7451
INSRR
No

710
1
160079100
160079716
ER_7493
ATP1A2
Yes

711
1
161646681
161647242
ER_7530
FCGR2B
No

712
1
2174671
2174976
ER_76
SKI
Yes

713
3
63922231
63922573
ER_76144
ATXN7
No

714
3
66519928
66520259
ER_77324
LRIG1
Yes

715
3
66543250
66543644
ER_77334
LRIG1
No

716
3
69942380
69942719
ER_77420
MITF
Yes

717
3
99721769
99722069
ER_78150
FILIP1L
Yes

718
1
170043984
170044284
ER_7816
KIFAP3
No

719
3
99833502
99833835
ER_78166
C3orf26
Yes

720
1
171226439
171226888
ER_7821
FMO1
Yes

721
3
112359914
112360214
ER_78589
CCDC80
No

722
3
122057821
122058174
ER_78781
CSTA
Yes

723
1
172608570
172609176
ER_7888
FASLG
No

724
3
124493174
124493574
ER_78936
ITGB5
No

725
3
126200679
126200979
ER_79014
UROC1
No

726
3
126678959
126679283
ER_79028
CHCHD6
Yes

727
3
129295579
129295977
ER_79235
PLXND1
No

728
3
133174831
133175237
ER_79397
BFSP2
Yes

729
3
160787663
160788036
ER_80595
PPM1L
No

730
3
168870086
168870443
ER_80932
MECOM
Yes

731
3
169758098
169758589
ER_80993
GPR160
No

732
3
183959578
183959923
ER_81693
VWA5B2
Yes

733
3
184052187
184052556
ER_81701
EIF4G1
No

734
3
195531202
195531699
ER_82319
MUC4
Yes

735
3
195603560
195603892
ER_82326
TNK2
Yes

736
3
196388403
196388727
ER_82377
LRRC33
No

737
4
680920
681252
ER_82472
MFSD7
Yes

738
4
686579
687011
ER_82474
MFSD7
No

739
4
757427
757729
ER_82479
PCGF3
Yes

740
4
965412
965736
ER_82491
DGKQ
Yes

741
4
987571
987878
ER_82493
IDUA
Yes

742
4
1239191
1239531
ER_82503
CTBP1
No

743
4
1729872
1730230
ER_82525
TACC3
Yes

744
4
1986291
1986591
ER_82549
WHSC2
Yes

745
4
2798112
2798444
ER_82594
SH3BP2
Yes

746
4
3341090
3341444
ER_82632
RGS12
Yes

747
4
3772680
3772980
ER_82661
ADRA2C
Yes

748
4
6928789
6929118
ER_82798
TBC1D14
Yes

749
4
7219719
7220019
ER_82822
SORCS2
No

750
4
8062466
8062766
ER_82882
ABLIM2
Yes

751
4
8130159
8130480
ER_82884
ABLIM2
Yes

752
4
8412544
8412844
ER_82916
ACOX3
No

753
4
48908995
48909295
ER_83945
OCIAD2
Yes

754
1
22773934
22774523
ER_859
ZBTB40
No

755
4
119910484
119910799
ER_85984
SYNPO2
No

756
4
184644416
184644716
ER_87970
TRAPPC11
Yes

757
5
373069
373625
ER_88159
AHRR
Yes

758
5
429733
430313
ER_88163
AHRR
Yes

759
5
610077
610670
ER_88173
CEP72
Yes

760
5
672776
673076
ER_88176
TPPP
No

761
5
1201313
1201673
ER_88195
SLC6A19
No

762
5
1207117
1207709
ER_88196
SLC6A19
Yes

763
5
1443109
1443488
ER_88204
SLC6A3
No

764
5
7851676
7852193
ER_88404
C5orf49
Yes

765
5
37840402
37840702
ER_89386
GDNF
No

766
5
43603889
43604448
ER_89613
NNT
Yes

767
5
52385969
52386507
ER_89737
ITGA2
Yes

768
1
201095792
201096337
ER_8996
TMEM9
No

769
1
201252202
201252787
ER_9013
PKP1
No

770
5
95226771
95227393
ER_91036
ELL2
No

771
1
203096814
203097402
ER_9205
ADORA1
No

772
1
203122931
203123270
ER_9215
ADORA1
No

773
5
138299601
138299901
ER_92405
SIL1
Yes

774
1
203297838
203298428
ER_9241
FMOD
Yes

775
5
138731543
138732087
ER_92433
SPATA24
Yes

776
5
139033837
139034156
ER_92444
CXXC5
Yes

777
5
139069265
139069565
ER_92451
CXXC5
Yes

778
5
140012702
140013307
ER_92479
CD14
No

779
1
203488468
203489054
ER_9257
OPTC
Yes

780
1
203594651
203595113
ER_9266
ATP2B4
No

781
5
148513872
148514255
ER_92755
ABLIM3
No

782
5
159687745
159688359
ER_93120
CCNJL
Yes

783
5
176239632
176240113
ER_93644
UNC5A
Yes

784
5
176816546
176817069
ER_93670
SLC34A1
No

785
5
176874917
176875279
ER_93675
PRR7-AS1
Yes

786
5
177029224
177029524
ER_93689
B4GALT7
Yes

787
5
177547872
177548392
ER_93712
N4BP3
No

788
5
180022475
180023061
ER_93819
SCGB3A1
Yes

789
5
180648505
180649224
ER_93834
MIR4638
No

790
6
379290
379637
ER_93850
IRF4
Yes

791
6
3139405
3139938
ER_93950
BPHL
Yes

792
6
3723005
3723548
ER_93997
PXDC1
No

793
6
10420655
10421048
ER_94256
TFAP2A
No

794
1
205633387
205633954
ER_9471
SLC45A3
Yes

795
6
30698796
30699096
ER_95075
FLOT1
No

796
6
31477979
31478310
ER_95117
MICB
No

797
6
31743735
31744048
ER_95131
VWA7
Yes

798
6
31744689
31744989
ER_95132
VWA7
Yes

799
6
31833129
31833429
ER_95141
SLC44A4
Yes

800
6
31868596
31868896
ER_95144
C2
No

801
6
32050977
32051543
ER_95152
TNXB
Yes

802
6
32078224
32078792
ER_95153
TNXB
Yes

803
6
32135005
32135578
ER_95158
EGFL8
Yes

804
6
33173123
33173423
ER_95186
HSD17B8
No

805
6
33288112
33288670
ER_95195
DAXX
Yes

806
6
33993601
33993995
ER_95239
GRM4
Yes

807
6
34511677
34512101
ER_95268
SPDEF
Yes

808
6
40363002
40363510
ER_95568
LRFN2
No

809
6
42108932
42109278
ER_95663
C6orf132
Yes

810
6
43463227
43463527
ER_95722
TJAP1
No

811
6
43603250
43603772
ER_95728
MAD2L1BP
No

812
6
52268606
52268913
ER_96019
PAQR8
Yes

813
6
56580975
56581555
ER_96201
RNU6-71
No

814
6
64281501
64282055
ER_96269
PTP4A1
No

815
1
25070145
25070811
ER_965
CLIC4
No

816
6
106576993
106577649
ER_97203
PRDM1
Yes

817
6
109267245
109267600
ER_97322
ARMC2
Yes

818
6
138425688
138426269
ER_98290
PERP
No

819
6
149806486
149806786
ER_98712
ZC3H12D
Yes

820
6
151937170
151937602
ER_98798
CCDC170
Yes

821
6
152124722
152125100
ER_98841
ESR1
Yes

822
6
152432244
152432773
ER_98857
ESR1
No

823
6
155052906
155053506
ER_98929
SCAF8
Yes

824
7
921963
922279
ER_99284
GET4
Yes

825
7
923724
924024
ER_99285
GET4
No

826
7
927894
928206
ER_99286
GET4
Yes

827
7
940841
941164
ER_99287
ADAP1
Yes

828
7
955121
955452
ER_99289
ADAP1
Yes

829
7
966854
967230
ER_99290
ADAP1
Yes

830
7
1004933
1005233
ER_99293
COX19
No

831
7
1026106
1026406
ER_99296
CYP2W1
No

832
7
1027843
1028143
ER_99297
CYP2W1
Yes

833
7
1054343
1054643
ER_99302
C7orf50
Yes

834
7
1139221
1139554
ER_99313
C7orf50
Yes

835
7
1286406
1286773
ER_99325
UNCX
No

836
7
1501011
1501415
ER_99342
MICALL2
Yes

837
7
1552985
1553309
ER_99348
INTS1
No

838
1
217886370
217887036
ER_9936
SPATA17
Yes

839
7
1659555
1659913
ER_99368
TFAMP1
No

840
7
1753468
1753819
ER_99371
ELFN1
No

841
7
1782517
1782817
ER_99372
ELFN1
No

842
7
1891218
1891549
ER_99377
MAD1L1
No

843
7
1931906
1932296
ER_99382
MAD1L1
No

844
7
1959815
1960115
ER_99385
MAD1L1
No

845
7
1961938
1962358
ER_99386
MAD1L1
Yes

846
7
1967307
1967639
ER_99387
MAD1L1
Yes

847
7
1968165
1968519
ER_99388
MAD1L1
No

848
7
2191489
2191835
ER_99406
MAD1L1
Yes

849
7
2673205
2673630
ER_99460
TTYH3
No

850
7
2702609
2702937
ER_99466
TTYH3
No

851
7
2731506
2731863
ER_99473
AMZ1
Yes

852
7
2760404
2760837
ER_99477
AMZ1
Yes

853
7
4876684
4876984
ER_99580
RADIL
No

854
7
5436737
5437105
ER_99601
TNRC18
Yes

855
7
5526272
5526875
ER_99612
FBXL18
Yes

856
7
6312965
6313265
ER_99654
CYTH3
No

In one example, the method of detecting differential methylation comprises detecting differential methylation at one or more CpG dinucleotide sequences within one or more regions set forth in rows 1-856 of Table 1 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences. For example, the method may comprise detecting differential methylation at a single CpG dinucleotide sequence within any one of the genomic regions defined in Table 1 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequence.

In another example, the method of detecting differential methylation comprises detecting differential methylation at two or more CpG dinucleotides within any genomic region defined in Table 1 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding two or more CpG dinucleotide sequences. For example, the method may comprise detecting differential methylation at two or more CpG dinucleotides, or three or more CpG dinucleotides, or four or more CpG dinucleotides, or five or more CpG dinucleotides, or six or more CpG dinucleotides, or seven or more CpG dinucleotides, or eight or more CpG dinucleotides, or nine or more CpG dinucleotides, or 10 or more CpG dinucleotides, or 20 or more CpG dinucleotides, or 30 or more CpG dinucleotides, or 40 or more CpG dinucleotides, or 50 or more CpG dinucleotides within a genomic region set forth in Table 1 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequences. The two or more CpG dinucleotides may be consecutive (i.e., contiguous) within a genomic region. Alternatively, the two or more CpG dinucleotides may not be consecutive (i.e., may not be contiguous) within any genomic region.

In yet another example, the method of detecting differential methylation comprises detecting differential methylation of at least one CpG dinucleotide within two or more different genomic regions set forth in Table 1 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequences. For example, the method may comprise detecting differential methylation at a CpG dinucleotide within two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more different genomic regions set forth in Table 1 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequences.

In one example, identifying differential methylation at one or more CpG dinucleotide sequences within one or more regions set forth in rows 1-856 of Table 1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject's likely response to endocrine therapy.

Detecting differential methylation at a single CpG dinucleotide sequence within any one of the genomic regions defined in Table 1 may be indicative of the subject's likely response to endocrine therapy.

Alternatively, detecting differential methylation at two or more CpG dinucleotides within any genomic region defined in Table 1 may be indicative of the subject's likely response to endocrine therapy. For example, detecting differential methylation at two or more CpG dinucleotides, or three or more CpG dinucleotides, or four or more CpG dinucleotides, or five or more CpG dinucleotides, or six or more CpG dinucleotides, or seven or more CpG dinucleotides, or eight or more CpG dinucleotides, or nine or more CpG dinucleotides, or 10 or more CpG dinucleotides, or 20 or more CpG dinucleotides, or 30 or more CpG dinucleotides, or 40 or more CpG dinucleotides, or 50 or more CpG dinucleotides within a genomic region set forth in Table 1 may be indicative of the subject's likely response to endocrine therapy. The two or more CpG dinucleotides may be consecutive (i.e., contiguous) within a genomic region. Alternatively, the two or more CpG dinucleotides may not be consecutive (i.e., may not be contiguous) within any genomic region.

Detecting differential methylation of at least one CpG dinucleotide within two or more different genomic regions set forth in Table 1 may be indicative of the subject's likely response to endocrine therapy. For example, detecting differential methylation at a CpG dinucleotide within two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more different genomic regions set forth in Table 1 may be indicative of the subject's likely response to endocrine therapy.

Detecting differential methylation at a single CpG dinucleotide sequence within any one of the genomic regions defined in Table 1 may be indicative of the subject having breast cancer which is refractory to endocrine therapy.

Alternatively, detecting differential methylation at two or more CpG dinucleotides within any genomic region defined in Table 1 may be indicative of the subject having breast cancer which is refractory to endocrine therapy. For example, detecting differential methylation at two or more CpG dinucleotides, or three or more CpG dinucleotides, or four or more CpG dinucleotides, or five or more CpG dinucleotides, or six or more CpG dinucleotides, or seven or more CpG dinucleotides, or eight or more CpG dinucleotides, or nine or more CpG dinucleotides, or 10 or more CpG dinucleotides, or 20 or more CpG dinucleotides, or 30 or more CpG dinucleotides, or 40 or more CpG dinucleotides, or 50 or more CpG dinucleotides within a genomic region set forth in Table 1 may be indicative of the subject having breast cancer which is refractory to endocrine therapy. The two or more CpG dinucleotides may be consecutive (i.e., contiguous) within a genomic region. Alternatively, the two or more CpG dinucleotides may not be consecutive (i.e., may not be contiguous) within any genomic region.

Detecting differential methylation of at least one CpG dinucleotide within two or more different genomic regions set forth in Table 1 may be indicative of the subject having breast cancer which is refractory to endocrine therapy. For example, detecting differential methylation at a CpG dinucleotide within two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more different genomic regions set forth in Table 1 may be indicative of the subject having breast cancer which is refractory to endocrine therapy.

Detecting differential methylation at a single CpG dinucleotide sequence within any one of the genomic regions defined in Table 1 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer.

Alternatively, detecting differential methylation at two or more CpG dinucleotides within any genomic region defined in Table 1 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. For example, detecting differential methylation at two or more CpG dinucleotides, or three or more CpG dinucleotides, or four or more CpG dinucleotides, or five or more CpG dinucleotides, or six or more CpG dinucleotides, or seven or more CpG dinucleotides, or eight or more CpG dinucleotides, or nine or more CpG dinucleotides, or 10 or more CpG dinucleotides, or 20 or more CpG dinucleotides, or 30 or more CpG dinucleotides, or 40 or more CpG dinucleotides, or 50 or more CpG dinucleotides within a genomic region set forth in Table 1 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. The two or more CpG dinucleotides may be consecutive (i.e., contiguous) within a genomic region. Alternatively, the two or more CpG dinucleotides may not be consecutive (i.e., may not be contiguous) within any genomic region.

Detecting differential methylation of at least one CpG dinucleotide within two or more different genomic regions set forth in Table 1 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. For example, detecting differential methylation at a CpG dinucleotide within two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more different genomic regions set forth in Table 1 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer.

In another example, the methods of the disclosure may comprise determining methylation status of one or more CpG dinucleotide sequences within one or more ESR1 binding sites which is/are intragenic. For example, the one or more CpG dinucleotide sequences may be within one or more of the 617 intragenic ESR1 binding sites set forth in Table 2. The 617 genomic regions listed in Table 2 encompass intragenic ESR1 binding sites that overlap estrogen responsive enhancer regions and which contain hypermethylated CpG dinucleotides in multiple models of endocrine resistance (i.e., MCF7-derived cell lines, tamoxifen-resistant (TAMR)10, fulvestrant-resistant (FASR)11 and estrogen deprivation resistant (MCF7X)12 cells), relative to ESR1-positive hormone sensitive MCF7 cells.

The ESR1 binding sites set forth in Table 2 are also defined with reference to hg19. Thus, the nucleotide sequences of each of the regions identified in Table 2 (or in any of the Tables disclosed herein) can be identified by reference to hg19, using the “start” and “end” positions described in Table 2 (or in any of the Tables disclosed herein). For each of the ESR1 binding sites set forth in Table 2, the following information is provided:

(i) Chromosome ID (Column 2);

(ii) genomic coordinates of ESR1-binding site with respect to hg19 (Columns 3-4);

(iii) ESR1-binding site name (Column 5);

(iv) name of gene which ESR1-binding site is most closely associated (Column 6);

(v) inverse correlation between methylation and gene expression in TCGA (Column 7);

(vi) spearman's RHO (Column 8);

(vii) correlation P-value (Column 9); and

(viii) number of HM450K probes per ESR1 binding site (Column 10).

TABLE 2

Hypermethylated ESR1 binding sites in estrogen enhancer regions which are intragenic

Inverse

No.

correlation

statistically

between

significant

methylation and

Correlation
probes

Row
Chromo-

ESR1

Expression

P-value
per ESR1

No.
some
Start
End
site name
Gene
in TCGA
Spearman's RHO
(P > 0.001)
binding site

1
7
27210940
27211286
ER_100307
HOXA-AS4
No
n/a
n/a
n/a

2
7
43288514
43288831
ER_100892
HECW1
No
n/a
n/a
n/a

3
7
44224085
44224425
ER_100934
GCK
No
n/a
n/a
n/a

4
7
47611478
47611980
ER_101074
TNS3
No
n/a
n/a
n/a

5
7
55620324
55620676
ER_101291
VOPP1
No
n/a
n/a
n/a

6
7
75279353
75279713
ER_101692
HIP1
No
n/a
n/a
n/a

7
7
75362309
75362707
ER_101699
HIP1
Yes
−0.25958627
2.59613E−08
1

8
7
87855579
87855879
ER_102057
SRI
No
n/a
n/a
n/a

9
1
224400118
224400678
ER_10231
NVL
No
n/a
n/a
n/a

10
7
98989800
98990147
ER_102479
ARPC1B
No
n/a
n/a
n/a

11
7
99719664
99719969
ER_102522
CNPY4
No
n/a
n/a
n/a

12
7
100463589
100463889
ER_102585
SLC12A9
No
n/a
n/a
n/a

13
7
100464090
100464390
ER_102586
SLC12A9
No
n/a
n/a
n/a

14
7
100800324
100800624
ER_102605
AP1S1
Yes
−0.308429704
2.87107E−11
1

15
7
101017180
101017480
ER_102622
EMID2
No
0.325866479
1.36233E−12
1

16
7
101180514
101180818
ER_102635
EMID2
No
0.471143615
3.03374E−26
1

17
7
101768479
101768882
ER_102709
CUX1
No
n/a
n/a
n/a

18
7
101959130
101959535
ER_102722
SH2B2
Yes
−0.285866301
7.82483E−10
1

19
7
102080614
102081003
ER_102733
ORAI2
Yes
−0.18527387
7.91545E−05
1

20
7
105313870
105314183
ER_102843
ATXN7L1
No
n/a
n/a
n/a

21
1
27240488
27241066
ER_1029
NR0B2
Yes
−0.224802698
1.45501E−06
1

22
7
107886705
107887046
ER_103011
NRCAM
No
n/a
n/a
n/a

23
1
228598287
228598587
ER_10389
TRIM17
Yes
−0.249438828
8.24599E−08
1

24
7
138560635
138560935
ER_104150
KIAA1549
No
n/a
n/a
n/a

25
7
139345059
139345423
ER_104192
HIPK2
Yes
−0.216367357
3.82399E−06
1

26
7
143106427
143106737
ER_104387
EPHA1-AS1
No
n/a
n/a
n/a

27
7
144104415
144104715
ER_104415
NOBOX
No
n/a
n/a
n/a

28
7
148901363
148901733
ER_104483
ZNF282
No
n/a
n/a
n/a

29
7
149569864
149570164
ER_104518
ATP6V0E2
No
n/a
n/a
n/a

30
7
151418719
151419146
ER_104616
PRKAG2
No
n/a
n/a
n/a

31
7
151424787
151425151
ER_104617
PRKAG2
No
n/a
n/a
n/a

32
7
151442252
151442633
ER_104619
PRKAG2
No
n/a
n/a
n/a

33
7
151493516
151493829
ER_104624
PRKAG2
No
n/a
n/a
n/a

34
7
151553656
151553979
ER_104631
PRKAG2
No
n/a
n/a
n/a

35
7
155600847
155601325
ER_104760
SHH
No
n/a
n/a
n/a

36
7
157916425
157916765
ER_104936
PTPRN2
Yes
−0.190965163
0.000045562
1

37
1
230895945
230896482
ER_10494
CAPN9
Yes
−0.361156359
2.60574E−15
1

38
1
231113407
231113970
ER_10502
TTC13
Yes
−0.215328471
0.000004263
1

39
8
1946769
1947125
ER_105037
KBTBD11
No
n/a
n/a
n/a

40
8
17394868
17395391
ER_105282
SLC7A2
Yes
−0.584122654
0
3

41
8
21948482
21948782
ER_105469
FAM160B2
Yes
−0.316480707
8.22104E−12
1

42
8
28223166
28223486
ER_105673
ZNF395
Yes
−0.30437003
5.3156E−11
1

43
8
37700545
37700845
ER_105921
GPR124
No
n/a
n/a
n/a

44
8
48739114
48739414
ER_106145
PRKDC
No
n/a
n/a
n/a

45
8
62567898
62568253
ER_106497
ASPH
Yes
−0.174035493
0.000211725
1

46
8
67425037
67425360
ER_106600
C8orf46
Yes
−0.446493874
1.96242E−23
1

47
8
74002442
74002818
ER_106800
SBSPON
No
n/a
n/a
n/a

48
8
90772144
90772507
ER_107685
RIPK2
No
n/a
n/a
n/a

49
8
98881668
98882017
ER_108508
MATN2
Yes
−0.199715554
2.05104E−05
1

50
8
101017572
101017872
ER_108722
RGS22
Yes
−0.424259141
4.35447E−21
1

51
1
249106283
249106588
ER_10898
SH3BP5L
No
n/a
n/a
n/a

52
8
102526632
102526954
ER_109025
GRHL2
Yes
−0.470255952
0
1

53
10
416280
416842
ER_10930
DIP2C
Yes
−0.266239142
1.1071E−08
1

54
10
579019
579393
ER_10949
DIP2C
Yes
−0.190689534
4.82507E−05
1

55
8
124193498
124193984
ER_110732
FAM83A
Yes
−0.43367174
4.64354E−22
1

56
8
124194626
124194965
ER_110733
FAM83A
Yes
−0.211481781
6.03929E−06
1

57
8
126082161
126082738
ER_111167
KIAA0196
Yes
−0.361311348
3.0008E−15
1

58
8
129103245
129103607
ER_111781
PVT1
Yes
−0.35335898
1.45588E−14
1

59
8
134224471
134224833
ER_112110
WISP1
No
n/a
n/a
n/a

60
8
134249814
134250165
ER_112114
NDRG1
No
n/a
n/a
n/a

61
8
135490722
135491122
ER_112226
ZFAT
No
n/a
n/a
n/a

62
8
142139549
142139849
ER_112470
DENND3
Yes
−0.208632932
8.48193E−06
1

63
8
142247703
142248043
ER_112484
SLC45A4
Yes
−0.18946273
5.40406E−05
2

64
8
143395413
143395760
ER_112563
TSNARE1
Yes
−0.175513031
0.000186653
1

65
8
143625782
143626082
ER_112589
BAI1
No
n/a
n/a
n/a

66
8
143763120
143763420
ER_112608
PSCA
No
n/a
n/a
n/a

67
8
143823681
143823981
ER_112619
SLURP1
Yes
−0.621291369
2.14036E−49
4

68
8
143851193
143851559
ER_112624
LYNX1
Yes
−0.204416812
1.29375E−05
1

69
8
143867136
143867477
ER_112628
LY6D
No
n/a
n/a
n/a

70
8
144129484
144129962
ER_112655
C8orf31
No
0.206690735
9.86107E−06
1

71
10
7984711
7985286
ER_11272
TAF3
No
n/a
n/a
n/a

72
8
145048592
145048911
ER_112777
PLEC
Yes
−0.233518914
5.8926E−07
1

73
8
145086414
145086732
ER_112784
SPATC1
Yes
−0.27978868
1.54356E−09
1

74
9
33443026
33443378
ER_113615
AQP3
Yes
−0.375082083
1.00675E−16
1

75
9
34372740
34373087
ER_113641
KIAA1161
Yes
−0.200056757
1.98428E−05
1

76
9
71660820
71661159
ER_113876
FXN
No
n/a
n/a
n/a

77
9
95834504
95834841
ER_115179
SUSD3
Yes
−0.290895198
3.83898E−10
1

78
1
3388133
3388514
ER_116
ARHGEF16
No
n/a
n/a
n/a

79
9
124979962
124980333
ER_116729
LHX6
No
0.267574721
9.30418E−09
2

80
9
129433798
129434130
ER_117042
LMX1B
Yes
−0.571087528
0
1

81
9
130484264
130484588
ER_117145
TTC16
No
n/a
n/a
n/a

82
9
130487106
130487456
ER_117146
TTC16
No
n/a
n/a
n/a

83
9
130524258
130524602
ER_117150
SH2D3C
No
0.368556421
6.03173E−16
1

84
9
130727796
130728104
ER_117181
FAM102A
No
n/a
n/a
n/a

85
9
133974376
133974700
ER_117471
AIF1L
No
n/a
n/a
n/a

86
9
136728378
136728709
ER_117634
VAV2
Yes
−0.239045592
3.12752E−07
1

87
9
137252760
137253089
ER_117681
RXRA
Yes
−0.244453224
1.65813E−07
1

88
9
137258516
137258927
ER_117685
RXRA
Yes
−0.22430142
1.63893E−06
1

89
9
137302122
137302482
ER_117702
RXRA
No
n/a
n/a
n/a

90
9
139734802
139735102
ER_117864
RABL6
No
n/a
n/a
n/a

91
9
140374065
140374394
ER_117940
PNPLA7
Yes
−0.227579527
1.14457E−06
1

92
9
140408341
140408692
ER_117943
PNPLA7
No
n/a
n/a
n/a

93
10
24496562
24497177
ER_11835
KIAA1217
Yes
−0.283127061
1.14663E−09
1

94
X
18708777
18709167
ER_118438
PPEF1
Yes
−0.26765505
9.20712E−09
2

95
X
40007163
40007510
ER_118823
BCOR
No
n/a
n/a
n/a

96
10
30316774
30317298
ER_12062
KIAA1462
No
n/a
n/a
n/a

97
X
153586498
153586870
ER_120632
FLNA
No
n/a
n/a
n/a

98
1
3471648
3472248
ER_122
MEGF6
No
0.163319391
0.000512684
1

99
1
37321565
37322055
ER_1299
GRIK3
No
0.530788416
4.53213E−34
1

100
10
71213510
71214074
ER_13175
TSPAN15
Yes
−0.424739404
3.89108E−21
1

101
10
73828553
73829113
ER_13269
SPOCK2
No
0.440229269
0
1

102
1
3614361
3614966
ER_133
TP73
No
0.428857492
0
1

103
10
76858883
76859398
ER_13395
DUSP13
Yes
−0.374994214
1.79367E−16
6

104
10
77872231
77872922
ER_13427
C10orf11
No
0.192369608
4.12673E−05
2

105
10
82189362
82189690
ER_13657
FAM213A
No
n/a
n/a
n/a

106
10
88475369
88475919
ER_13815
LDB3
Yes
−0.20457813
1.27322E−05
1

107
10
88703091
88703639
ER_13821
MMRN2
No
n/a
n/a
n/a

108
10
95228052
95228774
ER_14079
MYOF
Yes
−0.293781665
2.53507E−10
1

109
10
102792785
102793085
ER_14340
SFXN3
Yes
−0.550035375
0
1

110
10
105238778
105239360
ER_14449
CALHM3
Yes
−0.200511144
1.82534E−05
4

111
10
112259180
112259480
ER_14629
DUSP5
Yes
−0.47604403
0
1

112
1
42420202
42420763
ER_1481
HIVEP3
No
n/a
n/a
n/a

113
10
119102102
119102657
ER_14914
PDZD8
No
n/a
n/a
n/a

114
10
121079102
121079402
ER_15001
GRK5
No
n/a
n/a
n/a

115
10
121415148
121415773
ER_15018
BAG 3
Yes
−0.449812197
0
1

116
10
123900770
123901156
ER_15113
TACC2
Yes
−0.165628275
0.000425606
1

117
10
126211691
126211991
ER_15194
LHPP
No
n/a
n/a
n/a

118
10
126315723
126316102
ER_15199
FAM53B
Yes
−0.247233418
1.18974E−07
1

119
10
126700346
126700820
ER_15221
CTBP2
Yes
−0.267400497
9.51812E−09
1

120
1
44300618
44300993
ER_1532
ST3GAL3
Yes
−0.203979608
1.35099E−05
1

121
10
128606764
128607329
ER_15340
DOCK1
Yes
−0.286907293
6.76003E−10
1

122
10
134079116
134079490
ER_15434
STK32C
No
n/a
n/a
n/a

123
10
134224980
134225280
ER_15443
PWWP2B
Yes
−0.185156404
7.99969E−05
2

124
10
134261296
134261809
ER_15450
C10orf91
Yes
−0.370354575
4.46339E−16
3

125
10
134420347
134420647
ER_15478
INPP5A
Yes
−0.177054833
0.000163475
1

126
10
134498671
134499284
ER_15480
INPP5A
Yes
−0.207945257
9.09185E−06
1

127
10
134728825
134729477
ER_15487
TTC40
No
n/a
n/a
n/a

128
10
134729867
134730225
ER_15488
TTC40
No
n/a
n/a
n/a

129
10
135178534
135178842
ER_15502
ECHS1
No
n/a
n/a
n/a

130
11
849066
849366
ER_15551
TSPAN4
No
0.165317491
0.000436466
2

131
11
1275364
1275883
ER_15574
MUC5B
No
0.374119444
1.36781E−16
2

132
11
1460055
1460355
ER_15581
BRSK2
No
0.227080381
1.13056E−06
1

133
11
1507075
1507659
ER_15583
MOB2
Yes
−0.155896803
0.000917668
1

134
11
1777371
1777938
ER_15595
CTSD
Yes
−0.389507306
0
1

135
11
2004135
2004733
ER_15613
MRPL23
No
n/a
n/a
n/a

text missing or illegible when filed

136
11
2181411
2181715
ER_15620
INS
No
n/a
n/a
n/a

137
11
2181411
2181715
ER_15620
INS-IGF2
No
n/a
n/a
n/a

138
11
2431263
2431688
ER_15638
TRPM5
No
n/a
n/a
n/a

139
11
12309323
12309637
ER_15849
MICALCL
No
0.262835668
1.71706E−08
1

140
11
20063465
20064029
ER_16131
NAV2
No
n/a
n/a
n/a

141
11
33744676
33745230
ER_16397
CD59
Yes
−0.304313667
5.36089E−11
2

142
11
45928675
45929091
ER_16775
C11orf94
No
n/a
n/a
n/a

143
11
46373841
46374290
ER_16803
DGKZ
Yes
−0.292581725
3.01408E−10
1

144
11
61466759
61467374
ER_17096
DAGLA
Yes
−0.222108817
2.07767E−06
1

145
11
62437396
62437722
ER_17173
C11orf48
Yes
−0.162346349
0.000554124
2

146
11
62647962
62648262
ER_17194
SLC3A2
No
n/a
n/a
n/a

147
11
64034904
64035204
ER_17276
PLCB3
Yes
−0.229382466
9.37378E−07
2

148
11
64053773
64054355
ER_17285
GPR137
No
n/a
n/a
n/a

149
11
64322980
64323660
ER_17306
SLC22A11
No
n/a
n/a
n/a

150
11
66659547
66660139
ER_17525
PC
No
n/a
n/a
n/a

151
11
67165633
67165963
ER_17593
PPP1CA
No
n/a
n/a
n/a

152
11
67186225
67186704
ER_17595
CARNS1
No
n/a
n/a
n/a

153
11
67810737
67811051
ER_17643
TCIRG1
No
n/a
n/a
n/a

154
11
69061731
69062031
ER_17743
MYEOV
Yes
−0.161570899
0.000580565
1

155
11
69515571
69515871
ER_17789
FGF19
No
n/a
n/a
n/a

156
11
70917115
70917490
ER_17848
SHANK2
Yes
−0.315797247
9.15588E−12
3

157
11
71276995
71277590
ER_17878
KRTAP5-10
No
n/a
n/a
n/a

158
11
73000242
73000542
ER_17968
P2RY6
No
0.351070507
2.24479E−14
1

159
11
76371803
76372137
ER_18134
LRRC32
No
0.228162509
1.07319E−06
2

160
11
85468982
85469571
ER_18506
SYTL2
Yes
−0.459623471
0
3

161
1
6330275
6330725
ER_187
ACOT7
No
n/a
n/a
n/a

162
11
117300693
117300993
ER_19145
DSCAML1
No
n/a
n/a
n/a

163
11
118013408
118013932
ER_19187
SCN4B
No
0.166779885
0.000387521
1

164
1
6445555
6445855
ER_192
ACOT7
Yes
−0.346507797
3.85637E−14
1

165
11
118758401
118758701
ER_19225
CXCR5
No
0.327604866
1.01939E−12
1

166
11
119994594
119994894
ER_19291
TRIM29
No
n/a
n/a
n/a

167
11
119995602
119996221
ER_19292
TRIM29
No
n/a
n/a
n/a

168
12
2021757
2022057
ER_19684
CACNA2D4
No
n/a
n/a
n/a

169
12
48396469
48396769
ER_21327
COL2A1
No
n/a
n/a
n/a

170
12
52579425
52579733
ER_21620
KRT80
Yes
−0.156387208
0.000883714
1

171
12
53300210
53300676
ER_21729
KRT8
Yes
−0.358090657
5.78223E−15
1

172
12
53552932
53553480
ER_21786
CSAD
Yes
−0.462318596
0
1

173
12
57559601
57559901
ER_22058
LRP1
No
n/a
n/a
n/a

174
12
58160686
58160986
ER_22088
CYP27B1
Yes
−0.255597575
4.28052E−08
2

175
12
58209624
58209992
ER_22094
AVIL
No
n/a
n/a
n/a

176
12
63193501
63193816
ER_22367
PPM1H
Yes
−0.167030751
0.000379657
1

177
12
65043934
65044256
ER_22510
RASSF3
No
n/a
n/a
n/a

178
12
65066737
65067037
ER_22517
RASSF3
No
n/a
n/a
n/a

179
1
7705733
7706033
ER_240
CAMTA1
No
n/a
n/a
n/a

180
12
112207002
112207577
ER_24835
ALDH2
No
n/a
n/a
n/a

181
12
113547314
113547643
ER_24903
RASAL1
No
0.398144188
1.51701E−18
1

182
12
123446240
123446640
ER_25491
ABCB9
Yes
−0.188629804
5.83395E−05
1

183
12
123449086
123449400
ER_25493
ABCB9
Yes
−0.416828462
0
1

184
12
124844847
124845161
ER_25578
NCOR2
Yes
−0.158414149
0.000755335
2

185
12
124879925
124880326
ER_25592
NCOR2
Yes
−0.219796114
2.66153E−06
3

186
12
125004808
125005149
ER_25616
NCOR2
Yes
−0.17339707
0.000223506
1

187
12
131438531
131438856
ER_25787
GPR133
No
n/a
n/a
n/a

188
12
131622396
131622696
ER_25800
GPR133
No
0.383647195
0
2

189
12
132694663
132695050
ER_25875
GALNT9
No
0.176229524
0.000171659
1

190
13
34208323
34209020
ER_26636
STARD13
Yes
−0.292609379
3.0021E−10
1

191
13
41590103
41590403
ER_26934
ELF1
Yes
−0.28858071
4.43501E−10
1

192
13
51854732
51855052
ER_27435
FAM124A
No
0.197644104
2.50444E−05
1

193
13
113651709
113652104
ER_28798
MCF2L
Yes
−0.416344509
0
1

194
13
113677094
113677394
ER_28802
MCF2L
Yes
−0.233597269
5.84054E−07
1

195
13
113979939
113980263
ER_28813
GRTP1
Yes
−0.561680883
8.91715E−39
2

196
14
21494973
21495345
ER_28907
NDRG2
No
n/a
n/a
n/a

197
1
8823899
8824414
ER_290
RERE
Yes
−0.425083186
0
1

198
14
23623250
23623562
ER_29019
SLC7A8
Yes
−0.354356515
1.20238E−14
1

199
14
23623676
23623976
ER_29020
SLC7A8
Yes
−0.296853548
1.62175E−10
4

200
14
64932503
64932803
ER_31140
AKAP5
Yes
−0.214691628
4.5555E−06
2

201
14
68871364
68871773
ER_31488
RAD51B
No
n/a
n/a
n/a

202
14
74214079
74214466
ER_31871
C14orf43
Yes
−0.432667091
0
1

203
14
74238204
74238510
ER_31884
C14orf43
Yes
−0.195733971
3.00548E−05
1

204
1
1141942
1142242
ER_32
TNFRSF18
No
n/a
n/a
n/a

205
14
76447292
76447654
ER_32084
TGFB3
Yes
−0.303016542
6.51353E−11
3

206
14
88942632
88943092
ER_32519
PTPN21
Yes
−0.168486692
0.000336879
1

207
14
93412574
93412953
ER_32818
ITPK1
Yes
−0.361876948
2.66671E−15
2

208
14
93473727
93474077
ER_32832
ITPK1
Yes
−0.290394125
4.12381E−10
1

209
14
94392599
94392913
ER_32902
FAM181A-
No
n/a
n/a
n/a

text missing or illegible when filed

210
14
95047627
95047927
ER_32949
SERPINA5
Yes
−0.671013091
0
2

211
14
95078366
95078753
ER_32959
SERPINA3
Yes
−0.324660895
2.21327E−12
2

212
14
95615683
95616180
ER_32995
DICER1
Yes
−0.296402122
1.73236E−10
1

213
14
96691960
96692332
ER_33117
BDKRB2
Yes
−0.167100809
0.000377488
1

214
14
100055296
100055707
ER_33296
CCDC85C
Yes
−0.175193161
0.000191832
1

215
14
100618272
100618577
ER_33370
DEGS2
Yes
−0.394516582
0
1

216
14
103566340
103566640
ER_33567
EXOC3L4
No
n/a
n/a
n/a

217
14
103576070
103576413
ER_33570
EXOC3L4
No
n/a
n/a
n/a

218
14
104165149
104165544
ER_33615
XRCC3
No
n/a
n/a
n/a

219
14
104637698
104638043
ER_33656
KIF26A
No
0.430282487
0
1

220
14
105181841
105182228
ER_33695
INF2
Yes
−0.165915618
0.00041579
1

221
14
105434204
105434522
ER_33730
AHNAK2
Yes
−0.17137013
0.000265106
1

222
14
105823215
105823515
ER_33780
PACS2
No
n/a
n/a
n/a

223
15
32951599
32952135
ER_33988
SCG5
No
n/a
n/a
n/a

224
15
50519199
50519766
ER_34615
SLC27A2
Yes
−0.342165772
1.1351E−13
1

225
1
10075638
10076128
ER_353
RBP7
No
n/a
n/a
n/a

226
15
63074443
63074894
ER_35498
TLN2
No
n/a
n/a
n/a

227
15
63345507
63345850
ER_35518
TPM1
No
n/a
n/a
n/a

228
15
63671577
63672181
ER_35532
CA12
Yes
−0.663688479
0
1

229
15
63672612
63673165
ER_35533
CA12
Yes
−0.553637039
0
1

230
15
70384192
70384714
ER_35866
TLE3
Yes
−0.624063592
0
1

231
15
74232754
74233252
ER_36102
LOXL1
Yes
−0.219977053
2.6107E−06
1

232
15
74495260
74495560
ER_36118
STRA6
No
n/a
n/a
n/a

233
15
74537511
74538135
ER_36123
CCDC33
No
n/a
n/a
n/a

234
15
75974558
75974909
ER_36216
CSPG4
No
n/a
n/a
n/a

235
15
79223130
79223938
ER_36351
CTSH
No
0.30157074
8.08343E−11
1

236
15
80878455
80879031
ER_36421
ARNT2
Yes
−0.270986754
5.94211E−09
1

237
15
81596013
81596551
ER_36480
IL16
No
0.445491253
0
1

238
15
83781576
83782122
ER_36550
TM6SF1
No
n/a
n/a
n/a

239
15
86219506
86219806
ER_36659
AKAP13
No
0.155234149
0.000965466
1

240
15
89630906
89631554
ER_36798
ABHD2
No
0.239107665
2.85507E−07
2

241
15
90328102
90328625
ER_36856
ANPEP
No
n/a
n/a
n/a

242
15
90349924
90350224
ER_36858
ANPEP
No
n/a
n/a
n/a

243
15
96866722
96867218
ER_37215
NR2F2
Yes
−0.210236502
6.86762E−06
2

244
15
99236506
99236926
ER_37297
IGF1R
Yes
−0.537894014
0
1

245
15
99271960
99272504
ER_37301
IGF1R
Yes
−0.588333638
0
1

246
15
101548985
101549285
ER_37458
LRRK1
No
0.400507986
0
1

247
16
374320
374831
ER_37511
AXIN1
No
n/a
n/a
n/a

248
16
585341
586026
ER_37534
MIR5587
No
n/a
n/a
n/a

249
16
615200
615500
ER_37536
C16orf11
No
n/a
n/a
n/a

250
16
633459
633759
ER_37539
PIGQ
No
n/a
n/a
n/a

251
16
701631
702182
ER_37550
WDR90
No
0.194088004
0.000035121
1

252
16
863331
863631
ER_37577
PRR25
No
n/a
n/a
n/a

253
16
1138312
1138612
ER_37596
C1QTNF8
No
n/a
n/a
n/a

254
16
1209882
1210346
ER_37604
CACNA1H
No
n/a
n/a
n/a

255
16
1232244
1232599
ER_37605
CACNA1H
Yes
−0.232335435
6.7354E−07
1

256
16
1240629
1241133
ER_37606
CACNA1H
No
n/a
n/a
n/a

257
16
1430147
1430478
ER_37632
UNKL
No
n/a
n/a
n/a

258
16
1827853
1828153
ER_37665
SPSB3
Yes
−0.168403597
0.000339194
1

259
16
2004491
2004791
ER_37679
RPL3L
No
n/a
n/a
n/a

260
16
2047651
2048147
ER_37687
ZNF598
No
n/a
n/a
n/a

261
16
2285522
2286213
ER_37716
E4F1
No
n/a
n/a
n/a

262
16
2294337
2294666
ER_37717
ECI1
No
n/a
n/a
n/a

263
16
2879820
2880369
ER_37757
ZG16B
Yes
−0.331448616
7.19734E−13
5

264
16
3704538
3704838
ER_37830
DNASE1
Yes
−0.225124178
1.40433E−06
1

265
16
3707018
3707318
ER_37831
DNASE1
Yes
−0.190279859
4.85723E−05
1

266
16
4421377
4421694
ER_37887
CORO7
No
n/a
n/a
n/a

267
16
4425919
4426536
ER_37892
VASN
No
n/a
n/a
n/a

268
16
4478392
4478714
ER_37899
DNAJA3
No
n/a
n/a
n/a

269
16
4746859
4747392
ER_37919
ANKS3
No
n/a
n/a
n/a

270
16
4838430
4839026
ER_37923
Sep-12
Yes
−0.290559784
3.32936E−10
3

271
16
14030414
14030714
ER_38174
ERCC4
Yes
−0.286422287
7.23732E−10
1

272
16
14580747
14581084
ER_38229
PARN
Yes
−0.328987106
1.0858E−12
1

273
16
15602986
15603303
ER_38266
C16orf45
No
n/a
n/a
n/a

274
16
16090418
16090901
ER_38297
ABCC1
No
n/a
n/a
n/a

275
16
16108606
16109211
ER_38301
ABCC1
Yes
−0.199363421
2.12218E−05
1

276
16
24747980
24748466
ER_38621
TNRC6A
Yes
−0.363523046
1.88066E−15
1

277
16
28511293
28511661
ER_38732
IL27
No
n/a
n/a
n/a

278
16
28608240
28608616
ER_38739
SULT1A2
Yes
−0.449317709
0
1

279
16
30906082
30906783
ER_38861
BCL7C
No
n/a
n/a
n/a

280
16
68321665
68322263
ER_39511
SLC7A6
No
n/a
n/a
n/a

281
16
69358300
69358626
ER_39598
VPS4A
No
n/a
n/a
n/a

282
16
70472947
70473558
ER_39656
ST3GAL2
No
n/a
n/a
n/a

283
16
70687883
70688183
ER_39667
IL34
No
0.574054983
0
3

284
16
72994910
72995462
ER_39877
ZFHX3
Yes
−0.209502467
7.76642E−06
1

285
16
78991185
78991807
ER_40213
WWOX
No
n/a
n/a
n/a

286
16
81248258
81248721
ER_40354
PKD1L2
No
n/a
n/a
n/a

287
16
84628881
84629206
ER_40553
COTL1
No
0.234534492
5.25106E−07
2

288
16
84871395
84871718
ER_40585
CRISPLD2
No
n/a
n/a
n/a

289
16
85669299
85669599
ER_40724
KIAA0182
Yes
−0.459019682
0
2

290
16
85723351
85723651
ER_40737
GINS2
Yes
−0.29702843
1.58078E−10
2

291
16
85786954
85787520
ER_40750
C16orf74
Yes
−0.272675914
4.74847E−09
2

292
1
109373005
109373612
ER_4085
AKNAD1
Yes
−0.246004825
1.25377E−07
1

293
16
87778638
87778938
ER_40904
KLHDC4
No
n/a
n/a
n/a

294
16
87812661
87813207
ER_40908
KLHDC4
Yes
−0.172678581
0.000237498
1

295
16
87868043
87868626
ER_40912
SLC7A5
No
n/a
n/a
n/a

296
16
87890385
87890763
ER_40918
SLC7A5
Yes
−0.483398404
0
1

297
16
88110906
88111273
ER_40942
BANP
No
n/a
n/a
n/a

298
16
88548864
88549438
ER_40969
ZFPM1
No
n/a
n/a
n/a

299
16
88704888
88705257
ER_40987
IL17C
No
n/a
n/a
n/a

300
16
88988838
88989268
ER_41018
CBFA2T3
No
n/a
n/a
n/a

301
16
88991561
88992120
ER_41020
CBFA2T3
No
n/a
n/a
n/a

302
16
89004344
89004862
ER_41024
CBFA2T3
No
0.289451767
4.71623E−10
1

303
16
89043365
89043665
ER_41029
CBFA2T3
No
0.168608164
0.000327491
2

304
16
89648350
89648952
ER_41093
CPNE7
No
n/a
n/a
n/a

305
1
11020446
11021019
ER_411
C1orf127
Yes
−0.239407483
2.75616E−07
2

306
16
89900078
89900645
ER_41112
SPIRE2
Yes
−0.231466954
7.42637E−07
2

307
16
89927268
89927731
ER_41118
SPIRE2
No
0.337950574
2.37482E−13
1

308
17
151960
152279
ER_41149
RPH3AL
Yes
−0.376348525
6.43879E−17
3

309
17
1634282
1634616
ER_41197
WDR81
No
n/a
n/a
n/a

310
17
1901327
1901683
ER_41216
RTN4RL1
Yes
−0.201776075
1.67812E−05
2

311
17
1987743
1988066
ER_41224
SMG6
Yes
−0.176716395
0.000168319
1

312
17
3635535
3635874
ER_41267
ITGAE
No
0.203757714
1.38096E−05
1

313
17
4436972
4437323
ER_41315
SPNS2
No
0.441142392
0
1

314
17
4455826
4456126
ER_41316
MYBBP1A
No
n/a
n/a
n/a

315
17
14109320
14109680
ER_41695
COX10
Yes
−0.283019603
1.16385E−09
1

316
17
16322433
16322733
ER_41788
TRPV2
Yes
−0.369616508
4.66012E−16
1

317
17
16954969
16955288
ER_41817
MPRIP
No
n/a
n/a
n/a

318
17
17628505
17628831
ER_41872
RAI1
No
n/a
n/a
n/a

319
17
17718324
17718694
ER_41889
SREBF1
Yes
−0.37884006
1.97175E−17
1

320
17
18139202
18139554
ER_41939
LLGL1
Yes
−0.169416014
0.000311973
1

321
17
27295913
27296324
ER_42201
SEZ6
Yes
−0.183419447
9.10278E−05
1

322
17
29649808
29650164
ER_42279
NF1
Yes
−0.385262742
0
1

323
17
39577357
39577695
ER_42572
KRT37
Yes
−0.51802934
2.90057E−32
1

324
17
48048352
48048668
ER_42976
DLX4
No
n/a
n/a
n/a

325
17
48261929
48262229
ER_43003
COL1A1
No
n/a
n/a
n/a

326
17
55371434
55371787
ER_43290
MSI2
Yes
−0.576424707
0
1

327
17
55673703
55674091
ER_43343
MSI2
Yes
−0.341475332
1.28245E−13
1

328
1
12192916
12193471
ER_444
TNFRSF8
No
n/a
n/a
n/a

329
1
1609934
1610467
ER_45
SLC35E2B
No
n/a
n/a
n/a

330
17
59484187
59484499
ER_45541
TBX2
No
n/a
n/a
n/a

331
1
114218048
114218488
ER_4610
MAGI3
Yes
−0.376987343
5.0018E−17
1

332
17
65487266
65487622
ER_46920
PITPNC1
No
0.211843351
6.11506E−06
1

333
17
66291281
66291581
ER_46984
ARSG
Yes
−0.356520016
7.89327E−15
1

334
17
71612589
71612889
ER_47199
SDK2
No
n/a
n/a
n/a

335
17
72439108
72439488
ER_47235
GPRC5C
No
n/a
n/a
n/a

336
17
72732636
72732936
ER_47245
RAB37
No
0.157959167
0.000782549
1

337
17
72740901
72741201
ER_47248
RAB37
No
0.231474987
7.41968E−07
1

338
17
73500571
73500871
ER_47351
CASKIN2
No
n/a
n/a
n/a

339
17
73641528
73641923
ER_47388
RECQL5
Yes
−0.357417469
6.61148E−15
1

340
17
73696463
73696795
ER_47396
SAP30BP
Yes
−0.156918701
0.000848229
1

341
17
73805905
73806251
ER_47422
UNK
No
n/a
n/a
n/a

342
17
73872405
73872705
ER_47428
TRIM47
Yes
−0.203451408
1.42337E−05
2

343
17
74494140
74494511
ER_47474
RHBDF2
No
0.255975124
4.08406E−08
2

344
17
74684239
74684546
ER_47498
MXRA7
Yes
−0.158712948
0.000737942
1

345
17
75181779
75182110
ER_47526
SEC14L1
No
0.183371901
9.38792E−05
1

346
17
75473604
75473943
ER_47548
Sep-09
No
n/a
n/a
n/a

347
17
76498871
76499229
ER_47612
DNAH17
No
n/a
n/a
n/a

348
17
76522848
76523165
ER_47614
DNAH17
No
n/a
n/a
n/a

349
17
76858208
76858508
ER_47631
TIMP2
Yes
−0.172720063
0.000236668
1

350
17
76973124
76973427
ER_47643
LGALS3BP
Yes
−0.285188503
8.60401E−10
1

351
17
78522351
78522735
ER_47743
RPTOR
Yes
−0.211839663
6.11738E−06
1

352
17
78667839
78668195
ER_47754
RPTOR
Yes
−0.28719267
5.41578E−10
2

353
17
78791604
78791917
ER_47760
RPTOR
Yes
−0.235434315
4.73923E−07
1

354
17
78793449
78793890
ER_47761
RPTOR
No
n/a
n/a
n/a

355
17
78796720
78797088
ER_47762
RPTOR
No
n/a
n/a
n/a

356
17
79018691
79019077
ER_47773
BAIAP2
Yes
−0.250472546
8.04182E−08
1

357
17
79251153
79251492
ER_47785
SLC38A10
No
n/a
n/a
n/a

358
17
79961758
79962058
ER_47823
ASPSCR1
No
n/a
n/a
n/a

359
17
80162846
80163196
ER_47840
CCDC57
Yes
−0.224436269
1.61508E−06
1

360
17
80419732
80420082
ER_47870
NARF
No
n/a
n/a
n/a

361
18
3446546
3446906
ER_48111
TGIF1
No
n/a
n/a
n/a

362
1
114521332
114522214
ER_4835
OLFML3
No
n/a
n/a
n/a

363
18
74536197
74536522
ER_49880
ZNF236
Yes
−0.317765849
6.70861E−12
1

364
19
930678
930978
ER_49971
ARID3A
No
n/a
n/a
n/a

365
19
1169031
1169402
ER_49996
SBNO2
No
n/a
n/a
n/a

366
19
1496339
1496639
ER_50033
REEP6
Yes
−0.24403709
1.74201E−07
3

367
19
1907761
1908143
ER_50049
SCAMP4
Yes
−0.311880585
1.68785E−11
2

368
19
2167382
2167682
ER_50060
DOT1L
No
n/a
n/a
n/a

369
19
2624590
2624934
ER_50099
GNG7
No
n/a
n/a
n/a

370
19
3374719
3375065
ER_50124
NFIC
Yes
−0.335512035
2.66239E−13
1

371
19
6276448
6276748
ER_50272
MLLT1
No
n/a
n/a
n/a

372
19
7684955
7685267
ER_50326
XAB2
No
n/a
n/a
n/a

373
19
11617772
11618120
ER_50514
ECSIT
No
n/a
n/a
n/a

374
19
14066552
14066906
ER_50640
DCAF15
No
n/a
n/a
n/a

375
19
14544916
14545316
ER_50670
PKN1
Yes
−0.210598637
6.94619E−06
1

376
19
15590207
15590541
ER_50728
PGLYRP2
Yes
−0.526755523
1.7199E−33
4

377
19
15622532
15622947
ER_50734
CYP4F22
No
n/a
n/a
n/a

378
19
16603746
16604192
ER_50800
CALR3
No
n/a
n/a
n/a

379
19
17407033
17407362
ER_50855
ABHD8
No
n/a
n/a
n/a

380
19
33167485
33167785
ER_51224
RGS9BP
No
n/a
n/a
n/a

381
19
33624481
33624781
ER_51258
WDR88
No
n/a
n/a
n/a

382
19
35531859
35532247
ER_51354
HPN
Yes
−0.269976176
5.90098E−09
3

383
19
35800367
35800739
ER_51381
MAG
Yes
−0.511574789
2.22575E−31
1

384
19
35801186
35801551
ER_51382
MAG
Yes
−0.160341682
0.000640132
1

385
19
35940219
35940897
ER_51391
FFAR2
No
n/a
n/a
n/a

386
19
39222375
39222739
ER_51519
CAPN12
No
n/a
n/a
n/a

387
19
41633774
41634080
ER_51655
CYP2F1
No
n/a
n/a
n/a

388
19
45843797
45844097
ER_51894
KLC3
Yes
−0.288529952
5.37546E−10
2

389
19
45848214
45848514
ER_51895
KLC3
No
n/a
n/a
n/a

390
19
49059555
49059855
ER_52131
SULT2B1
No
n/a
n/a
n/a

391
19
50458158
50458475
ER_52226
SIGLEC11
No
n/a
n/a
n/a

392
19
50969943
50970265
ER_52248
FAM71E1
No
n/a
n/a
n/a

393
19
51568129
51568429
ER_52287
KLK13
Yes
−0.255470367
3.89102E−08
3

394
19
54600186
54600532
ER_52363
OSCAR
No
n/a
n/a
n/a

395
19
55880474
55880774
ER_52411
IL11
No
n/a
n/a
n/a

396
19
56047757
56048080
ER_52419
SBK2
Yes
−0.328477521
8.80677E−13
2

397
2
3452679
3453034
ER_52628
TRAPPC12
No
n/a
n/a
n/a

398
2
19555335
19555655
ER_53364
OSR1
No
0.211392327
6.09547E−06
1

399
2
25094778
25095111
ER_53566
ADCY3
No
n/a
n/a
n/a

400
2
25562754
25563086
ER_53585
DNMT3A
No
n/a
n/a
n/a

401
2
26199821
26200281
ER_53612
KIF3C
No
n/a
n/a
n/a

402
2
26947077
26947447
ER_53662
KCNK3
Yes
−0.3517283
1.49994E−14
3

403
2
27319112
27319450
ER_53686
KHK
No
n/a
n/a
n/a

404
2
28549119
28549437
ER_53751
BRE
No
n/a
n/a
n/a

405
2
45998277
45998784
ER_54551
PRKCE
Yes
−0.165247828
0.000438935
1

406
2
46361686
46362029
ER_54598
PRKCE
Yes
−0.154901111
0.000990347
1

407
2
47236009
47236309
ER_54678
TTC7A
No
0.203893879
0.000013625
1

408
2
54760101
54760460
ER_54825
SPTBN1
No
n/a
n/a
n/a

409
1
16074063
16074363
ER_550
TMEM82
Yes
−0.15474423
0.000990014
1

410
1
115211543
115212151
ER_5516
DENND2C
No
n/a
n/a
n/a

411
2
85280957
85281340
ER_55852
KCMF1
No
n/a
n/a
n/a

412
2
85621930
85622230
ER_55867
CAPG
No
n/a
n/a
n/a

413
1
16251091
16251574
ER_559
SPEN
No
n/a
n/a
n/a

414
2
95719289
95719611
ER_56072
MAL
No
0.264684405
1.18981E−08
1

415
2
97508396
97508742
ER_56156
ANKRD23
No
n/a
n/a
n/a

416
2
106007690
106008035
ER_56517
FHL2
Yes
−0.20727681
9.72463E−06
1

417
2
113875147
113875494
ER_56808
IL1RN
No
n/a
n/a
n/a

418
2
121036360
121036660
ER_56956
RALB
No
n/a
n/a
n/a

419
2
121036666
121036969
ER_56957
RALB
No
n/a
n/a
n/a

420
2
129066951
129067320
ER_57178
HS6ST1
No
n/a
n/a
n/a

421
1
116219030
116219646
ER_5750
VANGL1
Yes
−0.289419108
4.73818E−10
1

422
2
175499224
175499524
ER_59117
WIPF1
Yes
−0.320473352
4.35639E−12
2

423
1
16950505
16950951
ER_599
CROCCP2
No
n/a
n/a
n/a

424
2
197158771
197159125
ER_59918
HECW2
No
n/a
n/a
n/a

425
1
17035334
17035751
ER_603
ESPNP
No
n/a
n/a
n/a

426
2
216478034
216478350
ER_60685
LINC00607
No
n/a
n/a
n/a

427
2
220007041
220007402
ER_60937
NHEJ1
No
n/a
n/a
n/a

428
2
224624781
224625214
ER_61179
AP1S3
Yes
−0.292051154
3.25296E−10
1

429
2
236447665
236448023
ER_61661
AGAP1
Yes
−0.18315725
9.56943E−05
1

430
2
239169600
239169933
ER_61869
PER2
No
n/a
n/a
n/a

431
2
240186821
240187208
ER_61904
HDAC4
No
0.178834332
0.000140089
1

432
2
240241009
240241336
ER_61909
HDAC4
No
n/a
n/a
n/a

433
2
241807610
241808275
ER_61956
AGXT
Yes
−0.350729914
1.79926E−14
6

434
2
241832846
241833163
ER_61959
C2orf54
Yes
−0.342330286
8.10744E−14
1

435
2
241975925
241976252
ER_61978
SNED1
No
0.321003133
4.00127E−12
3

436
2
242138256
242138571
ER_61990
ANO7
Yes
−0.164860403
0.000452908
1

437
2
242500199
242500499
ER_62006
BOK
Yes
−0.326941071
1.52312E−12
1

438
20
17595352
17595711
ER_62469
RRBP1
No
n/a
n/a
n/a

439
20
18035798
18036353
ER_62490
OVOL2
Yes
−0.264697876
1.18771E−08
1

440
20
30432800
30433405
ER_62916
FOXS1
No
n/a
n/a
n/a

441
20
32888452
32889022
ER_63021
AHCY
Yes
−0.255937198
4.10339E−08
1

442
20
34205164
34205464
ER_63067
SPAG4
Yes
−0.253362831
5.64449E−08
1

443
20
35093680
35094313
ER_63095
DLGAP4
Yes
−0.226710124
1.25956E−06
2

444
20
36767493
36768090
ER_63162
TGM2
No
n/a
n/a
n/a

445
1
118727703
118728260
ER_6319
SPAG17
Yes
−0.468876303
5.62552E−26
9

446
1
17634485
17634785
ER_634
PADI4
Yes
−0.304565675
4.10341E−11
4

447
20
44048102
44048440
ER_63559
PIGT
Yes
−0.483985863
0
1

448
20
44330526
44330841
ER_63571
WFDC13
Yes
−0.372474463
2.94819E−16
1

449
20
47278241
47278894
ER_65136
PREX1
No
0.312514399
1.52976E−11
1

450
1
144989293
144989659
ER_6529
PDE4DIP
No
n/a
n/a
n/a

451
20
49411019
49411779
ER_65780
BCAS4
No
n/a
n/a
n/a

452
1
17887921
17888496
ER_662
ARHGEF10L
No
n/a
n/a
n/a

453
1
18006751
18007051
ER_669
ARHGEF10L
Yes
−0.203444297
1.42437E−05
1

454
20
58325746
58326345
ER_68282
PHACTR3
No
n/a
n/a
n/a

455
20
60510103
60510565
ER_68459
CDH4
No
n/a
n/a
n/a

456
20
60924954
60925254
ER_68486
LAMA5
Yes
−0.172436407
0.000242396
1

457
20
60932217
60932572
ER_68489
LAMA5
No
n/a
n/a
n/a

458
20
61451571
61451871
ER_68537
COL9A3
No
n/a
n/a
n/a

459
20
62282532
62282832
ER_68587
STMN3
No
n/a
n/a
n/a

460
1
2036450
2036863
ER_69
PRKCZ
Yes
−0.217921669
3.24696E−06
2

461
21
42212292
42212834
ER_69514
DSCAM
No
n/a
n/a
n/a

462
21
43107677
43108088
ER_69583
LINC00111
No
n/a
n/a
n/a

463
21
43735388
43735910
ER_69642
TFF3
Yes
−0.64300849
7.33107E−54
3

464
21
44897853
44898245
ER_69724
LINC00313
No
n/a
n/a
n/a

465
21
46321203
46321769
ER_69832
ITGB2
No
0.678975995
0
2

466
21
46325777
46326117
ER_69834
ITGB2
No
n/a
n/a
n/a

467
21
46331748
46332340
ER_69835
ITGB2
No
n/a
n/a
n/a

468
21
46409729
46410331
ER_69844
LINC00163
No
n/a
n/a
n/a

469
21
46953747
46954295
ER_69890
SLC19A1
No
n/a
n/a
n/a

470
22
18919539
18919839
ER_69990
PRODH
Yes
−0.498286049
1.29155E−29
1

471
22
19755289
19755589
ER_70033
TBX1
No
0.390493517
0
1

472
22
19879093
19879435
ER_70044
TXNRD2
No
n/a
n/a
n/a

473
1
151554445
151555180
ER_7042
TUFT1
Yes
−0.426720988
0
1

474
22
35695235
35695568
ER_70647
TOM1
No
n/a
n/a
n/a

475
22
38610003
38610303
ER_70793
MAFF
No
n/a
n/a
n/a

476
22
39759998
39760357
ER_70842
SYNGR1
Yes
−0.473134386
0
4

477
22
40404687
40405045
ER_70880
FAM83F
No
n/a
n/a
n/a

478
22
43525157
43525457
ER_70998
BIK
No
n/a
n/a
n/a

479
22
46921821
46922121
ER_71163
CELSR1
Yes
−0.358860373
4.95469E−15
1

480
22
50450954
50451412
ER_71287
IL17REL
No
n/a
n/a
n/a

481
22
50720235
50720566
ER_71300
PLXNB2
Yes
−0.26103668
2.16012E−08
3

482
22
50738759
50739090
ER_71305
PLXNB2
Yes
−0.165338693
0.000435717
1

483
3
8693628
8694000
ER_71572
C3orf32
No
n/a
n/a
n/a

484
3
9757534
9757834
ER_71643
CPNE9
No
n/a
n/a
n/a

485
3
11550571
11550871
ER_71746
ATG7
No
n/a
n/a
n/a

486
3
11643071
11643504
ER_71757
VGLL4
Yes
−0.222133443
2.07217E−06
2

487
3
12985852
12986194
ER_71863
IQSEC1
No
n/a
n/a
n/a

488
3
12994633
12995020
ER_71864
IQSEC1
Yes
−0.187690211
6.35759E−05
1

489
3
14920746
14921046
ER_72049
FGD5
No
n/a
n/a
n/a

490
3
15310870
15311256
ER_72070
SH3BP5
No
n/a
n/a
n/a

491
3
15687178
15687551
ER_72120
BTD
Yes
−0.367472893
7.79001E−16
1

492
1
154298772
154299072
ER_7259
ATP8B2
No
n/a
n/a
n/a

493
1
154377532
154377832
ER_7267
IL6R
Yes
−0.226436213
1.29803E−06
1

494
3
38067281
38067610
ER_73005
PLCD1
No
n/a
n/a
n/a

495
3
46734109
46734463
ER_73287
ALS2CL
No
n/a
n/a
n/a

496
3
48589861
48590161
ER_73356
PFKFB4
Yes
−0.248525803
1.01827E−07
1

497
1
155161639
155162099
ER_7336
MUC1
Yes
−0.415072063
3.62028E−20
3

498
3
52280070
52280428
ER_73463
PPM1M
Yes
−0.1589276
0.000725678
4

499
3
58028409
58028709
ER_73666
FLNB
Yes
−0.538338329
0
1

500
1
155912401
155912737
ER_7372
RXFP4
No
n/a
n/a
n/a

501
1
156095999
156096299
ER_7395
LMNA
Yes
−0.275981939
3.04831E−09
1

502
3
61793488
61793816
ER_73972
PTPRG
No
n/a
n/a
n/a

503
1
156822285
156822644
ER_7451
INSRR
No
0.245053419
1.40657E−07
1

504
1
161646681
161647242
ER_7530
FCGR2B
No
0.257178093
3.51452E−08
1

505
1
2174671
2174976
ER_76
SKI
No
n/a
n/a
n/a

506
3
63922231
63922573
ER_76144
ATXN7
No
n/a
n/a
n/a

507
3
66519928
66520259
ER_77324
LRIG1
Yes
−0.507588811
0
1

508
3
66543250
66543644
ER_77334
LRIG1
Yes
−0.352144949
1.83363E−14
1

509
3
69942380
69942719
ER_77420
MITF
No
n/a
n/a
n/a

510
3
99721769
99722069
ER_78150
FILIP1L
No
n/a
n/a
n/a

511
3
99833502
99833835
ER_78166
C3orf26
No
n/a
n/a
n/a

512
1
171226439
171226888
ER_7821
FMO1
No
0.188726331
5.78252E−05
1

513
3
112359914
112360214
ER_78589
CCDC80
No
n/a
n/a
n/a

514
3
122057821
122058174
ER_78781
CSTA
No
n/a
n/a
n/a

515
3
124493174
124493574
ER_78936
ITGB5
No
n/a
n/a
n/a

516
3
126200679
126200979
ER_79014
UROC1
No
n/a
n/a
n/a

517
3
126678959
126679283
ER_79028
CHCHD6
No
n/a
n/a
n/a

518
3
129295579
129295977
ER_79235
PLXND1
No
n/a
n/a
n/a

519
3
133174831
133175237
ER_79397
BFSP2
Yes
−0.17660393
0.000166181
1

520
3
160787663
160788036
ER_80595
PPM1L
No
0.259407701
2.65537E−08
1

521
3
168870086
168870443
ER_80932
MECOM
No
n/a
n/a
n/a

522
3
169758098
169758589
ER_80993
GPR160
Yes
−0.525559204
0
1

523
3
183959578
183959923
ER_81693
VWA5B2
Yes
−0.24189682
2.05298E−07
1

524
3
184052187
184052556
ER_81701
EIF4G1
No
n/a
n/a
n/a

525
3
195531202
195531699
ER_82319
MUC4
No
n/a
n/a
n/a

526
3
195603560
195603892
ER_82326
TNK2
No
n/a
n/a
n/a

527
3
196388403
196388727
ER_82377
LRRC33
No
0.496219142
0
2

528
4
680920
681252
ER_82472
MFSD7
Yes
−0.324124136
2.41555E−12
1

529
4
757427
757729
ER_82479
PCGF3
Yes
−0.384333816
0
1

530
4
965412
965736
ER_82491
DGKQ
Yes
−0.182120866
0.000104932
1

531
4
987571
987878
ER_82493
IDUA
Yes
−0.316721564
7.91431E−12
2

532
4
1239191
1239531
ER_82503
CTBP1
No
n/a
n/a
n/a

533
4
1729872
1730230
ER_82525
TACC3
No
n/a
n/a
n/a

534
4
1986291
1986591
ER_82549
WHSC2
Yes
−0.180201515
0.000124297
1

535
4
2798112
2798444
ER_82594
SH3BP2
No
n/a
n/a
n/a

536
4
3341090
3341444
ER_82632
RGS12
Yes
−0.390930721
0
1

537
4
6928789
6929118
ER_82798
TBC1D14
No
n/a
n/a
n/a

538
4
7219719
7220019
ER_82822
SORCS2
No
n/a
n/a
n/a

539
4
8062466
8062766
ER_82882
ABLIM2
No
0.165497975
0.000422976
3

540
4
8130159
8130480
ER_82884
ABLIM2
No
0.307594935
3.26131E−11
1

541
4
8412544
8412844
ER_82916
ACOX3
No
n/a
n/a
n/a

542
4
119910484
119910799
ER_85984
SYNPO2
No
n/a
n/a
n/a

543
5
373069
373625
ER_88159
AHRR
No
n/a
n/a
n/a

544
5
429733
430313
ER_88163
AHRR
No
n/a
n/a
n/a

545
5
610077
610670
ER_88173
CEP72
No
n/a
n/a
n/a

546
5
672776
673076
ER_88176
TPPP
Yes
−0.173994887
0.000208036
1

547
5
1207117
1207709
ER_88196
SLC6A19
No
0.176046676
0.000174395
1

548
5
1443109
1443488
ER_88204
SLC6A3
No
n/a
n/a
n/a

549
5
37840402
37840702
ER_89386
GDNF
No
n/a
n/a
n/a

550
5
43603889
43604448
ER_89613
NNT
Yes
−0.217287839
3.28333E−06
1

551
5
52385969
52386507
ER_89737
ITGA2
Yes
−0.21285208
5.51202E−06
1

552
1
201252202
201252787
ER_9013
PKP1
Yes
−0.160895926
0.000612605
5

553
5
95226771
95227393
ER_91036
ELL2
Yes
−0.204999399
1.22105E−05
1

554
1
203096814
203097402
ER_9205
ADORA1
Yes
−0.156376349
0.000873099
1

555
1
203122931
203123270
ER_9215
ADORA1
Yes
−0.17524604
0.000186865
1

556
5
138299601
138299901
ER_92405
SIL1
Yes
−0.199535932
2.08704E−05
1

557
5
139033837
139034156
ER_92444
CXXC5
Yes
−0.689848806
0
1

558
5
140012702
140013307
ER_92479
CD14
No
0.172842269
0.00023424
1

559
5
159687745
159688359
ER_93120
CCNJL
No
n/a
n/a
n/a

560
5
176239632
176240113
ER_93644
UNC5A
No
n/a
n/a
n/a

561
5
176816546
176817069
ER_93670
SLC34A1
Yes
−0.338284799
1.64743E−13
2

562
5
176874917
176875279
ER_93675
PRR7-AS1
No
n/a
n/a
n/a

563
5
177029224
177029524
ER_93689
B4GALT7
Yes
−0.203926933
1.35805E−05
1

564
5
177547872
177548392
ER_93712
N4BP3
Yes
−0.200270092
1.94359E−05
1

565
6
3139405
3139938
ER_93950
BPHL
Yes
−0.183369268
9.39013E−05
1

566
6
3723005
3723548
ER_93997
PXDC1
No
n/a
n/a
n/a

567
1
205633387
205633954
ER_9471
SLC45A3
No
n/a
n/a
n/a

568
6
30698796
30699096
ER_95075
FLOT1
Yes
−0.158565326
0.000746488
1

569
6
31477979
31478310
ER_95117
MICB
Yes
−0.294476318
2.29255E−10
2

570
6
31743735
31744048
ER_95131
VWA7
No
n/a
n/a
n/a

571
6
31744689
31744989
ER_95132
VWA7
No
n/a
n/a
n/a

572
6
31833129
31833429
ER_95141
SLC44A4
Yes
−0.191804667
4.35012E−05
3

573
6
31868596
31868896
ER_95144
C2
No
0.289384343
4.76165E−10
8

574
6
32050977
32051543
ER_95152
TNXB
No
0.160713551
0.000630719
1

575
6
32135005
32135578
ER_95158
EGFL8
Yes
−0.241470361
2.3573E−07
17

576
6
33173123
33173423
ER_95186
HSD17B8
Yes
−0.254003888
5.21528E−08
2

577
6
33288112
33288670
ER_95195
DAXX
Yes
−0.24031322
2.69879E−07
13

578
6
33993601
33993995
ER_95239
GRM4
Yes
−0.292034242
2.68505E−10
1

579
6
34511677
34512101
ER_95268
SPDEF
Yes
−0.522863026
0
1

580
6
40363002
40363510
ER_95568
LRFN2
Yes
−0.408390902
1.62215E−19
1

581
6
42108932
42109278
ER_95663
C6orf132
Yes
−0.240988721
2.28671E−07
1

582
6
43463227
43463527
ER_95722
TJAP1
No
n/a
n/a
n/a

583
6
43603250
43603772
ER_95728
MAD2L1BP
No
0.180749995
0.000118445
1

584
6
52268606
52268913
ER_96019
PAQR8
No
0.418197621
0
1

585
6
56580975
56581555
ER_96201
RNU6-71
No
n/a
n/a
n/a

586
6
64281501
64282055
ER_96269
PTP4A1
Yes
−0.259535604
2.31991E−08
5

587
6
109267245
109267600
ER_97322
ARMC2
Yes
−0.189528837
5.37126E−05
1

588
6
138425688
138426269
ER_98290
PERP
No
n/a
n/a
n/a

589
6
151937170
151937602
ER_98798
CCDC170
No
n/a
n/a
n/a

590
6
152124722
152125100
ER_98841
ESR1
Yes
−0.636001231
0
1

591
7
921963
922279
ER_99284
GET4
Yes
−0.206381595
0.000010638
1

592
7
923724
924024
ER_99285
GET4
Yes
−0.309385232
2.48011E−11
1

593
7
927894
928206
ER_99286
GET4
Yes
−0.265790218
1.17348E−08
2

594
7
940841
941164
ER_99287
ADAP1
No
n/a
n/a
n/a

595
7
955121
955452
ER_99289
ADAP1
No
n/a
n/a
n/a

596
7
966854
967230
ER_99290
ADAP1
No
0.267014125
1.00097E−08
3

597
7
1004933
1005233
ER_99293
COX19
No
n/a
n/a
n/a

598
7
1026106
1026406
ER_99296
CYP2W1
No
n/a
n/a
n/a

599
7
1027843
1028143
ER_99297
CYP2W1
No
n/a
n/a
n/a

600
7
1054343
1054643
ER_99302
C7orf50
No
n/a
n/a
n/a

601
7
1139221
1139554
ER_99313
C7orf50
No
n/a
n/a
n/a

602
1
217886370
217887036
ER_9936
SPATA17
Yes
−0.496347538
0
1

603
7
1753468
1753819
ER_99371
ELFN1
No
0.31719091
5.63452E−12
3

604
7
1782517
1782817
ER_99372
ELFN1
No
n/a
n/a
n/a

605
7
1891218
1891549
ER_99377
MAD1L1
No
n/a
n/a
n/a

606
7
1931906
1932296
ER_99382
MAD1L1
No
n/a
n/a
n/a

607
7
1959815
1960115
ER_99385
MAD1L1
No
n/a
n/a
n/a

608
7
1961938
1962358
ER_99386
MAD1L1
No
n/a
n/a
n/a

609
7
1967307
1967639
ER_99387
MAD1L1
No
n/a
n/a
n/a

610
7
1968165
1968519
ER_99388
MAD1L1
No
n/a
n/a
n/a

611
7
2191489
2191835
ER_99406
MAD1L1
No
n/a
n/a
n/a

612
7
2673205
2673630
ER_99460
TTYH3
Yes
−0.20153074
1.71888E−05
1

613
7
2702609
2702937
ER_99466
TTYH3
No
0.168197637
0.000344996
1

614
7
2731506
2731863
ER_99473
AMZ1
No
n/a
n/a
n/a

615
7
4876684
4876984
ER_99580
RADIL
No
n/a
n/a
n/a

616
7
5436737
5437105
ER_99601
TNRC18
No
n/a
n/a
n/a

617
7
5526272
5526875
ER_99612
FBXL18
No
n/a
n/a
n/a

text missing or illegible when filed

indicates data missing or illegible when filed

In one example, the method of detecting differential methylation comprises identifying differential methylation at one or more CpG dinucleotide sequences within one or more regions set forth in rows 1-617 of Table 2 in a subject suffering from ESR1 positive cancer relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences. For example, the method may comprise detecting differential methylation at a single CpG dinucleotide sequence within any one of the genomic regions defined in Table 2 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequence.

In another example, the method of detecting differential methylation comprises detecting differential methylation at two or more CpG dinucleotides within any genomic region defined in Table 2 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequences. For example, the method may comprise detecting differential methylation at two or more CpG dinucleotides, or three or more CpG dinucleotides, or four or more CpG dinucleotides, or five or more CpG dinucleotides, or six or more CpG dinucleotides, or seven or more CpG dinucleotides, or eight or more CpG dinucleotides, or nine or more CpG dinucleotides, or 10 or more CpG dinucleotides, or 20 or more CpG dinucleotides, or 30 or more CpG dinucleotides, or 40 or more CpG dinucleotides, or 50 or more CpG dinucleotides within a genomic region set forth in Table 2 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequences. The two or more CpG dinucleotides may be consecutive (i.e., contiguous) within a genomic region. Alternatively, the two or more CpG dinucleotides may not be consecutive (i.e., may not be contiguous) within any genomic region.

In yet another example, the method of detecting differential methylation comprises detecting differential methylation of at least one CpG dinucleotide within two or more different genomic regions set forth in Table 2 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequences. For example, the method may comprise detecting differential methylation at a CpG dinucleotide within two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more different genomic regions set forth in Table 2 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequences.

In one example, identifying differential methylation at one or more CpG dinucleotide sequences within one or more regions set forth in rows 1-617 of Table 2 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject's likely response to endocrine therapy.

Detecting differential methylation at a single CpG dinucleotide sequence within any one of the genomic regions defined in Table 2 may be indicative of the subject's likely response to endocrine therapy.

Alternatively, detecting differential methylation at two or more CpG dinucleotides within any genomic region defined in Table 2 may be indicative of the subject's likely response to endocrine therapy. For example, detecting differential methylation at two or more CpG dinucleotides, or three or more CpG dinucleotides, or four or more CpG dinucleotides, or five or more CpG dinucleotides, or six or more CpG dinucleotides, or seven or more CpG dinucleotides, or eight or more CpG dinucleotides, or nine or more CpG dinucleotides, or 10 or more CpG dinucleotides, or 20 or more CpG dinucleotides, or 30 or more CpG dinucleotides, or 40 or more CpG dinucleotides, or 50 or more CpG dinucleotides within a genomic region set forth in Table 2 may be indicative of the subject's likely response to endocrine therapy. The two or more CpG dinucleotides may be consecutive (i.e., contiguous) within a genomic region. Alternatively, the two or more CpG dinucleotides may not be consecutive (i.e., may not be contiguous) within any genomic region.

Detecting differential methylation of at least one CpG dinucleotide within two or more different genomic regions set forth in Table 2 may be indicative of the subject's likely response to endocrine therapy. For example, detecting differential methylation at a CpG dinucleotide within two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more different genomic regions set forth in Table 2 may be indicative of the subject's likely response to endocrine therapy.

Detecting differential methylation at a single CpG dinucleotide sequence within any one of the genomic regions defined in Table 2 may be indicative of the subject having breast cancer which is refractory to endocrine therapy.

Alternatively, detecting differential methylation at two or more CpG dinucleotides within any genomic region defined in Table 2 may be indicative of the subject having breast cancer which is refractory to endocrine therapy. For example, detecting differential methylation at two or more CpG dinucleotides, or three or more CpG dinucleotides, or four or more CpG dinucleotides, or five or more CpG dinucleotides, or six or more CpG dinucleotides, or seven or more CpG dinucleotides, or eight or more CpG dinucleotides, or nine or more CpG dinucleotides, or 10 or more CpG dinucleotides, or 20 or more CpG dinucleotides, or 30 or more CpG dinucleotides, or 40 or more CpG dinucleotides, or 50 or more CpG dinucleotides within a genomic region set forth in Table 2 may be indicative of the subject having breast cancer which is refractory to endocrine therapy. The two or more CpG dinucleotides may be consecutive (i.e., contiguous) within a genomic region. Alternatively, the two or more CpG dinucleotides may not be consecutive (i.e., may not be contiguous) within any genomic region.

Detecting differential methylation of at least one CpG dinucleotide within two or more different genomic regions set forth in Table 2 may be indicative of the subject having breast cancer which is refractory to endocrine therapy. For example, detecting differential methylation at a CpG dinucleotide within two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more different genomic regions set forth in Table 2 may be indicative of the subject having breast cancer which is refractory to endocrine therapy.

Detecting differential methylation at a single CpG dinucleotide sequence within any one of the genomic regions defined in Table 2 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer.

Alternatively, detecting differential methylation at two or more CpG dinucleotides within any genomic region defined in Table 2 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. For example, detecting differential methylation at two or more CpG dinucleotides, or three or more CpG dinucleotides, or four or more CpG dinucleotides, or five or more CpG dinucleotides, or six or more CpG dinucleotides, or seven or more CpG dinucleotides, or eight or more CpG dinucleotides, or nine or more CpG dinucleotides, or 10 or more CpG dinucleotides, or 20 or more CpG dinucleotides, or 30 or more CpG dinucleotides, or 40 or more CpG dinucleotides, or 50 or more CpG dinucleotides within a genomic region set forth in Table 2 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. The two or more CpG dinucleotides may be consecutive (i.e., contiguous) within a genomic region. Alternatively, the two or more CpG dinucleotides may not be consecutive (i.e., may not be contiguous) within any genomic region.

Detecting differential methylation of at least one CpG dinucleotide within two or more different genomic regions set forth in Table 2 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. For example, detecting differential methylation at a CpG dinucleotide within two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more different genomic regions set forth in Table 2 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer.

Of the ESR1 binding sites set forth in Table 1, hypermethylation of 328 of those sites correlated with reduced expression of the gene(s) with which the respective ESR1 binding sites were most closely associated (Table 3). These 328 ESR1 binding sites represented 291 unique genes. Accordingly, the method of the disclosure may comprise determining the methylation status of one or more CpG dinucleotide sequences within one or more of the ESR1 binding sites set forth in Table 3. The ESR1 binding sites set forth in Table 3 are also defined with reference to hg19. Thus, the nucleotide sequences of each of the regions identified in Table 3 (or in any of the Tables disclosed herein) can be identified by reference to hg19, using the “start” and “end” positions described in Table 3 (or in any of the Tables disclosed herein).

For each of the ESR1 binding sites set forth in Table 3, the following information is provided:

(i) Chromosome ID (Column 2);

TABLE 3

Hypermethylated ESR1 binding sites in estrogen enhancer regions correlating with reduced gene expression

Row No.
Chromosome
Start
End
ESR1 site name
Gene
Probe name
Spearman'sRHO
Correlation P-value

1
7
74024952
74024954
ER_101675
GTF2IRD1
cg13446584
−0.335580192
3.57E−13

2
7
75362553
75362555
ER_101699
HIP1
cg27531731
−0.25958627
2.60E−08

3
1
223854446
223854448
ER_10205
CAPN8
cg02313130
−0.510874625
2.77E−31

4
7
100800361
100800363
ER_102605
AP1S1
cg24080086
−0.308429704
2.87E−11

5
7
101959149
101959151
ER_102722
SH2B2
cg27275553
−0.285866301
7.82E−10

6
7
102080712
102080714
ER_102733
ORAI2
cg22402224
−0.18527387
7.92E−05

7
1
27240513
27240515
ER_1029
NR0B2
cg16762386
−0.224802698
1.46E−06

8
1
228598445
228598447
ER_10389
TRIM17
cg13472406
−0.249438828
8.25E−08

9
7
139345114
139345116
ER_104192
HIPK2
cg14630106
−0.216367357
3.82E−06

10
1
230882386
230882388
ER_10488
CAPN9
cg27412093
−0.512610884
1.61E−31

11
7
157916686
157916688
ER_104936
PTPRN2
cg27324698
−0.190965163
4.56E−05

12
1
230896050
230896052
ER_10494
CAPN9
cg18126320
−0.361156359
2.61E−15

13
1
231113418
231113420
ER_10502
TTC13
cg00018229
−0.215328471
4.26E−06

14
8
17394957
17394959
ER_105282
SLC7A2
cg16906456
−0.584122654
0

15
8
21948559
21948561
ER_105469
FAM160B2
cg20576064
−0.316480707
8.22E−12

16
8
28223456
28223458
ER_105673
ZNF395
cg09759289
−0.30437003
5.32E−11

17
8
62568171
62568173
ER_106497
ASPH
cg11390489
−0.174035493
0.000211725

18
8
67425315
67425317
ER_106600
C8orf46
cg12897502
−0.446493874
1.96E−23

19
8
98881926
98881928
ER_108508
MATN2
cg16202564
−0.199715554
2.05E−05

20
8
101017844
101017846
ER_108722
RGS22
cg16308540
−0.424259141
4.35E−21

21
1
246869081
246869083
ER_10884
SCCPDH
cg14094521
−0.6475097
0

22
8
102526826
102526828
ER_109025
GRHL2
cg04225551
−0.470255952
0

23
10
416314
416316
ER_10930
DIP2C
cg01117269
−0.266239142
1.11E−08

24
10
579258
579260
ER_10949
DIP2C
cg23596826
−0.190689534
4.83E−05

25
8
124179874
124179876
ER_110727
FAM83A
cg12002745
−0.372495492
2.94E−16

26
8
124193816
124193818
ER_110732
FAM83A
cg02715629
−0.43367174
4.64E−22

27
8
124194964
124194966
ER_110733
FAM83A
cg15425827
−0.211481781
6.04E−06

28
8
126082719
126082721
ER_111167
KIAA0196
cg16593865
−0.361311348
3.00E−15

29
10
5538777
5538779
ER_11143
CALML5
cg05876550
−0.271567366
5.50E−09

30
8
129103557
129103559
ER_111781
PVT1
cg19508622
−0.35335898
1.46E−14

31
8
142139838
142139840
ER_112470
DENND3
cg01287592
−0.208632932
8.48E−06

32
8
142247863
142247865
ER_112484
SLC45A4
cg27109043
−0.18946273
5.40E−05

33
8
143395440
143395442
ER_112563
TSNARE1
cg12692919
−0.175513031
0.000186653

34
8
143823733
143823735
ER_112619
SLURP1
cg20862496
−0.621291369
2.14E−49

35
8
143851426
143851428
ER_112624
LYNX1
cg17770910
−0.204416812
1.29E−05

36
8
145048766
145048768
ER_112777
PLEC
cg07598407
−0.233518914
5.89E−07

37
8
145086426
145086428
ER_112784
SPATC1
cg03288742
−0.27978868
1.54E−09

38
8
145721559
145721561
ER_112829
PPP1R16A
cg00994323
−0.166529283
0.000395527

39
8
145728629
145728631
ER_112830
GPT
cg19352605
−0.510988729
0

40
9
33443364
33443366
ER_113615
AQP3
cg14473416
−0.375082083
1.01E−16

41
9
34372820
34372822
ER_113641
KIAA1161
cg20241254
−0.200056757
1.98E−05

42
9
95834819
95834821
ER_115179
SUSD3
cg13916469
−0.290895198
3.84E−10

43
1
32039681
32039683
ER_1168
TINAGL1
cg03211098
−0.32389197
2.51E−12

44
1
3398885
3398887
ER_117
ARHGEF16
cg22007638
−0.482192011
0

45
9
129433968
129433970
ER_117042
LMX1B
cg14916924
−0.571087528
0

46
9
136728553
136728555
ER_117634
VAV2
cg14178533
−0.239045592
3.13E−07

47
9
137253048
137253050
ER_117681
RXRA
cg13677043
−0.244453224
1.66E−07

48
9
137258919
137258921
ER_117685
RXRA
cg13786567
−0.22430142
1.64E−06

49
9
139949062
139949064
ER_117897
ENTPD2
cg13981310
−0.367248102
8.21E−16

50
9
140374284
140374286
ER_117940
PNPLA7
cg01701837
−0.227579527
1.14E−06

51
10
24496597
24496599
ER_11835
KIAA1217
cg18829521
−0.283127061
1.15E−09

52
X
16889682
16889684
ER_118379
RBBP7
cg14719055
−0.328262296
1.22E−12

53
X
18708952
18708954
ER_118438
PPEF1
cg17198372
−0.26765505
9.21E−09

54
X
133792598
133792600
ER_120294
PLAC1
cg02831668
−0.351896922
1.45E−14

55
1
37503349
37503351
ER_1301
GRIK3
cg17815567
−0.292808764
2.40E−10

56
10
71213994
71213996
ER_13175
TSPAN15
cg06791973
−0.424739404
3.89E−21

57
10
74020427
74020429
ER_13281
DDIT4
cg16407699
−0.428856044
0

58
10
76859205
76859207
ER_13395
DUSP13
cg10963664
−0.374994214
1.79E−16

59
10
88475770
88475772
ER_13815
LDB3
cg19185706
−0.20457813
1.27E−05

60
10
95228649
95228651
ER_14079
MYOF
cg26808637
−0.293781665
2.54E−10

61
10
99331807
99331809
ER_14202
UBTD1
cg10343327
−0.162973315
0.000527081

62
10
102792834
102792836
ER_14340
SFXN3
cg15428620
−0.550035375
0

63
10
105238872
105238874
ER_14449
CALHM3
cg04804772
−0.200511144
1.83E−05

64
10
112259304
112259306
ER_14629
DUSP5
cg09357462
−0.47604403
0

65
10
121415188
121415190
ER_15018
BAG3
cg01303372
−0.449812197
0

66
10
123900860
123900862
ER_15113
TACC2
cg24181174
−0.165628275
0.000425606

67
10
126315760
126315762
ER_15199
FAM53B
cg24916358
−0.247233418
1.19E−07

68
10
126700660
126700662
ER_15221
CTBP2
cg04839409
−0.267400497
9.52E−09

69
1
44300941
44300943
ER_1532
ST3GAL3
cg09989037
−0.203979608
1.35E−05

70
10
128606856
128606858
ER_15340
DOCK1
cg26717418
−0.286907293
6.76E−10

71
10
134225119
134225121
ER_15443
PWWP2B
cg23622878
−0.185156404
8.00E−05

72
10
134261412
134261414
ER_15450
C10orf91
cg15834395
−0.370354575
4.46E−16

73
10
134332382
134332384
ER_15463
INPP5A
cg00149455
−0.161112302
0.000611156

74
10
134420622
134420624
ER_15478
INPP5A
cg00805619
−0.177054833
0.000163475

75
10
134499265
134499267
ER_15480
INPP5A
cg19649172
−0.207945257
9.09E−06

76
11
1507262
1507264
ER_15583
MOB2
cg11611244
−0.155896803
0.000917668

77
11
1777551
1777553
ER_15595
CTSD
cg25050723
−0.389507306
0

78
11
10764591
10764593
ER_15808
CTR9
cg09645818
−0.483304247
0

79
11
33744822
33744824
ER_16397
CD59
cg04188351
−0.304313667
5.36E−11

80
11
46374123
46374125
ER_16803
DGKZ
cg12087471
−0.292581725
3.01E−10

81
11
61467285
61467287
ER_17096
DAGLA
cg07011711
−0.222108817
2.08E−06

82
11
62437614
62437616
ER_17173
C11orf48
cg02375832
−0.162346349
0.000554124

83
11
64008173
64008175
ER_17271
FKBP2
cg03764062
−0.155263384
0.000963309

84
11
64034958
64034960
ER_17276
PLCB3
cg18375707
−0.229382466
9.37E−07

85
11
65582809
65582811
ER_17439
OVOL1
cg20077042
−0.282987867
1.17E−09

86
11
67914462
67914464
ER_17656
SUV420H1
cg27606671
−0.287097977
6.58E−10

87
11
69061910
69061912
ER_17743
MYEOV
cg22779330
−0.161570899
0.000580565

88
11
70917158
70917160
ER_17848
SHANK2
cg21810373
−0.315797247
9.16E−12

89
11
85469036
85469038
ER_18506
SYTL2
cg11429283
−0.459623471
0

90
1
6445631
6445633
ER_192
ACOT7
cg16429975
−0.346507797
3.86E−14

91
11
120106597
120106599
ER_19293
POU2F3
cg21886186
−0.250931346
7.60E−08

92
12
48340532
48340534
ER_21318
TMEM106C
cg26115531
−0.34357773
8.83E−14

93
12
51236581
51236583
ER_21514
TMPRSS12
cg02956274
−0.385063766
2.36E−17

94
12
52542741
52542743
ER_21611
KRT80
cg00230924
−0.278960719
2.03E−09

95
12
52579701
52579703
ER_21620
KRT80
cg23345977
−0.156387208
0.000883714

96
12
53300292
53300294
ER_21729
KRT8
cg26357344
−0.358090657
5.78E−15

97
12
53553085
53553087
ER_21786
CSAD
cg05242244
−0.462318596
0

98
12
58160842
58160844
ER_22088
CYP27B1
cg24722099
−0.255597575
4.28E−08

99
12
63193646
63193648
ER_22367
PPM1H
cg21177112
−0.167030751
0.000379657

100
12
117471699
117471701
ER_25133
FBXW8
cg04605980
−0.351039676
1.70E−14

101
12
122019524
122019526
ER_25371
KDM2B
cg03486832
−0.15698494
0.0008439

102
12
122458892
122458894
ER_25409
BCL7A
cg03496157
−0.226142681
1.34E−06

103
12
123446271
123446273
ER_25491
ABCB9
cg03131767
−0.188629804
5.83E−05

104
12
123449185
123449187
ER_25493
ABCB9
cg18473642
−0.416828462
0

105
12
124844873
124844875
ER_25578
NCOR2
cg13712197
−0.158414149
0.000755335

106
12
124880021
124880023
ER_25592
NCOR2
cg03905247
−0.219796114
2.66E−06

107
12
125004852
125004854
ER_25616
NCOR2
cg13146040
−0.17339707
0.000223506

108
13
34208893
34208895
ER_26636
STARD13
cg00527307
−0.292609379
3.00E−10

109
13
41590152
41590154
ER_26934
ELF1
cg20033981
−0.28858071
4.44E−10

110
13
113652068
113652070
ER_28798
MCF2L
cg06072822
−0.416344509
0

111
13
113677187
113677189
ER_28802
MCF2L
cg18050804
−0.233597269
5.84E−07

112
13
113980034
113980036
ER_28813
GRTP1
cg08351085
−0.561680883
8.92E−39

113
1
8824176
8824178
ER_290
RERE
cg20263853
−0.425083186
0

114
14
23623479
23623481
ER_29019
SLC7A8
cg05357695
−0.354356515
1.20E−14

115
14
23623683
23623685
ER_29020
SLC7A8
cg23543481
−0.296853548
1.62E−10

116
14
38054164
38054166
ER_29794
FOXA1
cg05555111
−0.477419115
0

117
14
64932677
64932679
ER_31140
AKAP5
cg26524899
−0.214691628
4.56E−06

118
14
69263266
69263268
ER_31542
ZFP36L1
cg20016914
−0.251562921
7.04E−08

119
14
74214182
74214184
ER_31871
C14orf43
cg22851561
−0.432667091
0

120
14
74238380
74238382
ER_31884
C14orf43
cg26217402
−0.195733971
3.01E−05

121
14
76447326
76447328
ER_32084
TGFB3
cg18298494
−0.303016542
6.51E−11

122
14
88942953
88942955
ER_32519
PTPN21
cg12215094
−0.168486692
0.000336879

123
14
93412665
93412667
ER_32818
ITPK1
cg16157895
−0.361876948
2.67E−15

124
14
93474065
93474067
ER_32832
ITPK1
cg05939041
−0.290394125
4.12E−10

125
14
95047654
95047656
ER_32949
SERPINA5
cg13984563
−0.671013091
0

126
14
95078598
95078600
ER_32959
SERPINA3
cg08057786
−0.324660895
2.21E−12

127
14
95615730
95615732
ER_32995
DICER1
cg01901579
−0.296402122
1.73E−10

128
14
96692186
96692188
ER_33117
BDKRB2
cg06616512
−0.167100809
0.000377488

129
14
100055668
100055670
ER_33296
CCDC85C
cg11773456
−0.175193161
0.000191832

130
14
100618490
100618492
ER_33370
DEGS2
cg20904336
−0.394516582
0

131
14
105181901
105181903
ER_33695
INF2
cg08043200
−0.165915618
0.00041579

132
14
105215643
105215645
ER_33701
ADSSL1
cg15983585
−0.301120862
6.94E−11

133
14
105434297
105434299
ER_33730
AHNAK2
cg09796640
−0.17137013
0.000265106

134
14
105992114
105992116
ER_33807
TMEM121
cg07037750
−0.284994395
8.84E−10

135
15
50519373
50519375
ER_34615
SLC27A2
cg05028948
−0.342165772
1.14E−13

136
15
63671691
63671693
ER_35532
CA12
cg08947167
−0.663688479
0

137
15
63673012
63673014
ER_35533
CA12
cg23931734
−0.553637039
0

138
15
70384231
70384233
ER_35866
TLE3
cg02009766
−0.624063592
0

139
15
74233032
74233034
ER_36102
LOXL1
cg16581800
−0.219977053
2.61E−06

140
15
80878743
80878745
ER_36421
ARNT2
cg08371852
−0.270986754
5.94E−09

141
15
89028368
89028370
ER_36781
MRPS11
cg01608635
−0.158819879
0.000731809

142
15
96866813
96866815
ER_37215
NR2F2
cg11122899
−0.210236502
6.87E−06

143
15
99236766
99236768
ER_37297
IGF1R
cg02613818
−0.537894014
0

144
15
99272175
99272177
ER_37301
IGF1R
cg12402183
−0.588333638
0

145
16
635622
635624
ER_37540
PIGQ
cg03804128
−0.400189696
0

146
16
1232362
1232364
ER_37605
CACNA1H
cg05272807
−0.232335435
6.74E−07

147
16
1275784
1275786
ER_37610
TPSG1
cg13997068
−0.53048809
5.01E−34

148
16
1353626
1353628
ER_37624
UBE2I
cg06908857
−0.192504589
4.07E−05

149
16
1479500
1479502
ER_37638
CCDC154
cg07000567
−0.210795649
6.48E−06

150
16
1828145
1828147
ER_37665
SPSB3
cg01676844
−0.168403597
0.000339194

151
16
2879943
2879945
ER_37757
ZG16B
cg05461841
−0.331448616
7.20E−13

152
16
3704736
3704738
ER_37830
DNASE1
cg08778316
−0.225124178
1.40E−06

153
16
3707038
3707040
ER_37831
DNASE1
cg04332526
−0.190279859
4.86E−05

154
16
4368123
4368125
ER_37883
GLIS2
cg04123578
−0.344446475
7.56E−14

155
16
4741494
4741496
ER_37916
MGRN1
cg06125591
−0.267451066
9.46E−09

156
16
4838558
4838560
ER_37923
Sep-12
cg11353547
−0.290559784
3.33E−10

157
16
14030551
14030553
ER_38174
ERCC4
cg03927470
−0.286422287
7.24E−10

158
16
14580862
14580864
ER_38229
PARN
cg03176203
−0.328987106
1.09E−12

159
16
16108801
16108803
ER_38301
ABCC1
cg00649632
−0.199363421
2.12E−05

160
16
22103595
22103597
ER_38518
VWA3A
cg02843201
−0.43583238
2.75E−22

161
16
24748338
24748340
ER_38621
TNRC6A
cg09018299
−0.363523046
1.88E−15

162
16
24856161
24856163
ER_38625
SLC5A11
cg04399565
−0.17392406
0.0002093

163
16
28608287
28608289
ER_38739
SULT1A2
cg00931491
−0.449317709
0

164
16
72995176
72995178
ER_39877
ZFHX3
cg16563255
−0.209502467
7.77E−06

165
16
83848108
83848110
ER_40473
HSBP1
cg08394248
−0.313956513
1.22E−11

166
16
84401246
84401248
ER_40523
ATP2C2
cg06786050
−0.192394761
4.12E−05

167
16
85669461
85669463
ER_40724
KIAA0182
cg09396032
−0.459019682
0

168
16
85723488
85723490
ER_40737
GINS2
cg08871354
−0.29702843
1.58E−10

169
16
85787023
85787025
ER_40750
C16orf74
cg07897180
−0.272675914
4.75E−09

170
1
109373103
109373105
ER_4085
AKNAD1
cg03884543
−0.246004825
1.25E−07

171
16
87813068
87813070
ER_40908
KLHDC4
cg05710032
−0.172678581
0.000237498

172
16
87890566
87890568
ER_40918
SLC7A5
cg03879320
−0.483398404
0

173
1
11020630
11020632
ER_411
C1orf127
cg21157465
−0.239407483
2.76E−07

174
16
89900193
89900195
ER_41112
SPIRE2
cg16769976
−0.231466954
7.43E−07

175
17
152088
152090
ER_41149
RPH3AL
cg04897931
−0.376348525
6.44E−17

176
17
1901436
1901438
ER_41216
RTN4RL1
cg19550533
−0.201776075
1.68E−05

177
17
1988011
1988013
ER_41224
SMG6
cg14439774
−0.176716395
0.000168319

178
17
3870414
3870416
ER_41284
ATP2A3
cg25112590
−0.33237346
6.16E−13

179
17
7283816
7283818
ER_41423
TNK1
cg02503376
−0.398328696
1.46E−18

180
17
14109670
14109672
ER_41695
COX10
cg26751588
−0.283019603
1.16E−09

181
17
16322481
16322483
ER_41788
TRPV2
cg26719625
−0.369616508
4.66E−16

182
17
17718524
17718526
ER_41889
SREBF1
cg19619576
−0.37884006
1.97E−17

183
17
18139505
18139507
ER_41939
LLGL1
cg09658183
−0.169416014
0.000311973

184
17
18280848
18280850
ER_41950
EVPLL
cg17549878
−0.460071152
5.93E−25

185
17
27296200
27296202
ER_42201
SEZ6
cg24778016
−0.183419447
9.10E−05

186
17
29649960
29649962
ER_42279
NF1
cg20368567
−0.385262742
0

187
17
39577628
39577630
ER_42572
KRT37
cg18068256
−0.51802934
2.90E−32

188
17
39662979
39662981
ER_42574
KRT13
cg10742225
−0.361827589
2.30E−15

189
17
39678106
39678108
ER_42579
KRT19
cg21513437
−0.217626424
3.35E−06

190
17
39685911
39685913
ER_42583
KRT19
cg08966188
−0.41138725
0

191
17
39694252
39694254
ER_42588
KRT19
cg10851010
−0.374811464
1.10E−16

192
17
40932198
40932200
ER_42648
WNK4
cg03777083
−0.473878767
1.43E−26

193
17
55371726
55371728
ER_43290
MSI2
cg26871347
−0.576424707
0

194
17
55673926
55673928
ER_43343
MSI2
cg26408224
−0.341475332
1.28E−13

195
1
114218185
114218187
ER_4610
MAGI3
cg20616821
−0.376987343
5.00E−17

196
17
64940744
64940746
ER_46841
CACNG4
cg07432111
−0.194033617
3.53E−05

197
17
64954455
64954457
ER_46847
CACNG4
cg22285671
−0.445992326
0

198
17
66291421
66291423
ER_46984
ARSG
cg08815110
−0.356520016
7.89E−15

199
17
73641808
73641810
ER_47388
RECQL5
cg07251887
−0.357417469
6.61E−15

200
17
73696508
73696510
ER_47396
SAP30BP
cg18563987
−0.156918701
0.000848229

201
17
73872583
73872585
ER_47428
TRIM47
cg10644544
−0.203451408
1.42E−05

202
17
74684503
74684505
ER_47498
MXRA7
cg27546012
−0.158712948
0.000737942

203
17
76858247
76858249
ER_47631
TIMP2
cg27549186
−0.172720063
0.000236668

204
17
76973327
76973329
ER_47643
LGALS3BP
cg26928788
−0.285188503
8.60E−10

205
17
78522608
78522610
ER_47743
RPTOR
cg02082642
−0.211839663
6.12E−06

206
17
78668007
78668009
ER_47754
RPTOR
cg13102028
−0.28719267
5.42E−10

207
17
78791722
78791724
ER_47760
RPTOR
cg27460531
−0.235434315
4.74E−07

208
17
79018697
79018699
ER_47773
BAIAP2
cg17027476
−0.250472546
8.04E−08

209
17
80163173
80163175
ER_47840
CCDC57
cg01777586
−0.224436269
1.62E−06

210
17
80174779
80174781
ER_47845
CCDC57
cg07013698
−0.235217952
4.86E−07

211
18
74536225
74536227
ER_49880
ZNF236
cg06398181
−0.317765849
6.71E−12

212
19
1496415
1496417
ER_50033
REEP6
cg02300825
−0.24403709
1.74E−07

213
19
1907971
1907973
ER_50049
SCAMP4
cg19254118
−0.311880585
1.69E−11

214
19
3374731
3374733
ER_50124
NFIC
cg14883993
−0.335512035
2.66E−13

215
19
7714474
7714476
ER_50328
STXBP2
cg07063348
−0.368882875
5.58E−16

216
19
14545200
14545202
ER_50670
PKN1
cg13922488
−0.210598637
6.95E−06

217
19
15590307
15590309
ER_50728
PGLYRP2
cg17752089
−0.526755523
1.72E−33

218
19
16045787
16045789
ER_50746
CYP4F11
cg03190825
−0.203840303
1.31E−05

219
19
35531858
35531860
ER_51354
HPN
cg21484586
−0.269976176
5.90E−09

220
19
35800588
35800590
ER_51381
MAG
cg02776658
−0.511574789
2.23E−31

221
19
35801214
35801216
ER_51382
MAG
cg04690840
−0.160341682
0.000640132

222
19
45843825
45843827
ER_51894
KLC3
cg03883348
−0.288529952
5.38E−10

223
19
51568259
51568261
ER_52287
KLK13
cg03307401
−0.255470367
3.89E−08

224
19
56047883
56047885
ER_52419
SBK2
cg00149708
−0.328477521
8.81E−13

225
2
26947125
26947127
ER_53662
KCNK3
cg11273176
−0.3517283
1.50E−14

226
2
45998547
45998549
ER_54551
PRKCE
cg23924526
−0.165247828
0.000438935

227
2
46361963
46361965
ER_54598
PRKCE
cg00518941
−0.154901111
0.000990347

228
1
16074113
16074115
ER_550
TMEM82
cg12703825
−0.15474423
0.000990014

229
2
102012903
102012905
ER_56390
RFX8
cg17654419
−0.224242571
1.55E−06

230
2
106007814
106007816
ER_56517
FHL2
cg02234235
−0.20727681
9.72E−06

231
1
116219478
116219480
ER_5750
VANGL1
cg04880253
−0.289419108
4.74E−10

232
2
175499282
175499284
ER_59117
WIPF1
cg03983223
−0.320473352
4.36E−12

233
2
224625079
224625081
ER_61179
AP1S3
cg02234653
−0.292051154
3.25E−10

234
2
236447791
236447793
ER_61661
AGAP1
cg10647547
−0.18315725
9.57E−05

235
2
241807746
241807748
ER_61956
AGXT
cg17461448
−0.350729914
1.80E−14

236
2
241832899
241832901
ER_61959
C2orf54
cg01588581
−0.342330286
8.11E−14

237
2
241936843
241936845
ER_61974
SNED1
cg16937168
−0.221441686
2.23E−06

238
2
242138540
242138542
ER_61990
ANO7
cg01583021
−0.164860403
0.000452908

239
2
242500483
242500485
ER_62006
BOK
cg24828208
−0.326941071
1.52E−12

240
20
18036012
18036014
ER_62490
OVOL2
cg02113429
−0.264697876
1.19E−08

241
20
32447029
32447031
ER_63011
CHMP4B
cg03217337
−0.193799476
3.61E−05

242
20
32888854
32888856
ER_63021
AHCY
cg00582941
−0.255937198
4.10E−08

243
20
34205181
34205183
ER_63067
SPAG4
cg27632911
−0.253362831
5.64E−08

244
20
35093927
35093929
ER_63095
DLGAP4
cg12992443
−0.226710124
1.26E−06

245
1
118727833
118727835
ER_6319
SPAG17
cg23257935
−0.468876303
5.63E−26

246
1
17634542
17634544
ER_634
PADI4
cg16091981
−0.304565675
4.10E−11

247
20
44048173
44048175
ER_63559
PIGT
cg21723559
−0.483985863
0

248
20
44330620
44330622
ER_63571
WFDC13
cg17890298
−0.372474463
2.95E−16

249
20
47448544
47448546
ER_65321
PREX1
cg18112953
−0.404365915
0

250
20
49345566
49345568
ER_65723
PARD6B
cg03326606
−0.55204559
0

251
20
49346623
49346625
ER_65724
PARD6B
cg07803218
−0.695503616
0

252
1
18006886
18006888
ER_669
ARHGEF10L
cg00172227
−0.203444297
1.42E−05

253
20
60925178
60925180
ER_68486
LAMA5
cg18668449
−0.172436407
0.000242396

254
1
2036507
2036509
ER_69
PRKCZ
cg17023856
−0.217921669
3.25E−06

255
21
43735411
43735413
ER_69642
TFF3
cg14283447
−0.64300849
7.33E−54

256
22
18919802
18919804
ER_69990
PRODH
cg11438552
−0.498286049
1.29E−29

257
1
151554823
151554825
ER_7042
TUFT1
cg20383521
−0.426720988
0

258
22
39760058
39760060
ER_70842
SYNGR1
cg06397161
−0.473134386
0

259
22
46921966
46921968
ER_71163
CELSR1
cg19206437
−0.358860373
4.95E−15

260
22
50720287
50720289
ER_71300
PLXNB2
cg00012194
−0.26103668
2.16E−08

261
22
50738889
50738891
ER_71305
PLXNB2
cg04089788
−0.165338693
0.000435717

262
3
11643367
11643369
ER_71757
VGLL4
cg08525922
−0.222133443
2.07E−06

263
3
12994820
12994822
ER_71864
IQSEC1
cg09361614
−0.187690211
6.36E−05

264
3
13517896
13517898
ER_71911
HDAC11
cg03190578
−0.488500124
0

265
3
15687270
15687272
ER_72120
BTD
cg21634628
−0.367472893
7.79E−16

266
1
19664275
19664277
ER_722
CAPZB
cg26157446
−0.160511147
0.00064087

267
1
154377620
154377622
ER_7267
IL6R
cg25853020
−0.226436213
1.30E−06

268
3
48590039
48590041
ER_73356
PFKFB4
cg20732160
−0.248525803
1.02E−07

269
1
155161678
155161680
ER_7336
MUC1
cg20949223
−0.415072063
3.62E−20

270
3
50639078
50639080
ER_73426
CISH
cg13519902
−0.25711001
3.54E−08

271
3
52280123
52280125
ER_73463
PPM1M
cg05406954
−0.1589276
0.000725678

272
3
58028596
58028598
ER_73666
FLNB
cg02026180
−0.538338329
0

273
1
156096177
156096179
ER_7395
LMNA
cg08881019
−0.275981939
3.05E−09

274
3
66519997
66519999
ER_77324
LRIG1
cg09716921
−0.507588811
0

275
3
66543262
66543264
ER_77334
LRIG1
cg24150385
−0.352144949
1.83E−14

276
3
133174925
133174927
ER_79397
BFSP2
cg05618222
−0.17660393
0.000166181

277
3
169758288
169758290
ER_80993
GPR160
cg12350863
−0.525559204
0

278
3
183959852
183959854
ER_81693
VWA5B2
cg24363374
−0.24189682
2.05E−07

279
4
681085
681087
ER_82472
MFSD7
cg23681017
−0.324124136
2.42E−12

280
4
686869
686871
ER_82474
MFSD7
cg21498785
−0.187877471
6.25E−05

281
4
757455
757457
ER_82479
PCGF3
cg26690744
−0.384333816
0

282
4
965629
965631
ER_82491
DGKQ
cg15639776
−0.182120866
0.000104932

283
4
987651
987653
ER_82493
IDUA
cg23332689
−0.316721564
7.91E−12

284
4
1986541
1986543
ER_82549
WHSC2
cg00248861
−0.180201515
0.000124297

285
4
3341176
3341178
ER_82632
RGS12
cg21343777
−0.390930721
0

286
4
48909056
48909058
ER_83945
OCIAD2
cg26134090
−0.19336451
3.76E−05

287
5
672844
672846
ER_88176
TPPP
cg22879098
−0.173994887
0.000208036

288
5
7851680
7851682
ER_88404
C5orf49
cg01035170
−0.394008422
3.66E−18

289
5
43604148
43604150
ER_89613
NNT
cg09489281
−0.217287839
3.28E−06

290
5
52386487
52386489
ER_89737
ITGA2
cg08874888
−0.21285208
5.51E−06

291
1
201096288
201096290
ER_8996
TMEM9
cg06714761
−0.236629383
4.13E−07

292
1
201252452
201252454
ER_9013
PKP1
cg17463149
−0.160895926
0.000612605

293
5
95226945
95226947
ER_91036
ELL2
cg19493250
−0.204999399
1.22E−05

294
1
203097233
203097235
ER_9205
ADORA1
cg12794758
−0.156376349
0.000873099

295
1
203123143
203123145
ER_9215
ADORA1
cg23257225
−0.17524604
0.000186865

296
5
138299676
138299678
ER_92405
SIL1
cg24650915
−0.199535932
2.09E−05

297
5
139034001
139034003
ER_92444
CXXC5
cg22885332
−0.689848806
0

298
5
139069448
139069450
ER_92451
CXXC5
cg10680621
−0.223444527
1.80E−06

299
1
203488763
203488765
ER_9257
OPTC
cg06122825
−0.223553288
1.67E−06

300
1
203595060
203595062
ER_9266
ATP2B4
cg21693907
−0.164075806
0.000482481

301
5
148513940
148513942
ER_92755
ABLIM3
cg16247269
−0.330183359
8.90E−13

302
5
176816678
176816680
ER_93670
SLC34A1
cg21145248
−0.338284799
1.65E−13

303
5
177029443
177029445
ER_93689
B4GALT7
cg03493123
−0.203926933
1.36E−05

304
5
177547972
177547974
ER_93712
N4BP3
cg17796323
−0.200270092
1.94E−05

305
6
3139626
3139628
ER_93950
BPHL
cg00266592
−0.183369268
9.39E−05

306
6
10420695
10420697
ER_94256
TFAP2A
cg17557766
−0.250606999
7.91E−08

307
6
30698986
30698988
ER_95075
FLOT1
cg16646298
−0.158565326
0.000746488

308
6
31478231
31478233
ER_95117
MICB
cg12989719
−0.294476318
2.29E−10

309
6
31833152
31833154
ER_95141
SLC44A4
cg23701033
−0.191804667
4.35E−05

310
6
32135026
32135028
ER_95158
EGFL8
cg05850971
−0.241470361
2.36E−07

311
6
33173133
33173135
ER_95186
HSD17B8
cg02802514
−0.254003888
5.22E−08

312
6
33288179
33288181
ER_95195
DAXX
cg03477252
−0.24031322
2.70E−07

313
6
33993821
33993823
ER_95239
GRM4
cg22971402
−0.292034242
2.69E−10

314
6
34512082
34512084
ER_95268
SPDEF
cg08392123
−0.522863026
0

315
6
40363183
40363185
ER_95568
LRFN2
cg18454510
−0.408390902
1.62E−19

316
6
42109175
42109177
ER_95663
C6orf132
cg24627621
−0.240988721
2.29E−07

317
6
64281603
64281605
ER_96269
PTP4A1
cg24631428
−0.259535604
2.32E−08

318
1
25070671
25070673
ER_965
CLIC4
cg10546487
−0.196885845
2.69E−05

319
6
109267292
109267294
ER_97322
ARMC2
cg13725172
−0.189528837
5.37E−05

320
6
149806731
149806733
ER_98712
ZC3H12D
cg14030904
−0.167456328
0.000360227

321
6
152124814
152124816
ER_98841
ESR1
cg08415493
−0.636001231
0

322
6
152432724
152432726
ER_98857
ESR1
cg10433043
−0.198527203
2.30E−05

323
7
922050
922052
ER_99284
GET4
cg07179981
−0.206381595
1.06E−05

324
7
923983
923985
ER_99285
GET4
cg25795026
−0.309385232
2.48E−11

325
7
927933
927935
ER_99286
GET4
cg03842205
−0.265790218
1.17E−08

326
7
1501247
1501249
ER_99342
MICALL2
cg22372849
−0.231820141
7.14E−07

327
1
217886460
217886462
ER_9936
SPATA17
cg10491122
−0.496347538
0

328
7
2673407
2673409
ER_99460
TTYH3
cg01827726
−0.20153074
1.72E−05

In one example, the method of detecting differential methylation comprises identifying differential methylation at one or more CpG dinucleotide sequences within one or more regions set forth in rows 1-328 of Table 3 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences. For example, the method may comprise detecting differential methylation at a single CpG dinucleotide sequence within any one of the genomic regions defined in Table 3 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequence.

In another example, the method of detecting differential methylation comprises detecting differential methylation at two or more CpG dinucleotides within any genomic region defined in Table 3 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequences. For example, the method may comprise detecting differential methylation at two or more CpG dinucleotides, or three or more CpG dinucleotides, or four or more CpG dinucleotides, or five or more CpG dinucleotides, or six or more CpG dinucleotides, or seven or more CpG dinucleotides, or eight or more CpG dinucleotides, or nine or more CpG dinucleotides, or 10 or more CpG dinucleotides, or 20 or more CpG dinucleotides, or 30 or more CpG dinucleotides, or 40 or more CpG dinucleotides, or 50 or more CpG dinucleotides within a genomic region set forth in Table 3 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequences. The two or more CpG dinucleotides may be consecutive (i.e., contiguous) within a genomic region. Alternatively, the two or more CpG dinucleotides may not be consecutive (i.e., may not be contiguous) within any genomic region.

In yet another example, the method of detecting differential methylation comprises detecting differential methylation of at least one CpG dinucleotide within two or more different genomic regions set forth in Table 3 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequences. For example, the method may comprise detecting differential methylation at a CpG dinucleotide within two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more different genomic regions set forth in Table 3 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding CpG dinucleotide sequences.

In one example, the method of detecting differential methylation comprise detecting hypermethylation of one or more CpG dinucleotides within the 328 ESR1 binding sites set forth in Table 3 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences. As hypermethylation of the one or more CpG dinucleotides within the 328 ESR1 binding sites set forth in Table 3 may alter expression of a gene within which the ESR1 binding site resides or with which the ESR1 binding site is closely associated, the method may further comprise detecting expression levels of genes associated with any of the ESR1 binding sites defined in Table 3 in the subject relative to a reference level of expression for the corresponding gene(s).

In one example, identifying differential methylation at one or more CpG dinucleotide sequences within one or more regions set forth in rows 1-328 of Table 3 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject's likely response to endocrine therapy.

Detecting differential methylation at a single CpG dinucleotide sequence within any one of the genomic regions defined in Table 3 may be indicative of the subject's likely response to endocrine therapy.

Alternatively, detecting differential methylation at two or more CpG dinucleotides within any genomic region defined in Table 3 may be indicative of the subject's likely response to endocrine therapy. For example, detecting differential methylation at two or more CpG dinucleotides, or three or more CpG dinucleotides, or four or more CpG dinucleotides, or five or more CpG dinucleotides, or six or more CpG dinucleotides, or seven or more CpG dinucleotides, or eight or more CpG dinucleotides, or nine or more CpG dinucleotides, or 10 or more CpG dinucleotides, or 20 or more CpG dinucleotides, or 30 or more CpG dinucleotides, or 40 or more CpG dinucleotides, or 50 or more CpG dinucleotides within a genomic region set forth in Table 3 may be indicative of the subject's likely response to endocrine therapy. The two or more CpG dinucleotides may be consecutive (i.e., contiguous) within a genomic region. Alternatively, the two or more CpG dinucleotides may not be consecutive (i.e., may not be contiguous) within any genomic region.

Detecting differential methylation of at least one CpG dinucleotide within two or more different genomic regions set forth in Table 3 may be indicative of the subject's likely response to endocrine therapy. For example, detecting differential methylation at a CpG dinucleotide within two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more different genomic regions set forth in Table 3 may be indicative of the subject's likely response to endocrine therapy.

Hypermethylation of one or more CpG dinucleotides within the 328 ESR1 binding sites set forth in Table 3 may alter expression of a gene within which the ESR1 binding site resides or with which the ESR1 binding site is closely associated. For example, hypermethylation of one or more CpG dinucleotides within the 328 ESR1 binding sites set forth in Table 3 may reduce expression of the gene(s) with which the respective ESR1 binding sites is/are most closely associated. Accordingly, expression levels of genes associated with any of the ESR1 binding sites defined in Table 3 may be indicative of the subject's likely response to endocrine therapy e.g., when that subject is suffering from ESR1 positive breast cancer.

Detecting differential methylation at a single CpG dinucleotide sequence within any one of the genomic regions defined in Table 3 may be indicative of the subject having breast cancer which is refractory to endocrine therapy.

Alternatively, detecting differential methylation at two or more CpG dinucleotides within any genomic region defined in Table 3 may be indicative of the subject having breast cancer which is refractory to endocrine therapy. For example, detecting differential methylation at two or more CpG dinucleotides, or three or more CpG dinucleotides, or four or more CpG dinucleotides, or five or more CpG dinucleotides, or six or more CpG dinucleotides, or seven or more CpG dinucleotides, or eight or more CpG dinucleotides, or nine or more CpG dinucleotides, or 10 or more CpG dinucleotides, or 20 or more CpG dinucleotides, or 30 or more CpG dinucleotides, or 40 or more CpG dinucleotides, or 50 or more CpG dinucleotides within a genomic region set forth in Table 3 may be indicative of the subject having breast cancer which is refractory to endocrine therapy. The two or more CpG dinucleotides may be consecutive (i.e., contiguous) within a genomic region. Alternatively, the two or more CpG dinucleotides may not be consecutive (i.e., may not be contiguous) within any genomic region.

Detecting differential methylation of at least one CpG dinucleotide within two or more different genomic regions set forth in Table 3 may be indicative of the subject having breast cancer which is refractory to endocrine therapy. For example, detecting differential methylation at a CpG dinucleotide within two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more different genomic regions set forth in Table 3 may be indicative of the subject having breast cancer which is refractory to endocrine therapy.

Hypermethylation of one or more CpG dinucleotides within the 328 ESR1 binding sites set forth in Table 3 may alter expression of a gene within which the ESR1 binding site resides or with which the ESR1 binding site is closely associated. For example, hypermethylation of one or more CpG dinucleotides within the 328 ESR1 binding sites set forth in Table 3 may reduce expression of the gene(s) with which the respective ESR1 binding sites is/are most closely associated. Accordingly, expression levels of genes associated with any of the ESR1 binding sites defined in Table 3 may be indicative of the subject having breast cancer which is refractory to endocrine therapy e.g., such as ESR1 positive breast cancer which is refractory to endocrine therapy.

Detecting differential methylation at a single CpG dinucleotide sequence within any one of the genomic regions defined in Table 3 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer.

Alternatively, detecting differential methylation at two or more CpG dinucleotides within any genomic region defined in Table 3 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. For example, detecting differential methylation at two or more CpG dinucleotides, or three or more CpG dinucleotides, or four or more CpG dinucleotides, or five or more CpG dinucleotides, or six or more CpG dinucleotides, or seven or more CpG dinucleotides, or eight or more CpG dinucleotides, or nine or more CpG dinucleotides, or 10 or more CpG dinucleotides, or 20 or more CpG dinucleotides, or 30 or more CpG dinucleotides, or 40 or more CpG dinucleotides, or 50 or more CpG dinucleotides within a genomic region set forth in Table 3 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. The two or more CpG dinucleotides may be consecutive (i.e., contiguous) within a genomic region. Alternatively, the two or more CpG dinucleotides may not be consecutive (i.e., may not be contiguous) within any genomic region.

Detecting differential methylation of at least one CpG dinucleotide within two or more different genomic regions set forth in Table 3 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. For example, detecting differential methylation at a CpG dinucleotide within two or more, or three or more, or four or more, or five or more, or six or more, or seven or more, or eight or more, or nine or more, or 10 or more different genomic regions set forth in Table 3 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer.

Hypermethylation of one or more CpG dinucleotides within the 328 ESR1 binding sites set forth in Table 3 may alter expression of a gene within which the ESR1 binding site resides or with which the ESR1 binding site is closely associated. For example, hypermethylation of one or more CpG dinucleotides within the 328 ESR1 binding sites set forth in Table 3 may reduce expression of the gene(s) with which the respective ESR1 binding sites is/are most closely associated. Accordingly, expression levels of genes associated with any of the ESR1 binding sites defined in Table 3 may be indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer e.g., in a patient suffering from ESR1 positive breast cancer and who is receiving, or about to receive, endocrine therapy.

Particular individual CpG dinucleotides within any of the genomic regions identified in Table 1, Table 2 or Table 3 may be particularly strong predictors of a subject's likely response to endocrine therapy. For example, identifying differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancer regions as defined in Table 1 associated with, or spanning, a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject's likely response to endocrine therapy. For example, differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 57, 111-113, 256-258, 288-289, 469-470, 805, 821-822 and 824-826 of Table 1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject's likely response to endocrine therapy.

In another example, identifying differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancer regions as defined in Table 1 associated with, or spanning, a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject's likely response to endocrine therapy. For example, differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 57, 111-113, 256-258, 460-470 and 805 of Table 1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject's likely response to endocrine therapy.

In yet another example, identifying differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancer regions as defined in Table 1 associated with, or spanning, a gene selected from FOXA1, ESR1 and/or GATA3 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject's likely response to endocrine therapy. For example, differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 277 and 821-822 of Table 1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject's likely response to endocrine therapy.

Particular individual CpG dinucleotides within any of the genomic regions identified in Table 1, Table 2 or Table 3 may have particularly strong diagnostic value in determining whether a subject suffering from ESR1-positive breast cancer is or will be refractory to endocrine therapy. For example, identifying differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancer regions as defined in Table 1 associated with, or spanning, a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject having breast cancer which is refractory to endocrine therapy. For example, differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 57, 111-113, 256-258, 288-289, 469-470, 805, 821-822 and 824-826 of Table 1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject having breast cancer which is refractory to endocrine therapy.

In another example, identifying differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancer regions as defined in Table 1 associated with, or spanning, a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject having breast cancer which is refractory to endocrine therapy. For example, differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 57, 111-113, 256-258, 460-470 and 805 of Table 1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject having breast cancer which is refractory to endocrine therapy.

In yet another example, identifying differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancer regions as defined in Table 1 associated with, or spanning, a gene selected from FOXA1, ESR1 and/or GATA3 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject having breast cancer which is refractory to endocrine therapy. For example, differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 277 and 821-822 of Table 1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the subject having breast cancer which is refractory to endocrine therapy.

Particular individual CpG dinucleotides within any of the genomic regions identified in Table 1, Table 2 or Table 3 may be particularly strong predictors of a subject's likely therapeutic outcome e.g., in a subject suffering from ESR1-positive cancer receiving endocrine therapy, and/or of the progression of the ESR1 positive breast cancer e.g., in a subject suffering from ESR1-positive cancer receiving endocrine therapy. For example, identifying differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancer regions e.g., as defined in Table 1, 2 or 3, associated with, or spanning, a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. For example, differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 57, 111-113, 256-258, 288-289, 469-470, 805, 821-822 and 824-826 of Table 1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer.

In another example, identifying differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancer regions e.g., as defined in Table 1, 2 or 3, associated with, or spanning, a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. For example, differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 57, 111-113, 256-258, 460-470 and 805 of Table 1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer.

In another example, identifying differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancer regions as defined in Table 1 associated with, or spanning, a gene selected from FOXA1, ESR1 and/or GATA3 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer. For example, differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 277 and 821-822 of Table 1 in a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences is indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer.

In accordance with any example described herein, the method of detecting differential methylation may comprise identifying differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancer regions as defined in Table 1 associated with, or spanning, a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences. For example, the method may comprise detecting differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 57, 111-113, 256-258, 288-289, 469-470, 805, 821-822 and 824-826 of Table 1 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences.

In another example, the method of detecting differential methylation may comprise identifying differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancer regions as defined in Table 1 associated with, or spanning, a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences. For example, the method may comprise detecting differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 57, 111-113, 256-258, 460-470 and 805 of Table 1 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences.

In yet another example, the method of detecting differential methylation may comprise identifying differential methylation at one or more CpG dinucleotide sequences within one or more estrogen responsive enhancer regions as defined in Table 1 associated with, or spanning, a gene selected from FOXA1, ESR1 and/or GATA3 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences. For example, the method may comprise detecting differential methylation at one or more CpG dinucleotide sequences selected from those defined in rows 277 and 821-822 of Table 1 in a subject suffering from ESR1 positive breast cancer relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences.

It will be understood that the methods described herein encompass determining methylation status of any combination of CpG dinucleotide sequences in any combination of genomic regions set forth in Table 1, Table 2 or Table 3, in any permutation. For example, the methods disclosed herein may comprise determining the methylation status of any one or more CpG dinucleotide sequences in any 2, or more, 3 or more, 4 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more genomic regions set forth in Table 1, Table 2 or Table 3, in any permutation.

Generally, the greater the number of CpG dinucleotides assessed for methylation status, the more reliable the diagnosis and/or prognosis of the subject. Thus, the greater the number of genomic regions defined in Table 1, Table 2 or Table 3 for which methylation status is determined in the methods disclosed herein, the more reliable the diagnosis or prognosis of the subject.

Breast Cancer Subtypes

The present disclosure provides (i) methods for predicting response to endocrine therapy in a subject suffering from ESR1 positive breast cancer, (ii) methods for diagnosing ESR1 positive breast cancer which is refractory to endocrine therapy, and (iii) methods for predicting the therapeutic outcome of and/or monitoring the progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy. Exemplary breast cancers which are ESR1 positive include basal breast cancer, Her2 positive breast cancer, progesterone receptor positive breast cancer, ductal carcinoma in situ, lobular carcinoma in situ, early breast cancer, invasive breast cancer, Paget's disease of the nipple, inflammatory breast cancer, locally advanced breast cancer and secondary breast cancer. Breast cancer may also be characterised according to various molecular subtypes which are typically categorized on an immunohistochemical basis. Exemplary molecular subtypes of breast cancer which are ESR1 positive are as follows:

(i) normal (ER+, PR+, HER2+, cytokeratin 5/6+, and HER1+);

(ii) luminal A (ER+ and/or PR+, HER2−); and

(iii) luminal B (ER+ and/or PR+, HER2+).

Detection of differential methylation e.g., hypermethylation, at combinations of the CpG dinucleotides within the ESR1 binding sites identified herein may be particularly useful in the diagnosis, prognosis and/or treatment management of any one or more of these known subtypes of breast cancer.

Diagnostic and/or Prognostic Assay Formats

1. Detection of Methylation of Nucleic Acid and Methods Therefor

The present inventors have identified CpG dinucleotide sequences within estrogen responsive enhancers which are differentially methylated in ESR1 positive breast cancer cells which are responsive to endocrine therapy compared to ESR1 positive breast cancer cells which are refractory to endocrine therapy. The present inventors have also shown that these differentially methylated CpG dinucleotide sequences reside within ESR1-binding sites e.g., as described in Table 1. The present inventors have also shown that a subset of these differentially methylated CpG dinucleotide sequences reside within ESR1-binding sites which are intragenic e.g., as described in Table 2. Furthermore, the present inventors have shown that a subset of these differentially methylated CpG dinucleotide sequences reside within ESR1-binding sites and that hypermethylation of those sites correlates with reduced expression of the gene with which the ESR1 binding site is most closely correlated e.g., as described in Table 3.

The inventors have demonstrated that the differentially methylated CpG dinucleotide sequences within estrogen responsive enhancers e.g., as defined in Tables 1, 2 or 3, are capable of predicting response to endocrine therapy in a subject suffering from ESR1 positive breast cancer. Accordingly, a method of predicting response to endocrine therapy in a subject suffering from ESR1 positive breast cancer shall be taken to include detecting methylation status of one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers e.g., as defined in Tables 1, 2 or 3, to determine whether or not the one or more CpG dinucleotide sequences is differentially methylated relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences, wherein differential methylation of said one or more CpG dinucleotide sequences in the subject relative to the reference level is indicative of the subject's likely response to endocrine therapy.

The inventors have also demonstrated that the differentially methylated CpG dinucleotide sequences within estrogen responsive enhancers e.g., as defined in Tables 1, 2 or 3, are capable of diagnosing ESR1 positive breast cancer which is refractory to endocrine therapy. Accordingly, a method of diagnosing ESR1 positive breast cancer which is refractory to endocrine therapy in a subject suffering from ESR1 positive breast cancer shall be taken to include detecting methylation status of one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers e.g., as defined in Tables 1, 2 or 3, to determine whether or not the one or more CpG dinucleotide sequences is differentially methylated relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences, wherein differential methylation of said one or more CpG dinucleotide sequences in the subject relative to the reference level is indicative of the subject having breast cancer which is refractory to endocrine therapy.

The inventors have also demonstrated that the differentially methylated CpG dinucleotide sequences within estrogen responsive enhancers e.g., as defined in Tables 1, 2 or 3, are capable of predicting the therapeutic outcome of and/or monitoring the progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy. Accordingly, a method of predicting the therapeutic outcome of and/or monitoring the progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy shall be taken to include detecting methylation status of one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers e.g., as defined in Tables 1, 2 or 3, to determine whether or not the one or more CpG dinucleotide sequences is differentially methylated relative to a reference level of methylation for the corresponding one or more CpG dinucleotide sequences, wherein differential methylation of said one or more CpG dinucleotide sequences in the subject relative to the reference level is indicative of the likely therapeutic outcome and/or of the progression of the ESR1 positive breast cancer.

Suitable methods for the detection of methylation status are known in the art and/or described herein.

The term “methylation” shall be taken to mean the addition of a methyl group by the action of a DNA methyl transferase enzyme to a CpG island of nucleic acid, e.g., genomic DNA. As described herein, there are several methods known to those skilled in the art for determining the level or degree of methylation of nucleic acid. By “differential methylation” of a nucleic acid it is meant that there is a deviation in the number of methylated CpG dinucleotides at a genomic region within the subject diagnosed compared to that detected within a corresponding genomic region in a suitable control sample i.e., which provides a reference level of methylation for that genomic region. The differentially methylated nucleic acid may have an increased level of methylation within a specific or defined region of nucleic acid e.g., such as hypermethylation, or a decreased level of methylation within a specific or defined region of nucleic acid e.g., such as hypomethylation.

The term “hypermethylation” shall be taken to mean that a plurality of CpG dinucleotides in a specific or defined region of nucleic acid are methylated relative to a reference level.

The term “hypomethylation” shall be taken to mean that a plurality of CpG dinucleotides in a specific or defined region of nucleic acid are unmethylated relative to a reference level.

The present disclosure is not to be limited by a precise number of methylated residues that are considered to be (i) predictive of a likely response to endocrine therapy in a subject suffering from ESR1 positive breast cancer (ii) or diagnostic of ESR1 positive breast cancer which is refractory to endocrine therapy, or (iii) predictive of the therapeutic outcome of and/or progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy, because some variation between patient samples will occur. Nor is the present disclosure to be limited by the specific positioning of the methylated residue within an estrogen responsive enhancer region.

In one example, the degree of methylation in a subject is determined for one or more CpG dinucleotides with one or more ESR1 binding sites set forth in Tables 1-3. In one example, the degree of methylation in a subject is determined for one or more CpG dinucleotides within one or more ESR1 binding sites set forth in Table 1. In one example, the degree of methylation in a subject is determined for one or more CpG dinucleotides within one or more ESR1 binding sites set forth in Table 2. In one example, the degree of methylation in a subject is determined for one or more CpG dinucleotides within one or more ESR1 binding sites set forth in Table 3. In one example, the degree of methylation in a subject is determined for one or more CpG dinucleotides within one or more estrogen responsive enhancers of a gene selected from DAXX, ESR1, RXRA, GET4, NCOR2, GATA3, MSI2, C8orf46 and/or ITPK1. In one example, the degree of methylation in a subject is determined for one or more CpG dinucleotides within one or more estrogen responsive enhancers of a gene selected from DAXX, RXRA, NCOR2, MSI2, and/or C8orf46. In one example, the degree of methylation in a subject is determined for one or more CpG dinucleotides within one or more estrogen responsive enhancers of a gene selected from FOXA1, ESR1 and/or GATA3.

a) Probe or Primer Design and/or Production

Several diagnostic and/or prognostic methods described herein use one or more probes and/or primers to detect methylation at a genomic region. Methods for designing probes and/or primers for use in, for example, PCR or hybridization are known in the art and described, for example, in Dieffenbach and Dveksler (Eds) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, N Y, 1995). Furthermore, several software packages are publicly available that design optimal probes and/or primers for a variety of assays, e.g. Primer 3 available from the Center for Genome Research, Cambridge, Mass., USA.

The potential use of the probe or primer should be considered during its design. For example, should the probe or primer be produced for use in, for example, a methylation specific PCR or ligase chain reaction (LCR) assay the nucleotide at the 3′ end (or 5′ end in the case of LCR) should correspond to a methylated nucleotide in a nucleic acid.

Probes and/or primers useful for detection of a marker associated with a breast cancer are assessed, for example, to determine those that do not form hairpins, self-prime or form primer dimers (e.g. with another probe or primer used in a detection assay).

Methods for producing/synthesizing a probe or primer of the present disclosure are known in the art. For example, oligonucleotide synthesis is described, in Gait (Ed) (In: Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford, 1984). For example, a probe or primer may be obtained by biological synthesis (e.g. by digestion of a nucleic acid with a restriction endonuclease) or by chemical synthesis. For short sequences (up to about 100 nucleotides) chemical synthesis is preferable.

Other methods for oligonucleotide synthesis include, for example, phosphotriester and phosphodiester methods (Narang, et al. Meth. Enzymol 68: 90, 1979) and synthesis on a support (Beaucage, et al Tetrahedron Letters 22: 1859-1862, 1981) as well as phosphoramidate technique, Caruthers, M. H., et al., “Methods in Enzymology,” Vol. 154, pp. 287-314 (1988), and others described in “Synthesis and Applications of DNA and RNA,” S. A. Narang, editor, Academic Press, New York, 1987, and the references cited therein.

Probes comprising locked nucleic acid (LNA) are synthesized as described, for example, in Nielsen et al, J. Chem. Soc. Perkin Trans., 1: 3423, 1997; Singh and Wengel, Chem. Commun. 1247, 1998. While, probes comprising peptide-nucleic acid (PNA) are synthesized as described, for example, in Egholm et al., Am. Chem. Soc., 114: 1895, 1992; Egholm et al., Nature, 365: 566, 1993; and Orum et al., Nucl. Acids Res., 21: 5332, 1993.

b) Methylation-Sensitive Endonuclease Digestion of DNA

In one example, the methylation status of one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers e.g., as defined in Tables 1, 2 or 3, in a sample is determined using a process comprising treating the nucleic acid with an amount of a methylation-sensitive restriction endonuclease enzyme under conditions sufficient for nucleic acid to be digested and then detecting the fragments produced. Exemplary methylation-sensitive endonucleases include, for example, HpaI or HpaII.

In one example, the digestion of nucleic acid is detected by selective hybridization of a probe or primer to the undigested nucleic acid. Alternatively, the probe selectively hybridizes to both digested and undigested nucleic acid but facilitates differentiation between both forms, e.g., by electrophoresis. Suitable detection methods for achieving selective hybridization to a hybridization probe include, for example, Southern or other nucleic acid hybridization (Kawai et al., Mol. Cell. Biol. 14, 7421-7427, 1994; Gonzalgo et al., Cancer Res. 57, 594-599, 1997).

The term “selectively hybridizable” means that the probe is used under conditions where a target nucleic acid hybridizes to the probe to produce a signal that is significantly above background (i.e., a high signal-to-noise ratio). The intensity of hybridization is measured, for example, by radiolabeling the probe, e.g. by incorporating [α-³⁵5] and/or [α-³²P]dNTPs, [γ-³²P]ATP, biotin, a dye ligand (e.g., FAM or TAMRA), a fluorophore, or other suitable ligand into the probe prior to use and then detecting the ligand following hybridization.

The skilled artisan will be aware that optimum hybridization reaction conditions should be determined empirically for each probe, although some generalities can be applied. Preferably, hybridizations employing short oligonucleotide probes are performed at low to medium stringency.

For the purposes of defining the level of stringency to be used in these diagnostic and/or prognostic assays, a low stringency is defined herein as being a hybridization and/or a wash carried out in about 6×SSC buffer and/or about 0.1% (w/v) SDS at about 28° C. to about 40° C., or equivalent conditions. A moderate stringency is defined herein as being a hybridization and/or washing carried out in about 2×SSC buffer and/or about 0.1% (w/v) SDS at a temperature in the range of about 45° C. to about 65° C., or equivalent conditions.

In the case of a GC rich probe or primer or a longer probe or primer a high stringency hybridization and/or wash is preferred. A high stringency is defined herein as being a hybridization and/or wash carried out in about 0.1×SSC buffer and/or about 0.1% (w/v) SDS, or lower salt concentration, and/or at a temperature of at least 65° C., or equivalent conditions. Reference herein to a particular level of stringency encompasses equivalent conditions using wash/hybridization solutions other than SSC known to those skilled in the art.

Generally, the stringency is increased by reducing the concentration of SSC buffer, and/or increasing the concentration of SDS and/or increasing the temperature of the hybridization and/or wash. Those skilled in the art will be aware that the conditions for hybridization and/or wash may vary depending upon the nature of the hybridization matrix used to support the sample DNA, and/or the type of hybridization probe used and/or constituents of any buffer used in a hybridization. For example, formamide reduces the melting temperature of a probe or primer in a hybridization or an amplification reaction.

Conditions for specifically hybridizing nucleic acid, and conditions for washing to remove non-specific hybridizing nucleic acid, are understood by those skilled in the art. For the purposes of further clarification only, reference to the parameters affecting hybridization between nucleic acid molecules is found in Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, ISBN 047150338, 1992), which is herein incorporated by reference.

In accordance with the present example, a difference in the fragments produced for a test sample and a control sample is indicative of (i) a subject's likely response to endocrine therapy, (ii) an ESR1 positive breast cancer which will be refractory to endocrine therapy, and/or (iii) a likely therapeutic outcome and/or progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy. Similarly, in cases where the control sample comprises data from a breast tumor, a breast cancer tissue or a breast cancerous cell, which is ESR1 positive and refractory to endocrine therapy, similarity, albeit not necessarily absolute identity, between the test sample and the control sample is indicative of (i) a subject's likely response to endocrine therapy, (ii) an ESR1 positive breast cancer which will be refractory to endocrine therapy, and/or (iii) a likely therapeutic outcome and/or progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy.

In an alternative example, the fragments produced by the restriction enzyme are detected using an amplification system, such as, for example, polymerase chain reaction (PCR), rolling circle amplification (RCA), inverse polymerase chain reaction (iPCR), in situ PCR (Singer-Sam et al., Nucl. Acids Res. 18, 687,1990), strand displacement amplification (SDA) or cycling probe technology.

Methods of PCR are known in the art and described, for example, by McPherson et al., PCR: A Practical Approach. (series eds, D. Rickwood and B. D. Hames), IRL Press Limited, Oxford. pp 1-253, 1991 and by Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, N Y, 1995), the contents of which are each incorporated in their entirety by way of reference. Generally, for PCR two non-complementary nucleic acid primer molecules comprising at least about 18 nucleotides in length, and more preferably at least 20-30 nucleotides in length are hybridized to different strands of a nucleic acid template molecule at their respective annealing sites, and specific nucleic acid molecule copies of the template that intervene the annealing sites are amplified enzymatically. Amplification products may be detected, for example, using electrophoresis and detection with a detectable marker that binds nucleic acids. Alternatively, one or more of the oligonucleotides are labeled with a detectable marker (e.g. a fluorophore) and the amplification product detected using, for example, a lightcycler (Perkin Elmer, Wellesley, Mass., USA).

Strand displacement amplification (SDA) utilizes oligonucleotide primers, a DNA polymerase and a restriction endonuclease to amplify a target sequence. The oligonucleotides are hybridized to a target nucleic acid and the polymerase is used to produce a copy of the region intervening the primer annealing sites. The duplexes of copied nucleic acid and target nucleic acid are then nicked with an endonuclease that specifically recognizes a sequence at the beginning of the copied nucleic acid. The DNA polymerase recognizes the nicked DNA and produces another copy of the target region at the same time displacing the previously generated nucleic acid. The advantage of SDA is that it occurs in an isothermal format, thereby facilitating high-throughput automated analysis.

Cycling Probe Technology uses a chimeric synthetic primer that comprises DNA-RNA-DNA that is capable of hybridizing to a target sequence. Upon hybridization to a target sequence the RNA-DNA duplex formed is a target for RNaseH thereby cleaving the primer. The cleaved primer is then detected, for example, using mass spectrometry or electrophoresis.

For primers that flank, or which are adjacent to a methylation-sensitive endonuclease recognition site, it is preferred that such primers flank only those sites that are hypermethylated in the ESR1 breast cancer to ensure that a diagnostic and/or prognostic amplification product is produced. In this regard, an amplification product will only be produced when the restriction site is not cleaved i.e., when it is methylated. Accordingly, detection of an amplification product indicates that the CpG dinucleotide/s of interest is/are methylated.

This form of analysis may be used to determine the methylation status of a plurality of CpG dinucleotides within a genomic region provided that each dinucleotide is within a methylation sensitive restriction endonuclease site.

In these methods, one or more of the primers may be labeled with a detectable marker to facilitate rapid detection of amplified nucleic acid, for example, a fluorescent label (e.g. Cy5 or Cy3) or a radioisotope (e.g. ³²P).

The amplified nucleic acids are generally analyzed using, for example, non-denaturing agarose gel electrophoresis, non-denaturing polyacrylamide gel electrophoresis, mass spectrometry, liquid chromatography (e.g. HPLC or dHPLC), or capillary electrophoresis. (e.g. MALDI-TOF). High throughput detection methods, such as, for example, matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, e.g. LC MS/MS), biosensor technology, evanescent fiber-optics technology or DNA chip technology (e.g., WO98/49557; WO 96/17958; Fodor et al., Science 767-773, 1991; U.S. Pat. No. 5,143,854; and U.S. Pat. No. 5,837,832, the contents of which are all incorporated herein by reference).

Alternatively, amplification of a nucleic acid may be continuously monitored using a melting curve analysis method as described herein and/or in, for example, U.S. Pat. No. 6,174,670, which is incorporated herein by reference.

c) Selective Mutagenesis of Non-Methylated DNA

In an alternative example of the present disclosure, the methylation status of a genomic region in a subject sample is determined using a process comprising treating the nucleic acid with an amount of a compound that selectively mutates a non-methylated cytosine residue within a CpG dinucleotide under conditions sufficient to induce mutagenesis.

Exemplary compounds mutate cytosine to uracil or thymidine, such as, for example, a salt of bisulfite, e.g., sodium bisulfite or potassium bisulfite (Frommer et al., Proc. Natl. Acad. Sci. USA 89, 1827-1831, 1992). Bisulfite treatment of DNA is known to distinguish methylated from non-methylated cytosine residues, by mutating cytosine residues that are not protected by methylation, including cytosine residues that are not within a CpG dinucleotide or that are positioned within a CpG dinucleotide that is not subject to methylation.

(i) Sequence Based Detection

In one example, the presence of one or more mutated nucleotides in a genomic region or the number of mutated sequences in a sample is determined by sequencing mutated DNA. One form of analysis comprises amplifying mutated nucleic acid or methylated nucleic acid using an amplification reaction described herein, for example, PCR. The amplified product is then directly sequenced or cloned and the cloned product sequenced. Methods for sequencing DNA are known in the art and include for example, the dideoxy chain termination method or the Maxam-Gilbert method (see Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd Ed., CSHP, New York 1989) or Zyskind et al., Recombinant DNA Laboratory Manual, (Acad. Press, 1988)).

As the treatment of nucleic acid with a compound, such as, for example, bisulfite results in non-methylated cytosines being mutated to uracil or thymidine, analysis of the sequence determines the presence or absence of a methylated nucleotide. For example, by comparing the sequence obtained using a control sample or a sample that has not been treated with bisulfite, or the known nucleotide sequence of the region of interest with a treated sample facilitates the detection of differences in the nucleotide sequence. Any thymine residue detected at the site of a cytosine in the treated sample compared to a control or untreated sample may be considered to be caused by mutation as a result of bisulfite treatment. Suitable methods for the detection of methylation using sequencing of bisulfite treated nucleic acid are described, for example, in Frommer et al., Proc. Natl. Acad. Sci. USA 89: 1827-1831, 1992 or Clark et al., Nucl. Acids Res. 22: 2990-2997, 1994. One example of a commercially available kit for carrying out such methods is the CpGenome™ DNA modification Kit (Millipore). Other suitable kits are available from MDX Health SA (Belgium).

In another example, the presence of a mutated or non-mutated nucleotide in a bisulfite treated sample is detected using pyrosequencing, such as, for example, as described in Uhlmann et al., Electrophoresis, 23: 4072-4079, 2002. Essentially this method is a form of real-time sequencing that uses a primer that hybridizes to a site adjacent or close to the site of a cytosine that is methylated in a cancer cell. Following hybridization of the primer and template in the presence of a DNA polymerase each of four modified deoxynucleotide triphosphates are added separately according to a predetermined dispensation order. Only an added nucleotide that is complementary to the bisulfite treated sample is incorporated and inorganic pyrophosphate (PPi) is liberated. The PPi then drives a reaction resulting in production of detectable levels of light. Such a method allows determination of the identity of a specific nucleotide adjacent to the site of hybridization of the primer.

A related method for determining the sequence of a bisulfite treated nucleotide is methylation-sensitive single nucleotide primer extension (Me-SnuPE) or SNaPmeth. Suitable methods are described, for example, in Gonzalgo and Jones Nucl. Acids Res., 25: 2529-2531 or Uhlmann et al., Electrophoresis, 23: 4072-4079, 2002.

Clearly other high throughput sequencing methods are encompassed by the present disclosure. Such methods include, for example, solid phase minisequencing (as described, for example, in Syvamen et al, Genomics, 13: 1008-1017, 1992), or minisequencing with FRET (as described, for example, in Chen and Kwok, Nucleic Acids Res. 25: 347-353, 1997).

(ii) Restriction Endonuclease-Based Assay Format

In one example, the presence of a non-mutated nucleic sequence is detected using combined bisulfite restriction analysis (COBRA) essentially as described in Xiong and Laird, Nucl. Acids Res., 25: 2532-2534, 2001. This method exploits the differences in restriction enzyme recognition sites between methylated and unmethylated nucleic acid after treatment with a compound that selectively mutates a non-methylated cytosine residue, e.g., bisulfite.

Following bisulfite treatment a genomic region of interest comprising one or more CpG dinucleotides that are methylated in a ESR1 positive cancer cell, and which are included in a restriction endonuclease recognition sequence, is amplified using an amplification reaction described herein, e.g., PCR. The amplified product is then contacted with the restriction enzyme that cleaves at the site of the CpG dinucleotide for a time and under conditions sufficient for cleavage to occur. A restriction site may be selected to indicate the presence or absence of methylation. For example, the restriction endonuclease Taql cleaves the sequence TCGA, following bisulfite treatment of a non-methylated nucleic acid the sequence will be TTGA and, as a consequence, will not be cleaved. The digested and/or non-digested nucleic acid is then detected using a detection means known in the art, such as, for example, electrophoresis and/or mass spectrometry. The cleavage or non-cleavage of the nucleic acid is indicative of cancer in a subject.

Clearly, this method may be employed in either a positive read-out or negative read-out system when performing a diagnostic and/or prognostic method of the disclosure.

(iii) Positive Read-Out Assay Format

In one example, the assay format of the disclosure comprises a positive read-out system in which hypermethylated DNA from a breast cancer sample e.g., an ESR1-positive breast cancer sample, that has been treated, for example, with bisulfite is detected as a positive signal if the breast cancer is, or is likely to be, refractory to endocrine therapy. For example, non-hypermethylated DNA from a healthy or normal control subject, or non-hypermethylated DNA from a breast cancer sample e.g., an ESR1 positive breast cancer sample, is not detected or only weakly detected and is likely to be or is responsive to endocrine therapy.

In one example, the enhanced methylation in a subject sample is determined using a process comprising:

(i) treating the nucleic acid with an amount of a compound that selectively mutates a non-methylated cytosine residue under conditions sufficient to induce mutagenesis thereby producing a mutated nucleic acid;

(ii) hybridizing a nucleic acid to a probe or primer comprising a nucleotide sequence that is complementary to a sequence comprising a methylated cytosine residue under conditions such that selective hybridization to the non-mutated nucleic acid occurs; and

(iii) Detecting the Selective Hybridization.

In this context, the term “selective hybridization” means that hybridization of a probe or primer to the non-mutated nucleic acid occurs at a higher frequency or rate, or has a higher maximum reaction velocity, than hybridization of the same probe or primer to the corresponding mutated sequence. Preferably, the probe or primer does not hybridize or detectably hybridize (e.g., does not hybridize at a level significantly above background) to the non-methylated sequence carrying the mutation(s) under the reaction conditions used.

In one example, the hybridization is detected using Southern, dot blot, slot blot or other nucleic acid hybridization means (Kawai et al., Mol. Cell. Biol. 14, 7421-7427, 1994; Gonzalgo et al., Cancer Res. 57, 594-599, 1997). Subject to appropriate probe selection, such assay formats are generally described herein above and apply mutatis mutandis to the presently described selective mutagenesis approach.

In one example, a ligase chain reaction format is employed to distinguish between a mutated and non-mutated nucleic acid. Ligase chain reaction (described in EP 320,308 and U.S. Pat. No. 4,883,750) uses at least two oligonucleotide probes that anneal to a target nucleic acid in such a way that they are juxtaposed on the target nucleic acid such that they can be linked using a ligase. The probes that are not ligated are removed by modifying the hybridization stringency. In this respect, probes that have not been ligated will selectively hybridize under lower stringency hybridization conditions than probes that have been ligated. Accordingly, the stringency of the hybridization can be increased to a stringency that is at least as high as the stringency used to hybridize the longer probe, and preferably at a higher stringency due to the increased length contributed by the shorter probe following ligation. One exemplary method melts the target-probe duplex, elute the dissociated probe and confirm that is has been ligated, e.g., by determining its length using electrophoresis, mass spectrometry, nucleotide sequence analysis, gel filtration, or other means known to the skilled artisan.

Methylation specific microarrays (MSO) are also useful for differentiating between a mutated and non-mutated sequence. A suitable method is described, for example, in Adorj et al, Nucl. Acids Res., 30: e21, 2002. MSO uses nucleic acid that has been treated with a compound that selectively mutates a non-methylated cytosine residue (e.g., bisulfite) as template for an amplification reaction that amplifies both mutant and non-mutated nucleic acid. The amplification is performed with at least one primer that comprises a detectable label, such as, for example, a fluorophore, e.g., Cy3 or Cy5. The labeled amplification products are then hybridized to oligonucleotides on the microarray under conditions that enable detection of single nucleotide differences. Following washing to remove unbound amplification product, hybridization is detected using, for example, a microarray scanner. Not only does this method allow for determination of the methylation status of a large number of CpG dinucleotides, it is also semi-quantitative, enabling determination of the degree of methylation at each CpG dinucleotide analyzed. As there may be some degree of heterogeneity of methylation in a single sample, such quantification may assist in the diagnosis of cancer.

In an alternative example, the hybridization is detected using an amplification system. In methylation-specific PCR formats (MSP; Herman et al. Proc. Natl. Acad. Sci. USA 93: 9821-9826, 1992), the hybridization is detection using a process comprising amplifying the bisulfite-treated DNA. By using one or more probe or primer that anneals specifically to the unmutated sequence under moderate and/or high stringency conditions an amplification product is only produced using a sample comprising a methylated nucleotide.

Any amplification assay format described herein can be used, such as, for example, polymerase chain reaction (PCR), rolling circle amplification (RCA), inverse polymerase chain reaction (iPCR), in situ PCR (Singer-Sam et al., Nucl. Acids Res. 18, 687,1990), strand displacement amplification, or cycling probe technology.

PCR techniques have been developed for detection of gene mutations (Kuppuswamy et al., Proc. Natl. Acad. Sci. USA 88:1143-1147, 1991) and quantitation of allelic-specific expression (Szabo and Mann, Genes Dev. 9: 3097-3108, 1995; and Singer-Sam et al., PCR Methods Appl. 1: 160-163, 1992). Such techniques use internal primers, which anneal to a PCR-generated template and terminate immediately 5′ of the single nucleotide to be assayed. Such as format is readily combined with ligase chain reaction as described herein above.

Methylation-specific melting-curve analysis (essentially as described in Worm et al., Clin. Chem., 47: 1183-1189, 2001) is also contemplated by the present disclosure. This process exploits the difference in melting temperature in amplification products produced using bisulfite treated methylated or unmethylated nucleic acid. In essence, non-discriminatory amplification of a bisulfite treated sample is performed in the presence of a fluorescent dye that specifically binds to double stranded DNA (e.g., SYBR Green I). By increasing the temperature of the amplification product while monitoring fluorescence the melting properties and thus the sequence of the amplification product is determined. A decrease in the fluorescence reflects melting of at least a domain in the amplification product. The temperature at which the fluorescence decreases is indicative of the nucleotide sequence of the amplified nucleic acid, thereby permitting the nucleotide at the site of one or more CpG dinucleotides to be determined. As the sequence of the nucleic acids amplified using the present disclosure

The present disclosure also encompasses the use of real-time quantitative forms of PCR, such as, for example, TaqMan (Holland et al., Proc. Natl Acad. Sci. USA, 88, 7276-7280, 1991; Lee et al., Nucleic Acid Res. 21, 3761-3766, 1993) to perform this embodiment. For example, the MethylLight method of Eads et al., Nucl. Acids Res. 28: E32, 2000 uses a modified TaqMan assay to detect methylation of a CpG dinucleotide.

Alternatively, rather than using a labeled probe that requires cleavage, a probe, such as, for example, a Molecular Beacon™ is used (see, for example, Mhlang and Malmberg, Methods 25: 463-471, 2001). Molecular beacons are single stranded nucleic acid molecules with a stem-and-loop structure. The loop structure is complementary to the region surrounding the one or more CpG dinucleotides that are methylated in a cancer sample and not in a control sample. The stem structure is formed by annealing two “arms” complementary to each other, which are on either side of the probe (loop). A fluorescent moiety is bound to one arm and a quenching moiety that suppresses any detectable fluorescence when the molecular beacon is not bound to a target sequence is bound to the other arm. Upon binding of the loop region to its target nucleic acid the arms are separated and fluorescence is detectable. However, even a single base mismatch significantly alters the level of fluorescence detected in a sample. Accordingly, the presence or absence of a particular base is determined by the level of fluorescence detected. Such an assay facilitates detection of one or more unmutated sites (i.e. methylated nucleotides) in a nucleic acid.

As exemplified herein, another amplification based assay useful for the detection of a methylated nucleic acid following treatment with a compound that selectively mutates a non-methylated cytosine residue makes use of headloop PCR technology (e.g., as described in published PCT Application No. PCT/AU03/00244; WO 03/072810). This form of amplification uses a probe or primer that comprises a region that binds to a nucleic acid and is capable of amplifying nucleic acid in an amplification reaction whether the nucleic acid is methylated or not. The primer additionally comprises a region that is complementary to a portion of the amplified nucleic acid enabling this region of the primer to hybridize to the amplified nucleic acid incorporating the primer thereby forming a hairpin. The now 3′ terminal nucleotide/s of the annealed region (i.e. the most 5′ nucleotide/s of the primer) hybridize to the site of one or more mutated cytosine residues (i.e., unmethylated in nucleic acid from a cancer subject). Accordingly, this facilitates self-priming of amplification products from unmethylated nucleic acid, the thus formed hairpin structure blocking further amplification of this nucleic acid. In contrast, the complementary region may or may not by capable of hybridizing to an amplification product from methylated (mutated) nucleic acid, but is unable to “self-prime” thereby enabling further amplification of this nucleic acid (e.g., by the inability of the now 3′ nucleotide to hybridize to the amplification product). This method may be performed using a melting curve analysis method to determine the amount of methylated nucleic acid in a biological sample from a subject.

Other amplification based methods for detecting methylated nucleic acid following treatment with a compound that selectively mutates a non-methylated cytosine residue include, for example, methylation-specific single stranded conformation analysis (MS-SSCA) (Bianco et al., Hum. Mutat., 14: 289-293, 1999), methylation-specific denaturing gradient gel electrophoresis (MS-DGGE) (Abrams and Stanton, Methods Enzymol., 212: 71-74, 1992) and methylation-specific denaturing high-performance liquid chromatography (MS-DHPLC) (Deng et al, Chin. J. Cancer Res., 12: 171-191, 2000). Each of these methods use different techniques for detecting nucleic acid differences in an amplification product based on differences in nucleotide sequence and/or secondary structure. Such methods are clearly contemplated by the present disclosure.

(iv) Negative Read-Out Assays

In an alternative example, the assay format comprises a negative read-out system in which non-hypermethylated DNA from a healthy or normal control subject, or non-hypermethylated DNA from a breast cancer sample e.g., a ESR1 positive breast cancer sample, which is responsive to endocrine therapy is detected as a positive signal and preferably, hypermethylated DNA from a breast cancer sample e.g., an ESR1-positive breast cancer sample, which is, or is likely to be, refractory to endocrine therapy, is not detected or is only weakly detected.

In one example, the non-hypermethylated DNA is determined using a process comprising:

(i) treating the nucleic acid with an amount of a compound that selectively mutates a non-methylated cytosine residue within a CpG island under conditions sufficient to induce mutagenesis thereby producing a mutated nucleic acid;

(ii) hybridizing the nucleic acid to a probe or primer comprising a nucleotide sequence that is complementary to a sequence comprising the mutated cytosine residue under conditions such that selective hybridization to the mutated nucleic acid occurs; and

(iii) Detecting the Selective Hybridization.

In this context, the term “selective hybridization” means that hybridization of a probe or primer to the mutated nucleic acid occurs at a higher frequency or rate, or has a higher maximum reaction velocity, than hybridization of the same probe or primer to the corresponding non-mutated sequence. In one example, the probe or primer does not hybridize or detectably hybridize to the methylated sequence (or non-mutated sequence) under the reaction conditions used.

The skilled artisan will be able to adapt a positive read-out assay described above to a negative read-out assay, e.g., by producing a probe or primer that selectively hybridizes to non-mutated DNA rather than mutated DNA.

d) Methylated DNA Immunoprecipitation (MeDiP)

In another example, the methylation status of a genomic region in a subject sample is determined using a process comprising physically isolating methylated DNA (e.g., hypermethylated DNA) from hypomethylated or non-methylated DNA in a sample, followed by sequencing of the physically-separated methylated DNA. Preferably, the physical separation of methylated DNA is accomplished using Methylated DNA Immunoprecipitation (MeDiP), a technique that has been described in the art (See e.g., Weber, M. et al. (2005) Nat. Genet. 37:853-862; and Rakyan, et al. (2008) Genome Res. 18:1518-1529; which are both expressly incorporated herein by reference).

In accordance with a method of the disclosure in which MeDiP is employed to physically separate methylated DNA (e.g., hypermethylated DNA), the input nucleic acid preparation (from a subject) is denatured, incubated with an antibody directed against 5-methylcytosine and then the methylated DNA is isolated by immunoprecipitation. For example, to accomplish immunoprecipitation, the anti-5-methylcytosine antibody can be coupled to a solid support (e.g., magnetic dynabeads, microscopic agarose beads or paramagnetic beads) to allow for precipitation of the methylated DNA from solution (direct immunoprecipitation). Alternatively, a secondary antibody or reagent can be used that recognizes the anti-5-methylcytosine antibody (e.g., the constant region of the antibody) and that is coupled to a solid support, to thereby allow for precipitation of the methylated DNA from solution (indirect immunoprecipitation). For direct or indirect immunoprecipitation, other approaches known in the art for physical separation of components within a sample, such as the biotin/avidin or biotin/streptavidin systems, can be used. For example, the anti-5-methylcytosine antibody can be coupled to biotin and then avidin or streptavidin coupled to a solid support can be used to allow for precipitation of the methylated DNA from solution. It will be apparent to the ordinarily skilled artisan that other variations known in the art for causing immunoprecipitation are also suitable for use in the method of the disclosure. Thus, as used herein, the term “Methylated DNA Immunoprecipitation” or “MeDiP” is intended to encompass any and all approaches in which an antibody that discriminates between hypermethylated DNA and hypomethylated or non-methylated DNA is contacted with a nucleic acid obtained from a subject suffering from ESR1 positive breast cancer, followed by precipitation of the hypermethylated DNA (i.e., the DNA that specifically binds to the antibody) out of solution. For example, an approach in which an antibody comprising a methylated DNA binding domain (MBD) or a bispecific molecule comprised of a MBD and an antibody or part thereof e.g., Fc portion), is clearly contemplated for use in a method of the disclosure for detecting and/or physically isolating methylated DNA from a sample. Techniques using antibodies and other proteins comprising MBD for detecting methylated DNA are described in US Patent Publication US200150267263 and in BLUEPRINT Consortium (2016) Nat. Biotechnol. Doi: 10.1038/nbt.3062; both of which are expressly incorporated herein by reference.

Typically after physical separation of the hypermethylated DNA from hypomethylated or non-methylated DNA, the hypermethylated DNA is then amplified. As used herein, the term “amplified” is intended to mean that additional copies of the DNA are made to thereby increase the number of copies of the DNA, which is typically accomplished using the polymerase chain reaction (PCR). One particular method for amplification of the hypermethylated DNA is ligation mediated polymerase chain reaction (LM-PCR), which has been described previously in the art (See e.g., Ren, B. et al. (2000) Science 22:2306-2309; and Oberley, M. J. et al. (2004) Methods Enzymol. 376:315-334; the contents of both of which are expressly incorporated herein by reference). In LM-PCR, linker ends are ligated onto a sample of DNA fragments through blunt-end ligation and then oligonucleotide primers that recognize the nucleotide sequences of the linker ends are used in PCR to thereby amplify the DNA fragments to which the linkers have been ligated. Thus, in an example of the method of the disclosure in which LM-PCR is used, DNA from a subject suffering from ESR1-positive breast cancer is fragmented (e.g., into fragments of approximately 300-800 bp), and linker arms are ligated onto the fragmented DNA by blunt-end ligation, after which the hypermethylated DNA is physically separated from the hypeomethylated DNA or non-methylated DNA (e.g., by MeDiP). Then, following physical separation of the hypermethylated DNA, the recovered hypermethylated DNA is subjected to PCR using oligonucleotide primers that recognized the linker ends that have been ligated onto the DNA. This results in amplification of the hypermethylated DNA sample.

The amplified hypermethylated DNA sample may then be sequenced using standard sequencing methodologies known in the art and described herein. Sequence data can then be used to determine the methylation status of a genomic region in a subject sample. Moreover, this form of analysis may be used to determine the methylation status of a plurality of CpG dinucleotides within a genomic region simultaneously.

e) Additional Method Steps

The methods disclosed herein may further comprise one or more steps of enriching methylated DNA in a sample. Thus, the methods disclosed herein may further comprise one or more steps of isolating methylated DNA from a sample. The enrichment/isolation step may be performed prior to or concomitant with any other step in the method for detecting the level of methylation of a CpG dinucleotide sequence within an estrogen responsive enhancer region as disclosed herein.

Any suitable enriching/isolating step known in the art may be used. For example, the methods disclosed herein may comprise a step of enriching methylated DNA in a sample using a commercially available kit such as the CpG MethylQuest DNA Isolation Kit (Millipore), which provides a recombinant protein comprising the methyl binding domain (MBD) of the mouse MBD2b protein fused to a glutathione-S-transferase (GST) protein from S. japonicum via a linker containing a thrombin cleavage site, the recombinant protein being immobilized to a magnetic bead. The MBD binds to methylated CpG sites with high affinity and in a sequence-independent manner, thereby allowing enrichment of methylated DNA in a sample.

It will be appreciated that alternative or additional methods known in the art for enrichment/isolation of methylated DNA in a sample can be used in the methods disclosed herein. For example, methods of enrichment/isolation of methylated DNA in a sample are described in Hsu et al., (2014) Methods Mol Biol, 1105:61-70, Serre et al., (2010) Nucleic Acids Res, 38:391-399, Rauch and Pfeifer (2005) Lab Invest, 85:1172-1180, Nair et al., (2011) Epigenetics, 6:34-44; and Robinson et al., (2010) Genome Res, 20:1719-1729.

A method disclosed herein according to any example may also comprise selecting a patient based on the result of a method disclosed herein and performing an additional diagnostic method or recommending performance of an additional diagnostic method. For example, for a patient diagnosed as suffering from ESR1 positive breast cancer which is, or is likely to become, refractory to endocrine therapy, the additional diagnostic method may be an ultrasound or a biopsy.

2. Detection of Reduced Gene Expression

Since methylation of a nucleic acid sequence affects its expression, the present inventors have also demonstrated that the level of expression of nucleic acids within any of a number of genomic regions described herein is varied (e.g., reduced or increased) in ESR1 positive breast cancer subjects and in ESR1 positive breast cancer cell lines. Thus, the methods disclosed herein may additionally or alternatively comprise determining the level of expression of any polynucleotides overlapping, spanning or closely associated with, any of the estrogen responsive enhancer regions identified in Tables 1-3 herein. In one example, the methods disclosed herein may additionally or alternatively comprise determining the level of expression of one or more polynucleotides overlapping, spanning or closely associated with, one or more of the estrogen responsive enhancer regions defined in Table 1. In one example, the methods disclosed herein may additionally or alternatively comprise determining the level of expression of one or more polynucleotides overlapping, spanning or closely associated with, one or more of the estrogen responsive enhancer regions defined in Table 2. In one example, the methods disclosed herein may additionally or alternatively comprise determining the level of expression of one or more polynucleotides overlapping, spanning or closely associated with, one or more of the estrogen responsive enhancer regions defined in Table 3. For example, detecting a reduced level of expression of one or more polynucleotides overlapping, spanning or closely associated with, one or more of the estrogen responsive enhancer regions defined in Tables 1-3 may be (i) predictive of a likely response to endocrine therapy in a subject suffering from ESR1 positive breast cancer, (ii) or diagnostic of ESR1 positive breast cancer which is refractory to endocrine therapy, or (iii) predictive of the therapeutic outcome and/or progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy.

a) Nucleic Acid Detection

In one example, the level of expression of a nucleic acid is determined by detecting the level of mRNA transcribed from genomic region described herein.

In one example, the mRNA is detected by hybridizing a nucleic acid probe or primer capable of specifically hybridizing to a transcript of a genomic region described herein to a nucleic acid in a biological sample derived from a subject and detecting the hybridization by a detection means. Preferably, the detection means is an amplification reaction, or a nucleic acid hybridization reaction, such as, for example, as described herein.

In this context, the term “selective hybridization” means that hybridization of a probe or primer to the transcript occurs at a higher frequency or rate, or has a higher maximum reaction velocity, than hybridization of the same probe or primer to any other nucleic acid. Preferably, the probe or primer does not hybridize to another nucleic acid at a detectable level under the reaction conditions used.

As transcripts of a gene or pseudogene described herein are detected using mRNA or cDNA derived therefrom, assays that detect changes in mRNA are preferred (e.g. Northern hybridization, RT-PCR, NASBA, TMA or ligase chain reaction).

Methods of RT-PCR are known in the art and described, for example, in Dieffenbach (ed) and Dveksler (ed) (In: PCR Primer: A Laboratory Manual, Cold Spring Harbour Laboratories, N Y, 1995). Essentially, this method comprises performing a PCR reaction using cDNA produced by reverse transcribing mRNA from a cell using a reverse transcriptase. Methods of PCR described supra are to be taken to apply mutatis mutandis to this embodiment of the disclosure.

Similarly LCR may be performed using cDNA. Preferably, one or more of the probes or primers used in the reaction specifically hybridize to the transcript of interest. Method of LCR are described supra and are to be taken to apply mutatis mutandis to this embodiment of the disclosure.

Methods of TMA or self-sustained sequence replication (3SR) use two or more oligonucleotides that flank a target sequence, a RNA polymerase, RNase H and a reverse transcriptase. One oligonucleotide (that also comprises a RNA polymerase binding site) hybridizes to an RNA molecule that comprises the target sequence and the reverse transcriptase produces cDNA copy of this region. RNase H is used to digest the RNA in the RNA-DNA complex, and the second oligonucleotide used to produce a copy of the cDNA. The RNA polymerase is then used to produce a RNA copy of the cDNA, and the process repeated.

NASBA systems relies on the simultaneous activity of three enzymes (a reverse transcriptase, RNase H and RNA polymerase) to selectively amplify target mRNA sequences. The mRNA template is transcribed to cDNA by reverse transcription using an oligonucleotide that hybridizes to the target sequence and comprises a RNA polymerase binding site at its 5′ end. The template RNA is digested with RNase H and double stranded DNA is synthesized. The RNA polymerase then produces multiple RNA copies of the cDNA and the process is repeated.

The present disclosure also contemplates the use of a microarray to determine the level of expression of one or more nucleic acids described herein. Such a method enables the detection of a number of different nucleic acids, thereby providing a multi-analyte test and improving the sensitivity and/or accuracy of the diagnostic assay of the disclosure.

b) Polypeptide Detection

In an alternative example, the level of expression of a genomic region is determined by detecting the level of a protein encoded by a nucleic acid within a genomic region described herein.

In this respect, the present disclosure is not necessarily limited to the detection of a protein comprising the specific amino acid sequence recited herein. Rather, the present disclosure encompasses the detection of variant sequences (e.g., having at least about 80% or 90% or 95% or 98% amino acid sequence identity) or the detection of an immunogenic fragment or epitope of said protein.

The amount, level or presence of a polypeptide is determined using any of a variety of techniques known to the skilled artisan such as, for example, a technique selected from the group consisting of, immunohistochemistry, immunofluorescence, an immunoblot, a Western blot, a dot blot, an enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, e.g. LC MS/MS), biosensor technology, evanescent fiber-optics technology or protein chip technology.

In one example, the assay used to determine the amount or level of a protein is a semi-quantitative assay. In another example, the assay used to determine the amount or level of a protein in a quantitative assay. As will be apparent from the preceding description, such an assay may require the use of a suitable control, e.g. from a normal individual or matched normal control.

Standard solid-phase ELISA or FLISA formats are particularly useful in determining the concentration of a protein from a variety of samples.

In one form such an assay involves immobilizing a biological sample onto a solid matrix, such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide). An antibody that specifically binds to a protein described herein is brought into direct contact with the immobilized biological sample, and forms a direct bond with any of its target protein present in said sample. This antibody is generally labeled with a detectable reporter molecule, such as for example, a fluorescent label (e.g. FITC or Texas Red) or a fluorescent semiconductor nanocrystal (as described in U.S. Pat. No. 6,306,610) in the case of a FLISA or an enzyme (e.g. horseradish peroxidase (HRP), alkaline phosphatase (AP) or β-galactosidase) in the case of an ELISA, or alternatively a second labeled antibody can be used that binds to the first antibody. Following washing to remove any unbound antibody the label is detected either directly, in the case of a fluorescent label, or through the addition of a substrate, such as for example hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indol-beta-D-galaotopyranoside (x-gal) in the case of an enzymatic label.

In another form, an ELISA or FLISA comprises immobilizing an antibody or ligand that specifically binds a protein described supra on a solid matrix, such as, for example, a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick or a glass support. A sample is then brought into physical relation with said antibody, and the polypeptide is bound or ‘captured’. The bound protein is then detected using a labeled antibody. For example, a labeled antibody that binds to an epitope that is distinct from the first (capture) antibody is used to detect the captured protein. Alternatively, a third labeled antibody can be used that binds the second (detecting) antibody.

In another example, the presence or level of a protein is detected in a body fluid using, for example, a biosensor instrument (e.g., BIAcore™, Pharmacia Biosensor, Piscataway, N.J.). In such an assay, an antibody or ligand that specifically binds a protein is immobilized onto the surface of a receptor chip. For example, the antibody or ligand is covalently attached to dextran fibers that are attached to gold film within the flow cell of the biosensor device. A test sample is passed through the cell. Any antigen present in the body fluid sample, binds to the immobilized antibody or ligand, causing a change in the refractive index of the medium over the gold film, which is detected as a change in surface plasmon resonance of the gold film.

In another example, the presence or level of a protein or a fragment or epitope thereof is detected using a protein and/or antibody chip. To produce such a chip, an antibody or ligand that binds to the antigen of interest is bound to a solid support such as, for example glass, polycarbonate, polytetrafluoroethylene, polystyrene, silicon oxide, gold or silicon nitride. This immobilization is either direct (e.g. by covalent linkage, such as, for example, Schiff's base formation, disulfide linkage, or amide or urea bond formation) or indirect.

To bind a protein to a solid support it is often necessary to treat the solid support so as to create chemically reactive groups on the surface, such as, for example, with an aldehyde-containing silane reagent or the calixcrown derivatives described in Lee et al, Proteomics, 3: 2289-2304, 2003. A streptavidin chip is also useful for capturing proteins and/or peptides and/or nucleic acid and/or cells that have been conjugated with biotin (e.g. as described in Pavlickova et al., Biotechniques, 34: 124-130, 2003). Alternatively, a peptide is captured on a microfabricated polyacrylamide gel pad and accelerated into the gel using microelectrophoresis as described in, Arenkov et al. Anal. Biochem. 278:123-131, 2000.

Other assay formats are also contemplated, such as flow-through immunoassays (PCT/AU2002/01684), a lateral flow immunoassay (US20040228761, US20040248322 or US20040265926), a fluorescence polarization immunoassay (FPIA) (U.S. Pat. Nos. 4,593,089, 4,492,762, 4,668,640, and 4,751,190), a homogeneous microparticles immunoassay (“HMI”) (e.g., U.S. Pat. Nos. 5,571,728, 4,847,209, 6,514,770, and 6,248,597) or a chemiluminescent microparticle immunoassay (“CMIA”).

3 Multiplex Assay Formats

The present disclosure also contemplates multiplex or multianalyte format assays to improve the accuracy or specificity of the diagnostic and/or prognostic methods described herein. Such assays may also improve the population coverage by an assay.

Methods for determining the sensitivity of an assay will be apparent to the skilled artisan. For example, an assay described herein is used to analyze a population of test subjects to determine those that will develop cancer. Post-mortem analysis is then used to determine those subjects that did actually determine breast cancer. The number of “true positives” (i.e., subjects that developed breast cancer and were positively identified using the method of the disclosure) and “true negatives” (i.e., subjects that did not develop breast cancer and were not identified using the method of the disclosure) are determined.

Sensitivity of the assay is then determined by the following formula:

No. of true positives/(No. of true positives+No. of false negatives).

In one example, a method of the disclosure has a high degree of sensitivity in predicting the likelihood of a subject suffering from ESR1 positive breast cancer responding to endocrine therapy. For example, in a test population of individuals, the method of the disclosure is able to predict that a subject will not respond to endocrine therapy, for example, in at least about 50% of subjects suffering from ESR1 positive breast cancer which do not respond to endocrine therapy, for example, in at least about 60% of subjects suffering from ESR1 positive breast cancer which do not respond to endocrine therapy, for example, in at least about 65% of subjects suffering from ESR1 positive breast cancer which do not respond to endocrine therapy, for example, in at least about 70% of subjects suffering from ESR1 positive breast cancer which do not respond to endocrine therapy, for example, in at least about 75% of subjects suffering from ESR1 positive breast cancer which do not respond to endocrine therapy, for example, in at least about 80% of subjects suffering from ESR1 positive breast cancer which do not respond to endocrine therapy, for example, in at least about 85% of subjects suffering from ESR1 positive breast cancer which do not respond to endocrine therapy, for example, in at least about 90% of subjects suffering from ESR1 positive breast cancer which do not respond to endocrine therapy, for example, in at least about 95% of subjects suffering from ESR1 positive breast cancer which do not respond to endocrine therapy.

In another example, a method of the disclosure has a high degree of sensitivity in detecting ESR1 positive breast cancer which is refractory to endocrine therapy. For example, in a test population of individuals, the method of the disclosure detects at least about 50% of subjects developing or suffering from ESR1 positive breast cancer which is refractory to endocrine therapy, for example, at least about 60% of subjects developing or suffering from ESR1 positive breast cancer which is refractory to endocrine therapy, for example, at least about 65% of subjects developing or suffering from ESR1 positive breast cancer which is refractory to endocrine therapy, for example, at least about 70% of subjects developing or suffering from ESR1 positive breast cancer which is refractory to endocrine therapy, for example, at least about 75% of subjects developing or suffering from ESR1 positive breast cancer which is refractory to endocrine therapy, for example, at least about 80% of subjects developing or suffering from ESR1 positive breast cancer which is refractory to endocrine therapy, for example, at least about 85% of subjects developing or suffering from ESR1 positive breast cancer which is refractory to endocrine therapy, for example, at least about 90% of subjects developing or suffering from ESR1 positive breast cancer which is refractory to endocrine therapy, for example, at least about 95% of subjects developing or suffering from ESR1 positive breast cancer which is refractory to endocrine therapy.

In another example, a method of the disclosure has a high degree of sensitivity in stratifying ESR1 positive breast cancer subtypes associated with prognostic profiles following endocrine therapy e.g., such as populations of ESR1 positive breast cancer patients with which are likely to respond to endocrine therapy and populations of ESR1 positive breast cancer patients with which are unlikely to respond to endocrine therapy. In this way, the method of the disclosure has a high degree of sensitivity in predicting a therapeutic outcome and/or progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy. For example, in a test population of individuals having ESR1 positive breast cancer, the method of the disclosure stratifies at least about 50% of subjects having ESR1 positive breast cancer according to a disease outcome, for example, at least about 60% of subjects having ESR1 positive breast cancer according to a disease outcome, for example, at least about 70% of subjects having ESR1 positive breast cancer according to a disease outcome, for example, at least about 80% of subjects having ESR1 positive breast cancer according to a disease outcome, for example, at least about 85% of subjects having ESR1 positive breast cancer according to a disease outcome, for example, at least about 90% of subjects having ESR1 positive breast cancer according to a disease outcome, for example, at least about 95% of subjects having ESR1 positive breast cancer according to a disease outcome. A disease outcome in accordance with this example is a likelihood that the breast cancer patient will survive at least 3 years following endocrine therapy, for example, at least 5 years following endocrine therapy, for example, at least 10 years following endocrine therapy.

Specificity is determined by the following formula:

No. of true negatives/(No. of true negatives+No. of false positives).

An exemplary multiplex assay for use in a method of the disclosure comprises, for example, detecting differential methylation of one or more CpG dinucleotides in a plurality of estrogen responsive enhancer regions set forth in Tables 1-3. In one example, the method comprises detecting the level of methylation of one or more CpG dinucleotides in a plurality of the estrogen responsive enhancer regions set forth in Tables 1-3 to predict response to endocrine therapy in a subject suffering from ESR1 positive breast cancer. In another example, the method comprises detecting the level of methylation of one or more CpG dinucleotides in a plurality of estrogen responsive enhancer regions set forth in Tables 1-3 to diagnose ESR1 positive breast cancer which is refractory to endocrine therapy. In yet another example, the method comprises detecting the level of methylation of one or more CpG dinucleotides in a plurality of estrogen responsive enhancer regions set forth in Tables 1-3 to stratify and/or predict a therapeutic outcome and/or progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy.

The multiplex assay of the disclosure is not to be limited to the detection of methylation at a single CpG dinucleotide within a region of interest i.e., each estrogen responsive enhancer region. Rather, the present disclosure contemplates detection of methylation at a sufficient number of CpG dinucleotides in each nucleic acid to provide a diagnosis/prognosis. For example, the disclosure contemplates detection of methylation at 1 or 2 or 3 or 4 or 5 or 7 or 9 or 10 or 15 or 20 or 25 or 30 CpG dinculeotides in each nucleic acid i.e., each estrogen responsive enhancer region. Preferably, the disclosure contemplates detection of methylation at more than 1 CpG dinculeotide in each nucleic acid i.e., each estrogen responsive enhancer region.

As will be apparent from the foregoing description, a methylation specific microarray is amenable to such high density analysis. Previously, up to 232 CpG dinucleotides have been analyzed using such a microarray (Adorján et al., Nucl. Acids Res. 30: e21, 2002).

A method of the disclosure may comprises one or more assays to determine the level of expression of a gene spanning, comprising or closely associated with at least one estrogen responsive enhancer region set forth in Tables 1-3 to predict response to endocrine therapy in a subject suffering from ESR1 positive breast cancer. A method of the disclosure may comprises one or more assays to determine the level of expression of a gene spanning, comprising or closely associated with at least one estrogen responsive enhancer region set forth in Table 1-3 to diagnose ESR1 positive breast cancer which is refractory to endocrine therapy A method of the disclosure may comprises one or more assays to determine the level of expression of a gene spanning, comprising or closely associated with at least one estrogen responsive enhancer region set forth in Tables 1-3 to stratify and/or predict a therapeutic outcome and/or progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy. For example, the method may comprise detecting the level of mRNA or protein corresponding to a gene spanning, comprising or closely associated with at least one estrogen responsive enhancer region set forth in Table 1-3. Alternatively, the level of mRNA transcribed from one or more genes spanning, comprising or closely associated with at least one estrogen responsive enhancer region set forth in Table 1-3 and the level of one or more proteins expressed by the same or different genes spanning, comprising or closely associated with at least one estrogen responsive enhancer region set forth in Table 1-3 may be determined.

Each of the previously described detection techniques can be used independently of one another in the diagnostic and/or prognostic methods described. Accordingly, a single sample may be analyzed to determine the level of methylation of one or more CpG dinculeotides in one or more estrogen responsive enhancer regions and to determine the level of expression of one or more nucleic acids and/or proteins. In accordance with this example, hypermethylation of one or more CpG dinucleotides within one or more estorgen enhancer regions defined in Tables 1-3, and reduced expression of one or more genes spanning, comprising or closely associated with at least one estrogen responsive enhancer region set forth in Tables 1-3, is indicative of (i) a subject's likely response to endocrine therapy e.g., non-response to endocrine therapy, (ii) a ESR1 positive breast cancer will be refractory to endocrine therapy, and/or (iii) a likely therapeutic outcome and/or progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy.

Based on the teachings provided herein, a variety of combinations of assays will be apparent to the skilled artisan.

The present disclosure also contemplates the use of a known diagnostic assay in combination with an assay described herein.

Samples

A sample useful for the method of the present disclosure is, for example, from a tissue suspected of comprising a ESR1 positive breast cancer or a ESR1 positive breast cancer cell. For example, the cell is from a region of a tissue thought to comprise a ESR1 positive breast cancer or a ESR1 positive breast cancer cell. This does not exclude cells that have originated in a particular tissue but are isolated from a remote source.

The sample may be taken from a subject suspected of having ESR1 positive breast cancer. For example, the sample may be taken from a subject having ESR1 positive breast cancer and suspected of having or being at risk of developing ESR1 positive breast cancer which is refractory to endocrine therapy. For example, the subject may have a family history of ESR1 positive breast cancer, including ESR1 positive breast cancer which is resistant, or develops resistance, to endocrine therapy. The subject may have been subjected to any other test for detecting any form of ESR1 positive breast cancer.

In one example, the sample comprises a body fluid or a derivative of a body fluid or a body secretion. For example, the body fluid is selected from the group consisting of whole blood, urine, saliva, breast milk, pleural fluid, sweat, tears and mixtures thereof. An example of a derivative of a body fluid is selected from the group consisting of plasma, serum or buffy coat fraction. In one example, the sample comprises a whole blood sample, a serum sample or a plasma sample.

In one example DNA is isolated from either; whole blood, plasma, serum, peripheral blood mononucleated cells (PBMC) or enriched epithelial cells derived from the blood of patients diagnosed with breast cancer or healthy controls. DNA may then be bisulfite converted and gene-specific methylated sequences may be detected by either; methylation specific headloop suppression PCR, MALDI-TOF mass spectrometry (sequenom) or other bisulfite based PCR assay.

Preferably, the sample comprises a nucleated cell or an extract thereof. More preferably, the sample comprises a breast cancer cell e.g., an ESR1 positive breast cancer cell, or an extract thereof.

In another example, the sample comprises nucleic acid and/or protein from a breast cancer cell e.g., a nucleic acid and/or protein from an ESR1 positive breast cancer cell. The nucleic acid and/or protein may be separate need not be isolated with a cell, but rather may be from, for example, a lysed cell.

As the present disclosure provides methods which are useful for early detection of ESR1 positive breast cancer which is refractory to endocrine therapy in the medium to long term, the term breast cancer cell is not to be limited by the stage of a cancer in the subject from which said breast cancer cell is derived (i.e. whether or not the patient is in remission or undergoing disease recurrence or whether or not the ESR1 positive breast cancer is a primary tumor or the consequence of metastases). Nor is the term “breast cancer cell”, “cancer cell” or similar to be limited by the stage of the cell cycle of said cancer cell.

In one example, the sample comprises a cell or a plurality of cells derived from a breast.

In one example, the biological sample has been isolated previously from the subject. In accordance with this example, a method of the present disclosure is performed ex vivo. In such cases, the sample may be processed or partially processed into a nucleic acid sample that is substantially free of contaminating protein. All such examples are encompassed by the present disclosure.

Methods for isolating a sample from a subject are known in the art and include, for example, surgery, biopsy, collection of a body fluid, for example, by paracentesis or thoracentesis or collection of, for example, blood or a fraction thereof. All such methods for isolating a biological sample shall be considered to be within the scope of providing or obtaining a sample.

For example, in the case of a breast cancer, a sample is collected, for example, using a fine needle aspiration biopsy, a core needle biopsy, or a surgical biopsy.

It will be apparent from the preceding description that methods provided by the present disclosure involve a degree of quantification to determine elevated or enhanced methylation of nucleic acid in tissue that is suspected of comprising a cancer cell or metastases thereof, or reduced gene expression in tissue that is suspected of comprising a cancer cell or metastases thereof. Such quantification is readily provided by the inclusion of appropriate control samples in the assays as described below.

As will be apparent to the skilled artisan, when internal controls are not included in each assay conducted, the control may be derived from an established data set.

Data pertaining to the control subjects are selected from the group consisting of:

- 1. a data set comprising measurements of the degree of methylation and/or gene expression for a typical population of subjects known to have ESR1 positive breast cancer which was responsive to endocrine therapy at the time of testing the subjects;
- 2. a data set comprising measurements of the degree of methylation and/or gene expression for the subject being tested wherein said measurements have been made previously, such as, for example, when the subject was known to be healthy or, in the case of a subject having ESR1 positive breast cancer, when the subject was at a stage in disease progression when the ESR1 positive breast cancer was responsive to endocrine therapy;
- 3. a data set comprising measurements of the degree of methylation and/or gene expression for a healthy individual or a population of healthy individuals;
- 4. a data set comprising measurements of the degree of methylation and/or gene expression for a normal individual or a population of normal individuals;
- 5. a data set comprising measurements of the degree of methylation and/or gene expression for an individual or a population of individuals diagnosed as having cancer other than a breast cancer characterized as being ESR1-negative subtype, or a ESR1-positive subtype which is refractory to endocrine therapy; and
- 6. a data set comprising measurements of the degree of methylation and/or gene expression from the subject being tested wherein the measurements are determined in a matched sample.

In a preferred example, the data comprising measurements of the degree of methylation and/or gene expression for a healthy subject, individual or population pertains to healthy breast epithelial cell(s) from the subject, individual or population.

Those skilled in the art are readily capable of determining the baseline for comparison in any diagnostic/prognostic assay of the present disclosure without undue experimentation, based upon the teaching provided herein.

In the present context, the term “typical population” with respect to subjects known to have ESR1 positive breast cancer which is responsive to endocrine therapy shall be taken to refer to a population or sample of subjects diagnosed with a specific form of ESR1 positive breast cancer that is representative of the spectrum of subjects suffering from ESR1 positive breast cancer. This is not to be taken as requiring a strict normal distribution of morphological or clinicopathopathological parameters in the population, since some variation in such a distribution is permissible. Preferably, a “typical population” will exhibit a spectrum of subtypes of ESR1 positive breast cancers at different stages of disease progression and with tumors at different stages and having different morphologies or degrees of differentiation.

In the present context, the term “healthy individual” shall be taken to mean an individual who is known not to suffer from breast cancer, such knowledge being derived from clinical data on the individual. It is preferred that the healthy individual is asymptomatic with respect to the any symptoms associated with breast cancer.

The term “normal individual” shall be taken to mean an individual having a normal level of methylation at a genomic region and/or gene expression as described herein in a particular sample derived from said individual.

As will be known to those skilled in the art, data obtained from a sufficiently large sample of the population will normalize, allowing the generation of a data set for determining the average level of a particular parameter. Accordingly, the level of methylation and/or gene expression as described herein can be determined for any population of individuals, and for any sample derived from said individual, for subsequent comparison to levels determined for a sample being assayed. Where such normalized data sets are relied upon, internal controls are preferably included in each assay conducted to control for variation.

The term “matched sample” shall be taken to mean that a control sample is derived from the same subject as the test sample is derived, at approximately the same point in time. In one example, the control sample shows little or no morphological and/or pathological indications of cancer. Matched samples are not applicable to blood-based or serum-based assays. Accordingly, it is preferable that the matched sample is from a region of the same tissue as the test sample e.g., breast tissue, such as breast epithelial tissue, however does not appear to comprise a cancer cell. For example, the matched sample does not include malignant cells or exhibit any symptom of the disease. For example, the sample comprises less than about 20% malignant cells, such as less than about 10% malignant cells, for example less than about 5% malignant cells, e.g., less than about 1% malignant cells. Morphological and pathological indications of malignant cells are known in the art and/or described herein.

In one example, the differential methylation of one or more CpG dinucleotides within one or more estrogen responsive enhancer regions defined in Tables 1-3 relative to the methylation status of a corresponding one or more CpG dinucleotides of a control is indicative of a subject's likely response to endocrine therapy. For example, hypermethylation of the one or more CpG dinucleotides is indicative that a subject having ESR1 positive breast cancer will be resistant to endocrine therapy. Alternatively, non-hypermethylation of the one or more CpG dinucleotides is indicative that a subject having ESR1 positive breast cancer which is responsive to endocrine therapy.

In another example, differential methylation of one or more CpG dinucleotides within one or more estrogen responsive enhancer regions defined in Tables 1-3 relative to the methylation status of a corresponding one or more CpG dinucleotides of a control is predictive of the therapeutic outcome and/or likely progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy. For example, hypermethylation of the one or more CpG dinucleotides is predictive that a subject suffering from ESR1 positive breast cancer who is receiving or about to receive endocrine therapy will not respond to the treatment and/or the cancer will progress to a worsening stage. Alternatively, non-hypermethylation of the one or more CpG dinucleotides is predictive that a subject suffering from ESR1 positive breast cancer who is receiving or about to receive endocrine therapy will have a good therapeutic outcome and/or the cancer will not progress to a worsening stage.

In an alternative example, the differential expression of a gene overlapping, spanning or closely associated with one or more CpG dinucleotides within one or more estrogen responsive enhancer regions defined in Tables 1-3 relative to a corresponding level of gene expression of a control is indicative of a subject's likely response to endocrine therapy. For example, reduced expression of a gene overlapping, spanning or closely associated with the one or more CpG dinucleotides is indicative that a subject having ESR1 positive breast cancer will be resistant to endocrine therapy. Alternatively, expression of a gene overlapping, spanning or closely associated with the one or more CpG dinucleotides which is not reduced is indicative that a subject having ESR1 positive breast cancer which is responsive to endocrine therapy.

In another alternative example, the differential expression of a gene overlapping, spanning or closely associated with one or more CpG dinucleotides within one or more estrogen responsive enhancer regions defined in Tables 1-3 relative to a corresponding level of gene expression of a control is diagnostic of ESR1 positive breast cancer which is refractory to endocrine therapy. For example, reduced expression of a gene overlapping, spanning or closely associated with the one or more CpG dinucleotides is diagnostic of ESR1 positive breast cancer which is refractory to endocrine therapy. Alternatively, expression of a gene overlapping, spanning or closely associated with the one or more CpG dinucleotides which is not reduced is diagnostic of ESR1 positive breast cancer is responsive to endocrine therapy.

In yet another alternative example, the differential expression of a gene overlapping, spanning or closely associated with one or more CpG dinucleotides within one or more estrogen responsive enhancer regions defined in Tables 1-3 relative to a corresponding level of gene expression of a control is predictive of the therapeutic outcome and/or likely progression of ESR1 positive breast cancer in a subject receiving or about to receive endocrine therapy. For example, reduced expression of a gene overlapping, spanning or closely associated with the one or more CpG dinucleotides is predictive that a subject suffering from ESR1 positive breast cancer who is receiving or about to receive endocrine therapy will not respond to the treatment and/or the cancer will progress to a worsening stage. Alternatively, expression of a gene overlapping, spanning or closely associated with the one or more CpG dinucleotides which is not reduced is predictive that a subject suffering from ESR1 positive breast cancer who is receiving or about to receive endocrine therapy will have a good therapeutic outcome and/or the cancer will not progress to a worsening stage.

The level(s) of differential methylation of the one or more CpG dinucleotides with the one or more estrogen responsive enhancer regions set forth in Tables 1-3 may be subjected to multivariate analysis to create an algorithm which enables the determination of an index of probability that a subject having ESR1 positive breast cancer will be resistant or responsive to endocrine therapy e.g., stratification of ESR1 positive breast cancer substypes, and/or that a subject having ESR1 positive breast cancer who is receiving or about to receive endocrine therapy will respond or is responding to endocrine therapy and/or that the ESR1 positive breast cancer will progress to a worsening stage following endocrine therapy. Hence, in one example, the present disclosure provides a rule based on the application of a comparison of levels of methylation biomarkers to control samples. In another example, the rule is based on application of statistical and machine learning algorithms. Such an algorithm uses the relationships between methylation biomarkers and disease status observed in training data (with known disease status) to infer relationships which are then used to predict the status of patients with unknown status. Practitioners skilled in the art of data analysis recognize that many different forms of inferring relationships in the training data may be used without materially changing the present disclosure.

The term “status” shall be taken to include whether or not a subject suffers from ESR1 positive breast cancer which is responsive or refractory to endocrine therapy (i.e., diagnostic status), whether or not an ESR1 positive breast cancer has responded to endocrine therapy and/or developed resistance thereto.

Analysis as described in the preceding paragraphs can also consider clinical parameters or traditional laboratory risk factors.

Information as discussed above can be combined and made more clinically useful through the use of various formulae, including statistical classification algorithms and others, combining and in many cases extending the performance characteristics of the combination beyond that of any individual data point. These specific combinations show an acceptable level of diagnostic/prognostic accuracy, and, when sufficient information from multiple markers is combined in a trained formula, often reliably achieve a high level of diagnostic/prognostic accuracy transportable from one population to another.

Several statistical and modeling algorithms known in the art can be used to both assist in biomarker selection choices and optimize the algorithms combining these choices. Statistical tools such as factor and cross-biomarker correlation/covariance analyses allow more rational approaches to panel construction. Mathematical clustering and classification tree showing the Euclidean standardized distance between the biomarkers can be advantageously used. Pathway informed seeding of such statistical classification techniques also may be employed, as may rational approaches based on the selection of individual biomarkers (e.g., such as those CpG dinucleotides within estrogen responsive enhancer regions set forth in Tables 1-3) based on their participation across in particular pathways or physiological functions or individual performance.

Ultimately, formulae such as statistical classification algorithms can be directly used to both select methylation biomarkers and to generate and train the optimal formula necessary to combine the results from multiple methylation biomarkers into a single index. Often techniques such as forward (from zero potential explanatory parameters) and backwards selection (from all available potential explanatory parameters) are used, and information criteria are used to quantify the tradeoff between the performance and diagnostic/prognostic accuracy of the panel and the number of methylation biomarkers used. The position of the individual methylation biomarkers on a forward or backwards selected panel can be closely related to its provision of incremental information content for the algorithm, so the order of contribution is highly dependent on the other constituent biomarkers in the panel.

Any formula may be used to combine methylation biomarker results into indices or indexes useful in the practice of the disclosure. As indicated herein, and without limitation, such indices may indicate, among the various other indications, the probability, likelihood, absolute or relative risk, time to or rate of disease, conversion from one to another disease states, or make predictions of future biomarker measurements of cancer. This may be for a specific time period or horizon, or for remaining lifetime risk, or simply be provided as an index relative to another reference subject population.

The actual model type or formula used may itself be selected from the field of potential models based on the performance and diagnostic accuracy characteristics of its results in a training population. The specifics of the formula itself may commonly be derived from biomarker results in the relevant training population. Amongst other uses, such formula may be intended to map the feature space derived from one or more biomarker inputs to a set of subject classes (e.g. useful in predicting class membership of subjects as normal, as having ESR1 positive breast cancer which is responsive or resistant/refractory to endocrine therapy or at risk of developing resistance to endocrine therapy), to derive an estimation of a probability function of risk using a Bayesian approach (e.g. the risk of ESR1 positive breast cancer which is resistant/refractory to endocrine therapy or at risk of developing resistance to endocrine therapy), or to estimate the class-conditional probabilities, then use Bayes' rule to produce the class probability function as in the previous case.

Following analysis and determination of an index of probability of the presence or absence of ESR1 positive breast cancer which is responsive or resistant/refractory to endocrine therapy or at risk of developing resistance to endocrine therapy, the index can be transmitted or provided to a third party, e.g., a medical practitioner for assessment. The index may be used by the practitioner to assess whether or not additional diagnostic methods are required, e.g., biopsy and histological analysis and/or other assays, or a change in treatment e.g., away from endocrine therapy, or commencement of treatment e.g., endocrine therapy.

Monitoring the Efficacy of Treatment and Disease Progression

As the methylation profile of ESR1-positive breast cancer can vary with the progression of the cancer, a subject suffering from ESR1-positive breast cancer who was previously responsive to endocrine therapy, or who has been previously identified as having a methylation profile which is indicative of responsiveness to endocrine therapy, may acquire (over time) a methylation profile which is indicative of resistance to endocrine therapy and thereby develop resistance to endocrine therapy. Accordingly, the methods described herein are useful for monitoring the progression of ESR1-positive breast cancer in a subject suffering therefrom and monitoring the efficacy of treatment. In this regard, the term “monitoring the progression of ESR1-positive breast cancer” and/or “monitoring the efficacy of treatment” includes, for example, determining whether a subject suffering from ESR1-positive breast cancer retains a methylation profile which is indicative of responsiveness to endocrine therapy or acquires a methylation profile which is indicative of resistance to endocrine therapy. For example, the method comprises determining differential methylation of one or more CpG dinucleotides with the one or more estrogen responsive enhancer regions set forth in Table 1, Table 2 and/or Table 3 in a sample from a subject relative to a reference level of methylation for the corresponding one or more CpG dinucleotides previously determined for the subject or a control sample.

In one example, an increase in methylation at one or more the CpG dinucleotides in the sample compared to the previously obtained sample may indicate that the ESR1-positive breast cancer has progressed to a worsening stage e.g., by acquiring resistance to endocrine therapy. In such circumstances, alternative or additional treatment of the breast cancer may be desired.

In another example, a decrease in methylation at one or more the CpG dinucleotides in the sample compared to the previously obtained sample may indicate that the ESR1-positive breast cancer has improved i.e., the subject is responding to treatment, and/or remains or has become responsive to endocrine therapy. For example, in circumstances where the subject has retained a methylation profile which is indicative of responsiveness to endocrine therapy and is already undergoing endocrine therapy, it may be desirable to continue endocrine therapy. For example, in circumstances where the subject was previously determined to have a methylation profile which is indicative of resistance to endocrine therapy and is therefore not undergoing endocrine therapy, it may be desirable to commence endocrine therapy.

Clearly, the detection of one or more additional biomarkers other than those set forth in Tables 1-3 is encompassed by this example of the disclosure.

Methods for detecting markers are described herein and are to be taken to apply mutatis mutandis to this example of the disclosure.

Methods of Treatment

The present disclosure additionally provides a method of treating ESR1-positive breast cancer. Such a method comprises, for example, diagnosing ESR1-positive breast cancer using a method of the disclosure described in any one or more examples described herein and, based on whether the subject is determined as being responsive or resistant to endocrine therapy, administering a suitable therapeutic compound or performing surgery or recommending treatment with a suitable therapeutic compound or recommending performance of surgery. For example, if, after performing the method of diagnosis or prognosis of the disclosure, the subject is determined as being responsive to endocrine therapy, the method may comprise commencing endocrine therapy e.g., by administering a therapeutic compound which blocks, alters or removes the activity of estrogen and/or progesterone, or recommending that the subject commence endocrine therapy. In another example, if, after performing the method of diagnosis or prognosis of the disclosure, the subject is determined as being resistance/refractory to endocrine therapy, the method may comprise commencing treatment other than endocrine therapy e.g., chemotherapy or radiotherapy and/or performing surgery, or recommending that the subject commences treatment other than endocrine therapy e.g., chemotherapy or radiotherapy, and/or recommending surgery.

Kits

The present disclosure additionally provides a kit for use in a method of the disclosure. In one embodiment, the kit comprises:

(i) one or more probes or primers (or isolated antibodies or ligands) that specifically hybridize to a biomarker (a CpG dinucleotide) described herein according to any example; and

(ii) detection means.

In another example, a kit additionally comprises a reference sample. Such a reference sample may for example, be a polynucleotide sample derived from a sample isolated from one or more subjects suffering from breast cancer. Alternatively, a reference sample may comprise a sample isolated from one or more normal healthy individuals.

In one example, the kit comprises a probe or primer. In one example, the probe or primer that is capable of selectively hybridizing to a CpG dinucleotide of an estrogen responsive enhancer region described herein according to any example.

In those cases where the probe is not already available, they must be produced. Apparatus for such synthesis is presently available commercially and techniques for synthesis of various nucleic acids are available in the literature. Methods for producing probes or primers are known in the art and/or described herein.

In one example, a probe or primer selectively hybridizes to a CpG dinucleotide of a estrogen responsive enhancer region set forth in Tables 1-3 that is selectively mutated by, for example, bisulphite treatment if the residue is not methylated. In another example, a probe or primer selectively hybridizes to a CpG dinucleotide of a genomic region set forth in Tables 1-3 that can be methylated in a ESR1 positive breast cancer cell.

The kit may further comprise instructions for the detection of methylation levels of any of the target genes disclosed herein and for the comparison of those methylation levels with a reference level. The instructions may provide one or a series of cut-off values demarcating the likelihood of risk of a subject having ESR1 positive breast cancer which is responsive or resistance to endocrine therapy.

The present disclosure additionally provides a kit or an article of manufacture comprising a compound for therapeutic treatment of ESR1 positive breast cancer packaged with instructions to perform a method substantially as described herein according to any example of the disclosure. For example, if the ESR1 positive breast cancer is determined as being responsive to endocrine therapy, the kit may comprise a therapeutic compound which blocks, alters or removes the activity of estrogen and/or progesterone e.g., anastrozole, exemestane, fulvestrant, goserelin, letrozole, leuprorelin, leuprolide acetate, megestrol, palbociclib, tamoxifen or toremifene. If, on the other hand, the ESR1 positive breast cancer is determined as being resistant to endocrine therapy, the kit may comprise a chemotherapeutic drug known in the art for treatment of breast cancer e.g., docetaxel, paclitaxel, platinum agents (cisplatin, carboplatin), vinorelbine, capecitabine, liposomal doxorubicin, gemcitabine, mitoxantrone, ixabepilone, albumin-bound paclitaxel or eribulin.

Knowledge-Based Systems

Knowledge-based computer software and hardware for implementing an algorithm of the disclosure also form part of the present disclosure. Such computer software and/or hardware are useful for performing a method of the disclosure. Thus, the present disclosure also provides software or hardware programmed to implement an algorithm that processes data obtained by performing the method of the disclosure via an univariate or multivariate analysis to provide a disease index value and provide or permit a diagnosis of ESR1 positive breast cancer which is responsive or resistance to endocrine therapy and/or for treatment management to determine progression or status of ESR1 positive breast cancer throughout treatment to determine whether there is likely to be a change in responsiveness or resistance to endocrine therapy, with the results of the disease index value in comparison with predetermined values.

FIG. 10 illustrates a computer system 100 for predicting response to endocrine therapy in a subject suffering from estrogen receptor 1 (ESR1) positive breast cancer. The computer system 100 comprises a processor 102 connected to a program memory 104, a data memory 106, a communication port 108 and a user port 110. The program memory 104 is a non-transitory computer readable medium, such as a hard drive, a solid state disk or CD-ROM. Software, that is, an executable program stored on program memory 104 causes the processor 102 to perform the methods disclosed herein. For example, processor 102 determines the methylation status of one or more CpG dinucleotide sequences within one or more estrogen responsive enhancers in the subject; and identifies differential methylation of said one or more CpG dinucleotide sequences in the subject relative to data for a reference level of methylation for the corresponding one or more CpG dinucleotide sequences. Differential methylation of said one or more CpG dinucleotide sequences in the subject relative to the reference level is indicative of the subject's likely response to endocrine therapy

As used in the context of a computer system 100 of the disclosure, the term “determines the methylation status”, “determining the methylation status” or similar refers to calculating, retrieving or receiving one or more data values indicative of the methylation status of the one or more CpG dinucleotide sequences in the subject. This also applies to related terms.

The processor 102 may then store the methylation status on data store 106, such as on RAM or a processor register. Processor 102 may also send the determined methylation status via communication port 108 to a server, such as a pathology server.

The processor 102 may receive data, such as sequencing data, from data memory 106 as well as from the communications port 108 and the user port 110, which is connected to a display 112 that shows a visual representation 114 of the predicted response to a user 116, such as a clinician. In one example, the processor 102 receives sequencing data from a sequencing machine via communications port 108, such as by using a local area network. Although communications port 108 and user port 110 are shown as distinct entities, it is to be understood that any kind of data port may be used to receive methylation status data, such as a network connection, a memory interface, a pin of the chip package of processor 102, or logical ports, such as IP sockets or parameters of functions stored on program memory 104 and executed by processor 102. These parameters may be stored on data memory 106 and may be handled by-value or by-reference, that is, as a pointer, in the source code.

The processor 102 may receive sequencing data through all these interfaces, which includes memory access of volatile memory, such as cache or RAM, or non-volatile memory, such as an optical disk drive, hard disk drive, storage server or cloud storage. The computer system 100 may further be implemented within a cloud computing environment, such as a managed group of interconnected servers hosting a dynamic number of virtual machines.

It is to be understood that any receiving step may be preceded by the processor 102 determining or computing the data that is later received. For example, the processor 102 determines the methylation status and stores the methylation status in data memory 106, such as RAM or a processor register. The processor 102 then requests the data from the data memory 106, such as by providing a read signal together with a memory address. The data memory 106 provides the data as a voltage signal on a physical bit line and the processor 102 receives the methylation status via a memory interface.

For example, processor 102 may receive sequencing data in the form of a file stored on a file system that is remote (cloud) or local including network attached storage (NAS) or server attached storage (SAN). Processor 102 analyses the sequencing data and identifies the presence of methylated cytosine nucleotides (5-methylcytosine or 5-MeC) and/or cytosine-to-uracil converted nucleotides (optionally identified as thymine nucleotides). Processor 102 may identify cytosine nucleotides which are methylated by comparing the received sequencing data to a reference and determining those cytosine nucleotides which are methylated and/or those cytosine nucleotides that have are not methylated (for example, those cytosines which have not been deaminated as a result of bisulphite treatment and thereby converted to uracil). Processor 102 stores the result of this identification in a separate file on the file system that may be the same or different to the file system on which the sequencing data is stored.

It is to be understood that throughout this disclosure unless stated otherwise, methylation status, sequences, methylation, level, patient, subject and the like may refer to data structures, which are physically stored on data memory 106 or processed by processor 102. Further, for the sake of brevity when reference is made to particular variable names, such as “differential methylation” or “methylation status” this can be understood to refer to values of variables stored as physical data in computer system 100.

The method for predicting response to endocrine therapy may be understood as a blueprint for the software program and may be implemented step-by-step, such that each step is represented by a function in a programming language, such as C++ or Java. The resulting source code may then be compiled and stored as computer executable instructions on program memory 104 or provided as executable source code such as PHP or Python.

Processor 102 may generate an output to indicate the predicted response to endocrine therapy. This output may comprise an electronic document, such as a PDF document. This output may also be rendered on a website that is remotely accessible by the clinician. Generating the output may then comprise generating HTML code and storing the HTML code on the data store of a webserver. This generation of the HTML code may occur dynamically and triggered by the clinician requesting the information. The predicted response to endocrine therapy may be stored on a database, such as a subject database, associated with the subject. The system 100 may be implemented using an Angular front-end for user interface generation and Flask backend for database management.

It should be understood that the techniques of the present disclosure might be implemented using a variety of technologies. For example, the methods described herein may be implemented by a series of computer executable instructions residing on a suitable computer readable medium. Suitable computer readable media may include volatile (e.g. RAM) and/or non-volatile (e.g. ROM, disk) memory, carrier waves and transmission media. Exemplary carrier waves may take the form of electrical, electromagnetic or optical signals conveying digital data steams along a local network or a publically accessible network such as the internet.

It should also be understood that, unless specifically stated otherwise as apparent from the following discussion, it is appreciated that throughout the description, discussions utilizing terms such as “estimating” or “processing” or “computing” or “calculating”, “optimizing” or “determining” or “displaying” or “maximising” or the like, in the context of a computer system 100, refer to the action and processes of a computer system, or similar electronic computing device, that processes and transforms data represented as physical (electronic) quantities within the computer system's registers and memories into other data similarly represented as physical quantities within the computer system memories or registers or other such information storage, transmission or display devices.

In one example, a method of the disclosure may be used in existing knowledge-based architecture or platforms associated with pathology services. For example, results from a method described herein are transmitted via a communications network (e.g. the internet) to a processing system in which an algorithm is stored and used to generate a predicted posterior probability value which translates to the index of disease probability (e.g., ESR1 positive breast cancer which is responsive to endocrine therapy or resistant to endocrine therapy) or responsiveness to treatment, which is then forwarded to an end user in the form of a diagnostic or predictive report.

The method of the disclosure may, therefore, be in the form of a kit or computer-based system which comprises the reagents necessary to detect the concentration of the biomarkers and the computer hardware and/or software to facilitate determination and transmission of reports to a clinician.

The assay of the present disclosure permits integration into existing or newly developed pathology architecture or platform systems. For example, the present disclosure contemplates a method of allowing a user to determine the status of a subject with respect to ESR1-positive breast cancer, the method including:

(a) receiving data in the form of levels of differential methylation of one or more CpG dinucleotides within one or more estrogen responsive enhancer regions set forth in Tables 1-3 for a test sample relative to a reference level of methylation, optionally in combination with another marker of breast cancer e.g., ESR1-positive breast cancer;

(b) processing the subject data via univariate and/or multivariate analysis to provide a disease index value;

(c) determining the status of the subject in accordance with the results of the disease index value in comparison with predetermined values; and

(d) transferring an indication of the status of the subject to the user via the communications network reference to the multivariate analysis includes an algorithm which performs the multivariate analysis function.

In one example, the method additionally includes:

(a) having the user determine the data using a remote end station; and

(b) transferring the data from the end station to the base station via the communications network.

The base station can include first and second processing systems, in which case the method can include:

(a) transferring the data to the first processing system;

(b) transferring the data to the second processing system; and

(c) causing the first processing system to perform the univariate or multivariate analysis function to generate the disease index value.

The method may also include:

(a) transferring the results of the univariate or multivariate analysis function to the first processing system; and

(b) causing the first processing system to determine the status of the subject.

In this case, the method also includes at least one of:

(a) transferring the data between the communications network and the first processing system through a first firewall; and

(b) transferring the data between the first and the second processing systems through a second firewall.

The second processing system may be coupled to a database adapted to store predetermined data and/or the univariate or multivariate analysis function, the method include:

(a) querying the database to obtain at least selected predetermined data or access to the multivariate analysis function from the database; and

(b) comparing the selected predetermined data to the subject data or generating a predicted probability index.

The second processing system can be coupled to a database, the method including storing the data in the database.

The method can also include having the user determine the data using a secure array, the secure array of elements capable of determining the level of biomarker and having a number of features each located at respective position(s) on the respective code. In this case, the method typically includes causing the base station to:

(a) determine the code from the data;

(b) determine a layout indicating the position of each feature on the array; and

The method can also include causing the base station to:

(a) determine payment information, the payment information representing the provision of payment by the user; and

(b) perform the comparison in response to the determination of the payment information.

The present disclosure also provides a base station for determining the status of a subject with respect to a ESR1 positive breast cancer, the base station including:

(a) a store method;

(b) a processing system, the processing system being adapted to:

- (i) receive subject data from the user via a communications network;
- (ii) determining the status of the subject in accordance with the results of the algorithmic function including the comparison; and

The processing system can be adapted to receive data from a remote end station adapted to determine the data.

The processing system may include:

(a) a first processing system adapted to:

- (i) receive the data; and
- (ii) determine the status of the subject in accordance with the results of the univariate or multivariate analysis function including comparing the data; and

(b) a second processing system adapted to:

- (i) receive the data from the processing system;
- (ii) perform the univariate or multivariate analysis function including the comparison; and
- (iii) transfer the results to the first processing system.

The base station typically includes:

(a) a first firewall for coupling the first processing system to the communications network; and

(b) a second firewall for coupling the first and the second processing systems.

The processing system can be coupled to a database, the processing system being adapted to store the data in the database.

The present disclosure is now described further in the following non-limiting examples.

EXAMPLES
Example 1—DNA Methylation Profiling of Enhancer Loci in Endocrine Resistant Cells

To interrogate DNA methylation remodelling as a critical component of acquired endocrine resistance, we performed methylation profiling in duplicate using the Infinium HumanMethylation 450 beadchip, on ESR1-positive hormone sensitive MCF7 cells, and three different well characterised endocrine resistant MCF7-derived cell lines; tamoxifen-resistant (TAMR)10, fulvestrant-resistant (FASR)11 and estrogen deprivation resistant (MCF7X)12 cells.

Cell Culture and HumanMethylation450K Array

MCF7 breast cancer cells and the corresponding endocrine resistant sub cell lines were provided by Dr Julia Gee (Cardiff University, UK). Briefly, MCF7 cells were maintained in RPMI-1640 based medium containing 5% (v/v) fetal calf serum (FCS). Tamoxifen-resistant MCF7 (TAMR) cells were generated by the long-term culture of MCF7 cells in phenol-red-free RPMI medium containing 5% charcoal stripped FCS and 4-OH-tamoxifen (1×10⁻⁷M) (TAM). Fulvestrant-resistant MCF-7 (FASR) cells were generated by the long-term culture of MCF7 cells in phenol-red-free RPMI medium containing 5% charcoal stripped FCS and fulvestrant (1×10⁻⁷M) (FAS). Long-term estrogen deprived MCF7 (MCF7X) cells were generated by the long-term culture of MCF7 cells in phenol-red-free RPMI medium containing 5% charcoal stripped FCS. Endocrine resistant sub lines were established and characterised following 6 months endocrine challenge/estrogen deprivation exposure^{10, 11, 12}. All cell lines were authenticated by short-tandem repeat (STR) profiling (Cell Bank, Australia) and cultured for less than 6 months after authentication. Genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue kit according to manufacturer's instructions. HumanMethylation450K arrays were carried out by the Australian Genome Research Facility (AGRF) (Melbourne, Australia). Cell line HumanMethylation450K array data is available online at GEO (GSE69118).

HM450 Analysis

Two biological replicates per condition—MCF7, TAMR, MCF7X, or FASR—were profiled on Illumina's HumanMethylation450K array. Raw HM450 data was pre-processed and background normalized with the Biconductor minfi package (Aryee et al., (2014) Bioinformatics 30:1363-1369) using preprocesslllumina (bg.correct=TRUE, normalize=“controls”, reference=1); resulting M-Values were used for statistical analyses and I3-Values for heatmap visualizations and clustering. Differential methylation analysis of the pre-processed data was performed using the Bioconductor limma package.

Results

Density plots showing the correlation between the DNA methylation profile of parent MCF7 cells and individual endocrine resistant cell lines indicate that the MCF7X and TAMR cells, which are both ESR1-positive (Knowlden et al., (2003) Endocrinology 144:1032-1044; Staka et al., (2005) Endocr Relat Cancer 12:S85-97), predominantly gained DNA methylation as indicated by the increased density of points above the trend line. In contrast, FASR cells, which are ESR1-negative (McClelland et al., (2001) Endocrinology, 142:2776-2788), exhibited both hyper and hypomethylation events relative to parent MCF7 cells as indicated by a symmetrical density distribution (FIG. 1a-c). We first sought to identify the common differential DNA methylation events present in each of the three uniquely derived endocrine resistant cell models by carrying out paired analyses (i.e. each endocrine resistant cell line vs MCF7 parent control) and overlapping the data (FIG. 1d). We found that across the individual resistant cell lines 14,749 CpG probes were commonly hypermethylated (FDR<0.01) whereas only 192 probes exhibited shared hypomethylation (FDR<0.01) (FIG. 1d).

Example 2—Characterisation of Functional Genomic Location of Differential Methylation at Enhancer Loci in Endocrine Resistant Cells

To comprehensively characterise the functional genomic location of differential methylation observed in the endocrine resistant cell models we used ChromHMM segmentation of the MCF7 genome as previously described in Taberlay et al., (2014) Genome Research, 24(9):1421-32.

Genomic Segmentation and Annotation

The ChromHMM segmentation of the MCF7 genome was obtained from Taberlay et al., (2014). Enhancer (“Enhancer” and “Enhancer+CTCF”) and Promoter categories (“Promoter”, “Promoter+CTCF”, and “Poised Promoter”) were collapsed into a single “Enhancer” and “Promoter” state respectively for the purposes of our analysis. RefSeq transcript annotations were obtained from UCSC genome browser (Kent et al. (2002) Genome Research 12:996-1006 (2002); Meyer et al. (2013) Nucleic Acids Res. 41:D64-69). Strikingly, significant enrichment of commonly hypermethylated probes was exclusively observed in enhancer regions of the genome (n=3932 probes, p<<0.0001; hypergeometric test) (FIG. 1e).

We next sought to determine whether the enhancer regions identified as being more heavily methylated in all endocrine resistance models were regulated by the estrogen receptor in the parental MCF7 cells. Using reprocessed, publically available ChIPSeq data for MCF7 ESR1 (Ross-Innes et al. (2012) Nature 481:389-393), GATA3 (Theodorou et al., (2013) Genome Res. 23:12-22) and FOXA1 (Hurtado et al., (2011) Nat. Genet. 43:27-33) (two transcription factors closely associated with ESR1-activity), we found that enhancer-specific CpG hypermethylated probes were enriched in ESR1 binding sites by approximately 6 fold, FOXA1 binding sites by 5 fold and GATA3 binding sites by 8 fold (p<<0.0001; hypergeometric test) (FIG. 2a). The greatest number of hypermethylated enhancer probes were found to overlap ESR1 binding sites (n=801), which represents approximately 20% of all hypermethylated probes in enhancer regions. Significantly, 47% (379 out of 801) of the hypermethylated enhancer probes that were located within an ESR1 binding site were also located within a FOXA1 and/or GATA3 binding site (FIG. 2b) which is particularly noteworthy since these transcription factors cooperatively modulate ESR1-transcriptional networks by forming a functional enhanceosome.

Example 3—Enhancer DNA Hypermethylation and Diminished ESR1 Binding

Having defined a subset of ESR1 binding sites that overlap enhancer regions that contain hypermethylated loci in multiple models of endocrine resistance (n=856 sites—Table 1), we sought to determine whether DNA methylation affected the intensity of ESR1 binding at these sites.

ChIP-Seq Data Acquisition and Analysis

Using MCF7 and TAMR ESR1 ChIP data previously described by Ross-Innes et al. (2012) Nature 481:389-393, we compared the change in ESR1 binding signal intensity at ESR1-enhancer sites that contained (a) hypermethylated probe(s) to that of all other ESR1-enhancer sites. Reads were mapped to genome build HG19 (GRCh37) with bowtie and mismatched (>3 mismatched bases), multiple mapping and duplicate reads were excluded from downstream analysis. ESR1 enrichment peaks were identified with the HOMER software suite (Heinz et al. (2010) Molecular cell 38:576-589) using the findPeaks utility (-style factor -fragLength 200 -size 300 -F 0 -L 0 -C 0 -poisson 1e⁻⁰⁶) on each experiment separately. The resulting peaks were merged to produce a ground set of 120,735 regions for subsequent analysis. Active ESR1 regions were identified in MCF7 by comparing the distribution of reads overlapping the ground set of ESR1 regions in the three MCF7 ESR1 experiments (GSM798423, GSM798424, and GSM798425) and MCF7 input experiment (GSM798440) with edegR (Robinson et al., (2010) Bioinformatics 26:139-140). This yielded 54,265 active ESR1 regions in MCF7 (FDR<0.05). A similar strategy was applied to TAMR data to yield 49,511 ESR1 regions in TAMR cells. Regions of differential ESR1 binding were identified by comparing the distribution of sequence reads in MCF7 and TAMR across the ground set of ESR1 regions using edgeR and potential variation in copy number was accounted for using DiffBind (Ross-Innes et al. (2012) Nature 481:389-393). This analysis resulted in 24,711 regions with statistical significant gain (FDR 5%) and 32,343 regions with statistical significant loss (FDR 5%) of ESR1 binding in TAMR cells as compared to MCF7 cells. ESR1 peaks overlapping HM450 probes were assigned to the nearest RefSeq transcript (<20 kb distance) for the purposes of gene expression analysis. Raw MCF7 GATA3 and FOXA1 ChIP-Seq data was obtained from Theodorou et al., (2013) Genome Res. 23:12-22 and Hurtado et al., (2011) Nat Genet 43:27-33 respectively. Data were processed in the same manner as outlined for ESR1 ChIP-seq previously described.

Results

At methylated ESR1-enhancer sites there was a 2.29 log fold reduction in ESR1 binding in TAMR compared to MCF7 cells. In contrast, at all other ESR1-enhancer binding sites, there was a 0.52 log fold reduction in ESR1 binding in TAMR compared to MCF7 cells. Thus, increased methylation at ESR1-enhancer sites is associated with reduction in ESR1 binding (p<<0.0001; t-test) (FIG. 2c). Four illustrative examples show the loss of ESR1 binding in the TAMR cells at enhancer regions that are more heavily methylated in the endocrine resistant versus the parent MCF7 (FIG. 2d). The examples include enhancer regions located within the gene body of death associated protein 6 (DAXX), golgi to ER traffic protein 4 homolog (GET4) (a member of the BAG6-UBL4A-GET4 DNA damage response/cell death complex), ESR1 itself and nuclear receptor co-repressor 2 (NCOR2) (FIG. 2d).

Example 4—Enhancer DNA Hypermethylation and Related Gene Expression

Since the vast majority of ESR1-enhancer binding sites identified as hypermethylated in the endocrine resistant cell lines compared to the parent MCF7 cells were intragenic i.e. 617 out of 856, 72% with at least partial overlap (Table 2), we next sought to determine if the DNA methylation of these regions correlated with the expression of the genes in which they were located (or closest TSS if intergenic) in human breast cancer.

TCGA Data Acquisition

DNA methylation analysis utilized clinical data available through the TCGA Breast Invasive Carcinoma (BRCA) cohort (TCGA (2012) Nature 490:61-70). Raw HM450 methylation data (level 1) were obtained from the TCGA data portal (normal samples=97, ESR1 positive tumours=353 and ESR1 negative=105). ESR1 positive tumours were further divided into luminal A (lumA=301) and luminal B (lumB=52) populations using progesterone receptor (PR) expression, such that lumA were ESR1+/PR+ and lumB were ESR1+/PR−. Processed RNA-Seq expression data (level 3) were obtained from TCGA data portal (588 ESR1 positive tumours with 73 matched normals and 174 ESR1 negative samples with 19 matched normals).

Gene Set Enrichment Analysis of TCGA Data

GSEA was performed against the Molecular Signatures Database v4.0 (MSigDB) (Subramanian et al. (2005) PNAS, 102:15545-15550) C2 Collection. Enrichment was assessed by hypergeometric testing as implemented in the R stats package.

Results

Using RNA-seq and HM450 methylation data derived from TCGA breast cohort (n=459 patients), we determined that out of the 856 ESR1-enhancer binding sites of interest, hypermethylation of 328 sites (i.e. 38% of ESR1-enhancer sites) correlated with the reduced expression of the genes with which they were most closely associated (Spearman's correlation coefficient; p<0.001) (Table 3). The 328 ESR1-enhancer binding sites represented 291 unique genes (including those presented in FIG. 2d). Gene set enrichment analysis revealed that these genes were over-represented in gene sets up-regulated by ESR1 activation, down-regulated in the acquisition of endocrine resistance and gene sets lowly expressed in basal vs luminal disease, thus suggesting that such genes were critical drivers of estrogen-driven tumours (FIG. 3a). Interestingly, using unsupervised clustering analysis, this gene set (n=291) stratifies ESR1-positive and ESR1-negative breast cancer patients (FIG. 3b). Cumulatively, this indicates that the methylation events occurring throughout the acquisition of endocrine resistance are serving to facilitate an estrogen-independent phenotype reflective of a breast cancer subtype that is refractory to endocrine therapy.

Example 5—ESR1-Enhancer Methylation Defines Breast Cancer Subtype

We next sought to determine whether ESR1-enhancer hypermethylation was indicative of breast cancer subtype. We assessed the median methylation of all hypermethylated ESR1-enhancer probes (n=801) in TCGA normal (n=97), luminal A (n=301), luminal B (n=52) and ESR1-negative (n=105) patient HM450 data.

Clinical Sample Acquisition and DNA Extraction

Formalin-fixed, paraffin-embedded (FFPE) breast cancer samples were obtained from the St. George Hospital, Kogarah, Australia. The de-identified haematoxylin-eosin stained sections were reviewed by a pathologist and representative tumour areas were marked and blocks were cored accordingly. Genomic DNA was extracted using the Qiagen AllPrep DNA/RNA FFPE kit according to the manufacturer's instructions.

Multiplex Bisulfite-PCR Resequencing of Clinical FFPE DNA

Bisulfite DNA conversions were performed using a manual protocol. For each conversion approximately 100 ng was bisulfite converted at a time. Conversion took place at 80° C. for 45 min in the presence of 0.3M NaOH, 3.75 mM quinone, and 2.32M sodium metabisulfite, as per the methodology described in Clark et al., (2006) Nat. Protoc. 1:2353-2364. The multiplex bisulfite PCR reaction was performed as detailed in Korbie et al., (2015) Clinical Epigenetics 7:28. Briefly, Promega HotStart GoTaq with Flexi-buffer (M5005) was used with the following components at the indicated concentrations: 5X green (1×), CES 5×, (0.5×, as described in Ralser et al., (2006) Biochem Biophys Res Commun 347:747-751), MgCl₂(4.5 mM), dNTP's (200 μM each), primers (forward and reverse at 100 mM), Hot Start Taq (0.025 U μL⁻¹), DNA (2 ng μL⁻¹). All primers used are listed in Table 4.

TABLE 4

rimer sequences for multiplex bisulfite-PCR resequencing of clinical FFPE DNA

Non-CpG

Primers
Primer sequence
Fusion primer sequences

GATA3_ct_f2
GAtAGAttAGAGGtAGtAAGGAA
ACACTGACGACATGGTTCTACAGAtAGAttAGAGGtAGtAAGGAA

GATA3_ct_r2
CTTTTCAaAAACACCTTaAAAaCTA
TACGGTAGCAGAGACTTGGTCTCTTTTCAaAAACACCTTaAAAaCTA

ESR1_ct_f1
TTGtAGGGTTTAGGATGAAGT
ACACTGACGACATGGTTCTACATTGtAGGGTTTAGGATGAAGT

ESR1_ct_r1
CTTTACAATCTCTCTTTTTCCATT
TACGGTAGCAGAGACTTGGTCTCTTTACAATCTCTCTTTTTCCATT

ESR1_ct_f2
GGTGTGGAAGGtAAGGGAA
ACACTGACGACATGGTTCTACAGGTGTGGAAGGtAAGGGAA

ESR1_ct_r2
CTaaaCATTaCAaaCTTaTTCAAATAT
TACGGTAGCAGAGACTTGGTCTCTaaaCATTaCAaaCTTaTTCAAATAT

GET4_ct_f1
GTTGGTGTttTTGGATATGTG
ACACTGACGACATGGTTCTACAGTTGGTGTttTTGGATATGTG

GET4_ct_r1
CCATCCATaaaaCAAaaTCAaCT
TACGGTAGCAGAGACTTGGTCTCCATCCATaaaaCAAaaTCAaCT

ITPK1_ct_f1
GAAAGtTGGtTTTtTGGttTtAGT
ACACTGACGACATGGTTCTACAGAAAGtTGGtTTTtTGGttTtAGT

ITPK1_ct_r2
CATCATCATCAACAACCAaACA
TACGGTAGCAGAGACTTGGTCTCATCATCATCAACAACCAaACA

MSI2_ct_f2
GAGtATtTGGtTTTtATTTTTAAGTG
ACACTGACGACATGGTTCTACAGAGtATtTGGtTTTtATTTTTAAGTG

MSI2_ct_r2
CCCAAaAATAAaCTCAACTCCTT
TACGGTAGCAGAGACTTGGTCTCCCAAaAATAAaCTCAACTCCTT

C8orf46_ga_f1
CCAaCATCAaAaAAaaaAaCACC
ACACTGACGACATGGTTCTACACCAaCATCAaAaAAaaaAaCACC

C8orf46_ga_r1
GGGtAGATTGAtTtTGtAGtTG
TACGGTAGCAGAGACTTGGTCTGGGtAGATTGAtTtTGtAGtTG

DAXX_ga_f2
aCATATTTaaAaATaACCTCATCCA
ACACTGACGACATGGTTCTACAaCATATTTaaAaATaACCTCATCCA

DAXX_ga_r2
ttTTtAAGGGtTGAGTGtTtTGA
TACGGTAGCAGAGACTTGGTCTttTTtAAGGGtTGAGTGtTtTGA

NCOR2_ga_f1
CTCCCAaAaCCACACCCT
ACACTGACGACATGGTTCTACACTCCCAaAaCCACACCCT

NCOR2_ga_r1
TTTTGGAGGtAAAGttAGTGG
TACGGTAGCAGAGACTTGGTCTTTTTGGAGGtAAAGttAGTGG

RXRA_ga_f1
aAaCTTTTaaTaTaCTaCCCACC
ACACTGACGACATGGTTCTACAaAaCTTTTaaTaTaCTaCCCACC

RXRA_ga_r1
GATGAGTtAGATGGtAGGG
TACGGTAGCAGAGACTTGGTCTGATGAGTtAGATGGtAGGG

Cycling conditions were: 94° C., 5 mins; 12 cycles of (95° C., 20 s; 60° C., 1 min); 12 cycles of (94° C., 20 s; 65° C., 1 min 30 s); 65° C., 3 mins, 10° C. hold. Agencourt XP beads were using to clean-up and concentrate the multiplex reaction for subsequent barcoding (i.e., addition of Illumina p5/p7 sequences and sample specific DNA barcodes). The barcoding PCR used the following reagents at the indicated final concentrations in a 100 μl reaction: 1× GoTaq Green Flexi buffer; 0.25×CES; 4.5 mM MgCl₂; 200 μM dNTPs; 0.05 U μL⁻¹HotStart Taq; 25 μL of pooled template after Agencourt XP bead cleanup; and 20 μl MiSeq (Fluidigm PN FLD-100-3771). Cycling conditions were: 94° C., 5 mins; 9 cycles of (97° C., 15 s; 60° C., 30 s; 72° C., 2 mins); 72° C., 2 mins; 6° C., 5 mins. MiSeq sequencing was performed used the MiSeq Reagent Kit v2, 300 cycle; PN MS-102-2002. Bioinformatic analysis started with adaptor trimming using Trim galore (options: --length 100). Mapping used the Bismark methylation mapping program (Krueger et al., (2011) Bioinformatics 27:1571-1572) running Bowtie2 (Langmead and Salzberg (2012) Nat. Methods, 9:357-359) (options: --bowtie2 -N 1 -L 15 --bam -p 2 --score L, −0.6, −0.6 --non_directional; bismark_methylation_extractor -s -merge_non_CpG -comprehensive --cytosine_report). To reduce computational overhead mapping took place against only those genomic regions which were being investigated, plus an additional 100 bp-1 kb of flanking sequence.

Results

In normal breast tissue (which is reported to be approximately 7% ESR1-positive e.g., as described in Petersen et al, (1987) Cancer Research 47:5748-5751), the median methylation of the ESR1-enhancer sites was highest, while median DNA methylation was significantly reduced in luminal A disease (p<<0.0001; Mann-Whitney U test), which is indicative of its endocrine-responsive state. Interestingly, median ESR1-enhancer methylation was greater in luminal B patients compared to luminal A patients (p=0.017; Mann-Whitney U test), who are almost twice as likely to acquire endocrine resistance. In ESR1-negative disease, median methylation was higher than in luminal disease (vs luminal A, p<<0.0001; vs luminal B, p<<0.0001; Mann-Whitney U test) (FIG. 4a). A heatmap highlights the hypomethylated status of the ESR1-enhancer sites in luminal A disease relative to normal breast tissue and the other breast cancer subtypes (FIG. 4b). This trend is clearly illustrated at the DAXX enhancer region in which each CpG within the ESR1 binding site was hypomethylated in luminal A disease compared to normal tissue and luminal B and ESR1-negative cancer (FIG. 4c). Critically, no such variability was apparent at the DAXX promoter region (1000 bp upstream and 100 bp downstream of the transcription start site) (FIG. 4c), suggesting a significant regulatory effect of increased methylation at the enhancer locus.

Example 6—ESR1-Enhancer Hypermethylation Predicts Endocrine Failure

Given that ESR1-enhancer hypermethylation is prevalent in acquired endocrine resistance in vitro (FIG. 1e and FIGS. 2a-d) and in molecular sub-classifications of breast cancer that are intrinsically less responsive to endocrine therapy (FIGS. 4a-c), we next sought to determine the methylation status of a panel of these loci in ESR1-positive (luminal A) breast cancer samples from patients with different outcomes.

Primary samples were sourced from patients that received endocrine therapy for five years and either experienced relapse-free survival (RFS) (>14 years) or those that had relapsed (<6 years), defined as no relapse-free survival (n/RFS). Matched local relapse samples were also compared to the primary n/RFS patient samples. All patients received the same endocrine therapy (tamoxifen) Patient data is provide in Table 5).

TABLE 5

Patient details

Histological
Ellis

Mol
ER
PR

PatientID
AgeAtOperation
SizeOfTumour
VascularInvasion
HistType
Grade
Grade
Margin
subtype
Status
Status

RFS

1
44
20
No
Ductal
High
3
Clear
Luminal A
2
1

2
72
19
No
Lobular
Medium
2
Clear
Luminal A
2
1

3
50
20
Yes
Ductal
High
3
Clear
Luminal A
2
2

n/RFS

1
38
15
No
Ductal
High
3
Clear
Luminal A
2
2

2
37
16
No
Lobular
High
3
Clear
Luminal A
2
2

3
45
30
No
Ductal
High
3
Clear
Luminal A
2
2

Local

Date of
Date of

Adj
Adj
Localised
Recurrence
Date of
last
local

PatientID
Tamoxifen
chemo
Radiotherapy
in Breast
Diagnosis
follow up
Recurrence

RFS

1
Adjuvant
Adjuvant
Y
N
July 1998
September 2012
—

2
Adjuvant
N
Y
N
November 1998
June 2013
—

3
Adjuvant
Adjuvant
Y
N
February 1998
January 2014
—

n/RFS

1
Adjuvant
Adjuvant
Y
Y
December 1999
—
November 2005

2
Adjuvant
Adjuvant
Y
Y
September 2001
—
September 2004

3
Adjuvant
Adjuvant
Y
Y
June 1998
—
June 2002

Using a multiplex bisulphite-PCR resequencing methodology specifically devised for FFPE derived DNA (Korbie et al., (2015) Clinical Epigenetics 7:28), the methylation of multiple CpG sites across a panel of 9 estrogen-responsive enhancer regions was interrogated (technical duplicate correlates for all amplicons investigated are shown in FIG. 4). These enhancer regions included those located within DAXX, MSI2, NCOR2, RXRA and C8orf46 (FIG. 5a-e) and enhancer regions located within GATA3, ITPK1, ESR1 and GET4 (FIG. 6a-d). The assay was repeated with DNA extracted from biological duplicates of the endocrine resistant cell lines and the parent MCF7 cells to ensure its viability (FIG. 7a-i; technical duplicate correlates for all amplicons investigated are shown in FIG. 8). The average methylation levels detected at all enhancer loci were significantly higher in the recurrent tumours compared to the matched primary (n/RFS) tumours (DAXX; p<0.0001, ESR1; p<0.0005, RXRA; p<0.005, GET4, NCOR2, GATA3, MSI2; p<0.01, C8orf46, ITPK1; p<0.05; t-test), confirming that DNA methylation at ESR1-responsive enhancers is acquired in resistant disease (FIGS. 6 and 9). The difference in DNA methylation between RFS and n/RFS primary tumours was less considerable, although a statistically significant difference was observed for DAXX; p<0.0001, RXRA; p<0.01, C8orf46; p=0.01, NCOR2 and MSI2 (p<0.05; t-test) enhancer regions (FIG. 9).

Conclusions Drawn from Examples 1-6

The results provided herein support a model whereby ESR1-responsive enhancer DNA methylation is a fundamental unifying characteristic that defines endocrine sensitivity in breast cancer. This study is the first to combine in depth MCF7 ChromHMM annotation and genome wide methylation data from multiple resistance models to more comprehensively characterise global differential methylation across diverse genomic regions. This study shows for the first time that the methylation status of enhancers is associated with the inhibition of ESR1 binding in vitro and with the reduced expression of critical regulators and effectors of ESR1-activity in human disease. The identification of ESR1-responsive enhanceosome hypermethylation is both novel and considerably pertinent in the context of endocrine resistance, since genome wide positional analyses defining the set of cis-regulatory elements that recruit ESR1 in breast cancer cells have revealed its predominant recruitment to enhancers as opposed to promoter regions. In this study, the majority of ESR1-regulated enhancer regions identified as hypermethylated in the resistant cells were located within gene bodies. Strikingly, hypermethylation of these enhancer regions was frequently correlated with reduced expression of the host gene. Examples of genes whose expression inversely correlated with ESR1-enhancer DNA methylation include DAXX and GET4, each of which are reported to have roles in apoptosis. It is possible that the loss of expression of genes associated with pro-apoptotic functions facilitates the progression of endocrine resistance by reducing the efficacy of apoptotic signalling pathways activated by endocrine therapies.

Importantly, the ESR1-responsive enhancer hypermethylation events identified in the endocrine-resistant cell lines were also differentially methylated in endocrine sensitive and endocrine-resistant breast cancer patient samples. Therefore, ESR1-responsive enhancer methylation status may be reflective of endocrine dependence and could be used to stratify patients as responders to endocrine therapy. For example, NCOR2, a gene whose expression has previously been associated with metastasis free survival in 620 lymph node-negative patients with ESR1-positive breast cancer, was shown to negatively correlate with ESR1-enhancer methylation. In the present study, NCOR2 enhancer methylation was significantly higher in the poor (non relapse-free) prognosis patients, compared to the good (relapse-free) prognosis primary luminal A breast cancer patients.

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