Patent Application
20170081723
This application claims the benefit of priority of Singapore application No. 10201400876T, filed 21 Mar. 2014, the contents of it being hereby incorporated by reference in its entirety for all purposes.
The present invention is in the field of cancer biomarkers, in particular fusion genes as prognostic biomarkers for cancer.
Cancer is a class of diseases characterized by a group of cells that has lost its normal control mechanisms resulting in unregulated growth. Cancerous cells are also called malignant cells and can develop from any tissue within any organ. As cancerous cells grow and multiply, they form a tumour that invades and destroys normal adjacent tissues. Cancerous cells from the primary site can also spread throughout the body.
An example of a cancer is gastric cancer (GC). Most GCs are diagnosed at an advanced stage, which limits the current treatment strategies with the overall 5-year survival rate for distant or metastatic disease of ˜3%.
On the molecular level, GC is heterogeneous and currently the only therapeutic target is the amplified receptor tyrosine-protein kinase ERBB2.
While recent whole-genome and exome sequencing studies have identified recurrently mutated genes genome rearrangements in GC have not been studied in great detail. Genomic rearrangements, can have dramatic impact on gene function by amplification, deletion and gene disruption, and can create fusion genes with new functions.
Therefore, there is a need to identify the prognostic factors and markers that can be used to reliably determine the prognosis of patients suffering from cancer, such as gastric cancer, to allow identification of high risk and low risk cancer patients to allow different treatment approaches.
In one aspect, there is provided a method of determining or making of a prognosis if a patient has cancer or is at an increased risk of having cancer, the method comprising testing for the presence of one or more cancer-associated fusion genes, or proteins derived thereof, in a sample obtained from a patient, wherein said presence of one or more cancer-associated fusion genes in the sample indicates that said patient has cancer, or is at an increased risk of cancer, wherein the cancer-associated fusion genes are selected from the group consisting of CLEC16A-EMP2 (SEQ ID NO.: 97, 99 or 101), SNX2-PRDM6 (SEQ ID NO.: 113 or 115), MLL3-PRKAG2 (SEQ ID NO.: 121, 123 or 125) and DUS2L-PSKH1 (SEQ ID NO.: 131 or 133), or wherein the cancer-associated fusion genes are selected from the group consisting of CLEC16A-EMP2 (SEQ ID NO.: 97, 99 or 101), SNX2-PRDM6 (SEQ ID NO.: 113 or 115), MLL3-PRKAG2 (SEQ ID NO.: 121, 123 or 125) and DUS2L-PSKH1 (SEQ ID NO.: 131 or 133) in combination with CLDN18-ARHGAP26 (SEQ ID NO: 107).
In one aspect, there is provided a method of determining if a patient has cancer or is at an increased risk of having cancer, the method comprising testing for the presence of one or more cancer-associated fusion genes, or proteins derived thereof, in a sample obtained from a patient, wherein said presence of one or more cancer-associated fusion genes in the sample is indicative of cancer, or an increased risk of cancer, in said patient, wherein the cancer-associated fusion genes are selected from a group consisting of CLEC16A-EMP2 (SEQ ID NO.: 97, 99 or 101), SNX2-PRDM6 (SEQ ID NO.: 113 or 115), MLL3-PRKAG2 (SEQ ID NO.: 121, 123 or 125), DUS2L-PSKH1 (SEQ ID NO.: 131 or 133) and CLDN18-ARHGAP26 (SEQ ID NO: 107).
In one aspect, there is provided a method of determining if a patient has cancer or is at increased risk of developing cancer, wherein said method comprises detecting one or more cancer-associated fusion genes selected from the group consisting of CLEC16A-EMP2 (SEQ ID NO.: 97, 99 or 101), SNX2-PRDM6 (SEQ ID NO.: 113 or 115), MLL3-PRKAG2 (SEQ ID NO.: 121, 123 or 125) and DUS2L-PSKH1 (SEQ ID NO.: 131 or 133) in a sample obtained from a patient, or detecting one or more cancer-associated fusion genes selected from the group consisting of CLEC16A-EMP2 (SEQ ID NO.: 97, 99 or 101), SNX2-PRDM6 (SEQ ID NO.: 113 or 115), MLL3-PRKAG2 (SEQ ID NO.: 121, 123 or 125) and DUS2L-PSKH (SEQ ID NO.: 131 or 133) in combination with CLDN18-ARHGAP26 (SEQ ID NO: 107), wherein the presence of one or more cancer-associated fusion genes in the sample indicates that the patient has cancer or is at an increased risk of developing cancer.
In one aspect, there is provided a method of determining if a patient has cancer or is at increased risk of developing cancer, wherein said method comprises detecting one or more cancer-associated fusion genes selected from a group consisting of CLEC16A-EMP2 (SEQ ID NO.: 97, 99 or 101), SNX2-PRDM6 (SEQ ID NO.: 113 or 115), MLL3-PRKAG2 (SEQ ID NO.: 121, 123 or 125), DUS2L-PSKH1 (SEQ ID NO.: 131 or 133) and CLDN18-ARHGAP26 (SEQ ID NO: 107) in a sample obtained from a patient, wherein the presence of one or more cancer-associated fusion genes in the sample indicates that the patient has cancer or is at an increased risk of developing cancer.
In one aspect, there is provided an expression vector comprising a nucleic acid sequence encoding any one of CLEC16A-EMP2 (SEQ ID NO.: 97, 99 or 101), SNX2-PRDM6 (SEQ ID NO.: 113 or 115), MLL3-PRKAG2 (SEQ ID NO.: 121, 123 or 125), DUS2L-PSKH1 (SEQ ID NO.: 131 or 133) or CLDN18-ARHGAP26 (SEQ ID NO: 107).
In one aspect, there is provided a cell transformed with the expression vector as disclosed herein.
In one aspect, there is provided a method for producing a polypeptide, comprising culturing the transformed cell as disclosed herein under conditions suitable for polypeptide expression and collecting the amount of said polypeptide from the cell.
In one aspect, there is provided a use of a cancer-associated fusion gene in the determination or prognosis of cancer in a patient, wherein the presence of one or more cancer-associated fusion genes in a sample obtained from the patient indicates that the patient has cancer or is at an increased risk of developing cancer, wherein the cancer-associated fusion genes are selected from a group consisting of CLEC16A-EMP2 (SEQ ID NO.: 97, 99 or 101), SNX2-PRDM6 (SEQ ID NO.: 113 or 115), MLL3-PRKAG2 (SEQ ID NO.: 121, 123 or 125) and DUS2L-PSKH1 (SEQ ID NO.: 131 or 133), or wherein the cancer-associated fusion genes selected from the group consisting of CLEC16A-EMP2 (SEQ ID NO.: 97, 99 or 101), SNX2-PRDM6 (SEQ ID NO.: 113 or 115), MLL3-PRKAG2 (SEQ ID NO.: 121, 123 or 125) and DUS2L-PSKH1 (SEQ ID NO.: 131 or 133) in combination with CLDN18-ARHGAP26 (SEQ ID NO: 107).
In one aspect, there is provided a use of a cancer-associated fusion gene in determining if a patient has cancer or is at an increased risk of cancer, wherein the presence of one or more cancer-associated fusion genes is in a sample obtained from the patient indicates that the patient has cancer or is at an increased risk of developing cancer, wherein the cancer-associated fusion genes are selected from a group consisting of CLEC16A-EMP2 (SEQ ID NO.: 97, 99 or 101), SNX2-PRDM6 (SEQ ID NO.: 113 or 115), MLL3-PRKAG2 (SEQ ID NO.: 121, 123 or 125) and DUS2L-PSKH1 (SEQ ID NO.: 131 or 133), or wherein the cancer-associated fusion genes selected from the group consisting of CLEC16A-EMP2 (SEQ ID NO.: 97, 99 or 101), SNX2-PRDM6 (SEQ ID NO.: 113 or 115), MLL3-PRKAG2 (SEQ ID NO.: 121, 123 or 125) and DUS2L-PSKH1 (SEQ ID NO.: 131 or 133) in combination with CLDN18-ARHGAP26 (SEQ ID NO: 107).
In one aspect, there is provided a kit when used in the method as disclosed herein comprising:
The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:
The following words and terms used herein shall have the meaning indicated:
As used herein, the term “prognosis” or grammatical variants thereof refers to a prediction of the probable course and outcome of a clinical condition or disease. A prognosis of a patient is usually made by evaluating factors or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease. The term “prognosis” does not refer to the ability to predict the course or outcome of a condition with 100% accuracy. Instead, the term “prognosis” refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition. For example, the course or outcome of a condition may be predicted with 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55% and 50% accuracy.
An example of prognosis is testing a sample for the presence of a marker wherein the presence of the marker indicates a favourable or an unfavourable disease outcome. Another example of prognosis is testing a sample for the presence of a marker wherein the presence of the marker indicates that a patient is a candidate for a type of treatment.
As used herein, the term “differential treatment plan” refers to a tailored treatment plan specific to a patient or disease subtype. For example, presence of a cancer marker in a patient sample indicates that the patient is a candidate for a differential treatment plan, wherein the differential treatment plan is targeted cancer therapy.
The term “sample” or “biological sample” as used herein refers to a cell, tissue or fluid that has been obtained from, removed or isolated from the subject. An example of a sample is a tumour tissue biopsy. Samples may be frozen fresh tissue, paraffin embedded tissue or formalin fixed paraffin embedded (FFPE) tissue. Another example of a sample is a cell line. An example of fluid samples include but is not limited to blood, serum, saliva, urine, cerebrospinal fluid and bone marrow fluid.
The term “testing for the presence” in relation to a gene, fusion gene or protein product derived thereof refers to screening for the presence or absence of a gene, fusion gene or protein derived thereof in a sample. The term “testing for the presence” in relation to a gene, fusion gene or protein product derived thereof also refers to quantifying expression of the gene, fusion gene or protein product derived thereof in a sample. It will be understood that quantifying expression includes quantifying the absolute expression of the gene, fusion gene or protein product in a sample.
The term “fusion gene” as used herein refers to a hybrid gene formed from two or more separate genes. Full-length or fragments of the coding sequence, non-coding sequence or both may be fused. Fusion may occur by one or more of the processes of chromosomal rearrangement, including but not limited to chromosomal translocation, inversion, duplication or deletion. The two or more genes may be on the same chromosome, different chromosomes or a combination of both. The two or more fused genes may be fused in-frame or out of frame.
It will be understood that fusion genes may gain the functions of one of the original unfused genes, or lose the functions of one of the original unfused genes or both. It will also be understood that fusion genes may gain functions that are not present in any of the unfused genes. For illustration, a fusion gene that is fused from gene A and gene B may gain the function(s) of gene A only, and lose the function(s) of gene B. Alternatively, the fusion gene that is fused from gene A and gene B may gain functions not found in gene A or gene B.
It will therefore be understood that a cell with a fused gene may have properties not found in a cell without the fused gene.
As used herein, the term “cancer-associated fusion genes” refer to fusion genes that are associated with cancer. It will be understood that one or more fusion genes may be associated with a cancer. For example, the presence of one or more cancer-associated fusion genes in a patient sample may indicate that the subject has cancer or that the subject has an increased risk of cancer. The detection of one or more cancer-associated fusion genes in a patient sample may also indicate that the subject qualifies for a targeted cancer treatment plan. Examples of cancer-associated fusion genes include but are not limited to CLEC16A-EMP2, SNX2-PRDM6, MLL3-PRKAG2, DUS2L-PSKH1 and CLDN18-ARHGAP26. It will be understood that the fusion genes may be detected alone or in combination. Without being bound by theory, it is understood that the presence of a combination of more than one cancer-associated fusion genes is correlated with a poorer prognosis or disease outcome relative to the presence of a single cancer-associated fusion gene. As such, it will be understood that the presence of a combination of more than one cancer-associated fusion genes is predictive of disease outcome or prognosis. For example, the fusion genes may be selected from the group consisting of CLEC16A-EMP2, SNX2-PRDM6, MLL3-PRKAG2 and DUS2L-PSKH1 in combination with CLDN18-ARHGAP26. It will be understood that 0, 1, 2, 3, 4, 5 or more fusion genes may be detected in a sample. For example, CLEC16A-EMP2 may be detected in a sample, or CLEC16A-EMP2 in combination with CLDN18-ARHGAP26 may be detected in a sample. In one example, CLDN18-ARHGAP26 shows loss of CLDN18 function and gain of ARHGAP26 function.
It will be understood that variations may exist between nucleotide and amino acid sequences of fusion genes in different subject. These genetic variations may be due to mutation, polymorphism or splice variants. It will also be understood that genetic variations may result in a phenotypic change in a subject or sample or may have no change in phenotype.
Proteins derived from a fusion gene may be functional or non-functional. Proteins derived from a fusion gene may be elongated or truncated. As used herein, a “functional protein” refers to a polypeptide that has biological activity. It will be understood that the biological activity or property of a functional protein derived from a fusion gene may be the same as a functional protein derived from one of the original unfused genes. It will also be understood that the biological activity or property of a functional protein derived from a fusion gene may be different to the biological activity or property of the unfused gene.
As used herein, “truncated protein” refers to a protein or polypeptide that has a reduced number of amino acids than a full length, untruncated protein.
As used herein, “elongated protein” refers to a protein that has an increased number of amino acids than a full length, untruncated protein.
It will also be understood that a fusion gene may confer different a biological property to a cell. For example, a fusion gene may result in a cell having an enhanced migration rate, pro-metastatic feature or changes in cell shape. A fusion gene may also result in a cell losing its epithelial phenotype, having impaired epithelial barrier properties and impaired wound healing properties.
It will be understood to one of skill in the art that the presence of fusion genes may be detected by a variety of methods. Examples include but are not limited to polymerase chain reaction (PCR), quantitative PCR, microarray, RT-PCR, Southern blot, Northern blot, fluorescence in situ hybridization (FISH) and DNA sequencing. DNA sequencing includes but is not limited to DNA-Paired-end tags (DNA-PET) sequencing and Next-Generation sequencing, SOLiD™ sequencing.
It will also be understood to one of skill in the art that a variety of detection agents may be used to detect fusion genes. Examples of detection agents include but are not limited to primers, probes and complementary nucleic acid sequences that hybridise to the fusion gene.
The term “primer” is used herein to mean any single-stranded oligonucleotide sequence capable of being used as a primer in, for example, PCR technology. Thus, a “primer” according to the disclosure refers to a single-stranded oligonucleotide sequence that is capable of acting as a point of initiation for synthesis of a primer extension product that is substantially identical to the nucleic acid strand to be copied (for a forward primer) or substantially the reverse complement of the nucleic acid strand to be copied (for a reverse primer). A primer may be suitable for use in, for example, PCR technology.
The term “probe” as used herein refers to any nucleic acid fragment that hybridizes to a target sequence. A probe may be labeled with radioactive isotopes, fluorescent tags, antibodies or chemical labels to facilitate detection of the probe.
As used herein, “hybridise” means that the primer, probe or oligonucleotide forms a noncovalent interaction with the target nucleic acid molecule under standard stringency conditions. The hybridising primer or oligonucleotide may contain non-hybridising nucleotides that do not interfere with forming the noncovalent interaction, e.g., a 5′ tail or restriction enzyme recognition site to facilitate cloning.
Furthermore, as used herein, any “hybridisation” is performed under stringent conditions. The term “stringent conditions” means any hybridisation conditions which allow the primers to bind specifically to a nucleotide sequence within the allelic expansion, but not to any other nucleotide sequences. For example, specific hybridisation of a probe to a nucleic acid target region under “stringent” hybridisation conditions, include conditions such as 3×SSC, 0.1% SDS, at 50° C. It is within the ambit of the skilled person to vary the parameters of temperature, probe length and salt concentration such that specific hybridisation can be achieved. Hybridisation and wash conditions are well known in the art.
It will be understood to one of skill in the art that fusion proteins may be detected by a variety of methods. Examples of methods to detect fusion proteins include but are not limited to immunohistochemistry (IHC), immunofluorescence labelling, Western blot, ELISA and SDS-PAGE.
It will also be understood to one of skill in the art that there are a variety of detection agents to quantify fusion protein expression. Examples of detection agents include but are not limited to antibodies and ligands that specifically bind to the fusion protein.
As mentioned above, detection of one or more fusion genes in a sample obtained from a patient is indicative of cancer, or an increased risk of cancer.
As used herein, “increased risk of cancer” means that a subject has not been diagnosed to have cancer but has an increased probability of having cancer relative to a control or reference that does not have the one or more fusion genes.
The terms “reference”, “control” or “standard” as used herein refer to samples or subjects on which comparisons to determine prognosis be performed. Examples of a “reference”, “control” or “standard” include a non-cancerous sample obtained from the same subject, a sample obtained from a non-metastatic tumour, a sample obtained from a subject that does not have cancer or a sample obtained from a subject that has a different cancer subtype. The terms “reference”, “control” or “standard” as used herein may also refer to the average expression levels of a gene or protein in a patient cohort. The terms “reference”, “control” or “standard” as used herein may also refer to the presence or absence of a fusion gene or protein in a cell line or plurality of cell lines. The terms “reference”, “control” or “standard” as used herein may also refer to a subject who is not suffering from cancer or who is suffering from a different type of cancer. An example of a reference or control is a patient without any one or more of the cancer-associated fusion genes.
As used herein, “cancer” refers to an epithelial cancer. Examples of epithelial cancers include but are not limited to gastric cancer, lung cancer, breast cancer, urogenital cancer, colon cancer, prostate cancer and cervical cancer.
A fusion polypeptide may be obtained by inserting a fusion gene into an expression vector. As used herein, “expression vector” refers to a plasmid that is used to introduce a specific gene into a target cell. Expression vectors may be transient expression vectors or stable expression vectors.
It will be understood that a cell may be transformed with an expression vector. Methods for transforming a cell will be understood by one of skill in the art. For example, a cell may be transformed by electroporation, heat shock, chemical or viral transfection.
The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims and non-limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
Exemplary, non-limiting embodiments of a method of determining or making of a prognosis if a patient has cancer or is at an increased risk of having cancer will now be disclosed.
The method comprises testing for the presence of one or more cancer-associated fusion genes, or proteins derived thereof, in a sample obtained from a patient, wherein said presence of one or more cancer-associated fusion genes in the sample indicates that said patient has cancer, or is at an increased risk of cancer, wherein the cancer-associated fusion genes are selected from the group consisting of CLEC16A-EMP2, SNX2-PRDM6, MLL3-PRKAG2 and DUS2L-PSKH1, or wherein the cancer-associated fusion genes are selected from the group consisting of CLEC16A-EMP2, SNX2-PRDM6, MLL3-PRKAG2 and DUS2L-PSKH1 in combination with CLDN18-ARHGAP26.
In one embodiment, the cancer-associated fusion gene is CLEC16A-EMP2, SNX2-PRDM6, MLL3-PRKAG2, DUS2L-PSKH1 or CLDN18-ARHGAP26. In a preferred embodiment, the cancer-associated fusion gene is CLEC16A-EMP2. In one embodiment, 2, 3 or 4 of the fusion genes are selected from the group consisting of CLEC16A-EMP2, SNX2-PRDM6, MLL3-PRKAG2 and DUS2L-PSKH1 in combination with CLDN18-ARHGAP26.
In one embodiment, CLEC16A-EMP2 is in combination with CLDN18-ARHGAP26. In one embodiment, SNX2-PRDM6 is in combination with CLDN18-ARHGAP26. In one embodiment, MLL3-PRKAG2 is in combination with CLDN18-ARHGAP26. In one embodiment, DUS2L-PSKH1 is in combination with CLDN18-ARHGAP26. In a preferred embodiment, CLEC16A-EMP2 is in combination with CLDN18-ARHGAP26. In a preferred embodiment, MLL3-PRKAG2 is in combination with CLDN18-ARHGAP26.
The method disclosed herein is suitable for determining or making a prognosis of cancer. The cancer may be a carcinoma, a sarcoma, leukaemia, lymphoma, myeloma or a cancer of the central nervous system.
In one embodiment the cancer is an epithelial cancer or carcinoma. The epithelial cancer is preferably selected from the group consisting of skin cancer, lung cancer, gastric cancer, breast cancer, urogenital cancer, colon cancer, prostate cancer, cervical cancer, skin cancer, ovarian cancer, liver cancer and renal cancer. In a preferred embodiment, the cancer is gastric cancer.
The method as described herein is suitable for use in a sample of fresh tissue, frozen tissue, paraffin-preserved tissue and/or ethanol preserved tissue. The sample may be a biological sample. Non-limiting examples of biological samples include whole blood or a component thereof (e.g. plasma, serum), urine, saliva lymph, bile fluid, sputum, tears, cerebrospinal fluid, bronchioalveolar lavage fluid, synovial fluid, semen, ascitic tumour fluid, breast milk and pus. In one embodiment, the sample is obtained from blood, amniotic fluid or a buccal smear. In a preferred embodiment, the sample is a tissue biopsy.
A biological sample as contemplated herein includes tissue samples, cultured biological materials, including a sample derived from cultured cells, such as culture medium collected from cultured cells or a cell pellet. Accordingly, a biological sample may refer to a lysate, homogenate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof. A biological sample may also be modified prior to use, for example, by purification of one or more components, dilution, and/or centrifugation.
Well-known extraction and purification procedures are available for the isolation of nucleic acid from a sample. The nucleic acid may be used directly following extraction from the sample or, more preferably, after a polynucleotide amplification step (e.g. PCR). The amplified polynucleotide is ‘derived’ from the sample.
Preferably, the nucleic acid sequence is denatured prior to amplification. In one embodiment, the denaturation comprises heat treatment. Preferably, the heat treatment is carried out at a temperature in the range selected from the group consisting of from about 70-110° C.; about 75-105° C.; about 80-100° C. and about 85-95° C. Preferably, the denaturation step is carried out at 94° C.
In another embodiment, the denaturation step is carried out for a period selected from the group consisting of from about 1-30 minutes; about 2-25 minutes and about 3-10 minutes. Preferably, the denaturation step is carried out for 3 minutes.
In a preferred embodiment, the amplification step comprises a polymerase chain reaction (PCR). Preferably, the PCR comprises 15 cycles at 94° C. for 20 seconds, 58° C. for 30 seconds and 68° C. for 10 minutes, and 20 cycles of 94° C. for 20 seconds, 55° C. for 30 seconds and 68° C. for 10 minutes and a final extension step at 68° C. for 15 minutes.
The one or more further amplicons may be analysed by capillary electrophoresis, melt curve analysis, on a DNA chip or next generation sequencing.
The primers according to the disclosure may additionally comprise a detectable label, enabling the probe to be detected. Examples of labels that may be used include: fluorescent markers or reporter dyes, for example, 6-carboxyfluorescein (6FAM™), NED™ (Applera Corporation), HEX™ or VIC™ (Applied Biosystems); TAMRA™ markers (Applied Biosystems, Calif., USA); chemiluminescent markers, for example Ruthenium probes.
Alternatively the label may be selected from the group consisting of electroluminescent tags, magnetic tags, affinity or binding tags, nucleotide sequence tags, position specific tags, and or tags with specific physical properties such as different size, mass, gyration, ionic strength, dielectric properties, polarisation or impedance.
Well-known extraction and purification procedures are available for the isolation of protein from a sample. The protein may be used directly following extraction from the sample. Protein extraction may be by physical cell disruption or detergent based cell lysis. Extracted proteins may be analysed by Western blot, Coomasie stain, Bradford assay and BCA assay.
The method disclosed herein is suitable for determining if a patient is a candidate for a differential treatment plan. A differential treatment plan may comprise of one or more types of treatment selected from the group consisting of chemotherapy, immunotherapy, radiation therapy, targeted therapy and transplantation. A differential treatment plan may also include a combination of one or more therapies. A differential treatment plan may comprise one or more therapies applied simultaneously or sequentially. In a preferred embodiment, the differential therapy is targeted therapy. In another preferred embodiment, the differential therapy is targeted therapy in combination with chemotherapy. In one embodiment, the differential treatment plan is transtuzumab or ramucirumab. In another embodiment, the differential treatment plan is transtuzumab or ramucirumab in combination with chemotherapy.
The method disclosed herein is suitable for determining or making of a prognosis if a person is at risk of cancer. As previously described, a person at risk of cancer has an increased probability of having cancer relative to a control or reference that does not have the one or more fusion genes. In one embodiment, a person or patient has a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% increased risk of cancer.
The nucleotide sequence of the one or more fusion genes may be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected from the group consisting of CLEC16A-EMP2 (SEQ ID NO.: 97, 99 or 101), SNX2-PRDM6 (SEQ ID NO. 115), MLL3 PRKAG2 (SEQ ID NO.: 121, 123 or 125), DUS2L-PSKH1 (SEQ ID NO.: 131 or 133) and CLDN18-ARHGAP26 (SEQ ID NO: 107). In one example, the nucleotide sequence of CLEC16A-EMP2 is 70% identical to SEQ ID NO.: 97. In another example, the nucleotide sequence of CLDN18-ARHGAP26 is 95% identical to SEQ ID NO: 107. In yet another example, wherein the cancer-associated fusion gene is CLEC16A-EMP2 in combination with CLDN18-ARHGAP26, CLEC16A-EMP2 is 80% identical to SEQ ID NO. 97 and CLDN18-ARHGAP26 is 85% identical to SEQ ID NO. 107.
There is also provided an expression vector comprising the coding sequence of any of the fusion genes disclosed herein. In one embodiment, the expression vector is a mammalian expression vector. Suitable expression vectors include but are not limited to pMXs-Puro, pVSVG, pEGFP and pCMVmyc.
There is also provided a cell transformed with an expression vector as disclosed herein. Transformation may be by electroporation, heat shock, chemical or viral transfection. In one embodiment, the cell is transformed by chemical transfection. In another embodiment, the chemical transfection is by Lipofectamine 2000. In another embodiment, transformation is by viral transfection. In yet another embodiment, viral transfection is lentiviral or retroviral transfection.
There is also provided a method for producing a polypeptide, comprising culturing the transformed cell in Eagle's Minimum Essential Medium or Dulbecco's Modified Eagle's Medium or RPMI with 10% bovine serum, 2 mM Glutamine, 1% non essential amino acids and 1% penicillin/streptomycin in a humidified chamber at 5% CO2 and 37° C. for polypeptide expression and collecting the amount of said polypeptide from the cell. It is within the ambit of the skilled person to vary the parameters of the culture conditions to optimize production and extraction of the polypeptide.
Also disclosed is a use of a cancer-associated fusion gene in the determination or prognosis of cancer in a patient, wherein the presence of one or more cancer-associated fusion genes in a sample obtained from the patient indicates that the patient has cancer or is at an increased risk of developing cancer.
Non-limiting examples of the invention and comparative examples will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.
Materials and Methods
Clinical Tumor Samples
Patient samples and clinical information were obtained from patients who had undergone surgery for gastric cancer at the National University Hospital, Singapore, and Tan Tock Seng Hospital, Singapore. Informed consent was obtained from all subjects and the study was approved by the Institutional Review Board of the National University of Singapore (reference code 05-145) as well as the National Healthcare Group Domain Specific Review Board (reference code 2005/00440).
DNA/RNA Extraction from Samples
Genomic DNA and total RNA extraction from tissue samples was performed using Allprep DNA/RNA Mini Kit (Qiagen). Genomic DNA was extracted from blood samples with Blood & Cell Culture DNA kit (Qiagen).
Primers and Oligonucleotides
The primers and oligonucleotides used in this study are described in Table 1.
ATG
TTTGGCCGCTCGCGGAG
TCCTCAGTATCAG
ATG
GCCGTGACTGCCTGTCA
CACGTAATTCTCA
CC
CGATTGATTCTGG
ATG
ATTTTGAATAGCCTCTC
Antibodies and Reagents
Primary and secondary commercial antibodies and reagents are described in Table 2.
RT-PCR Screen for the Presence of a Fusion Gene
1 μg of total RNA is reverse transcribed to cDNA using the SuperScript III kit (Invitrogen) according to the manufacturer's recommendations. JumpStart RED AccuTaq LA DNA Polymerase kit (Sigma) was used with the following protocol:
Cycling conditions are as follows: 94° C. for 3 min, (94° C. for 20 seconds, 58° C. for 30 seconds, 68° C. for 10 min)×15 cycles, (94° C. for 20 seconds, 55° C. for 30 seconds, 68° C. for 10 min)×20 cycles, 68° C. for 15 min.
Cell Culture Conditions and Transfections
MDCK II, HeLa, HGC27 and TMK1 cell lines were cultured according to standard conditions. Transient and stable transfections experiments were carried using JetPrimePolyPlus transfection kit according to manufacturer's instructions. Stable transfectants were generated with G418 selection.
DNA-PET Libraries Construction, Sequencing, Mapping and Data Analysis
DNA-PET library construction of 10 kb fragments of genomic DNA, sequencing, mapping and data analysis were performed with refined bioinformatics filtering. The short reads were aligned to the NCBI human reference genome build 36.3 (hg18) using Bioscope (Life Technologies). DNA-PET data of TMK1 and tumors 17, 26, 28 and 38 have been previously described (NCBI Gene Expression Omnibus (GEO) accession no. GSE26954) and of tumors 82 and 92 (NCBI GEO accession number GSE30833). The SOLID sequencing data of the eight additional tumor/normal pairs can be accessed at NCBI's Sequence Read Archive (SRA) under BioProject ID PRJNA234469. Procedures for the identification of recurrent genomic breakpoints of CLDN18-ARHGAP26, filtering of germline structural variations (SV) in cancer genomes and breakpoint distribution analyses are described as follows.
For 10 of the 15 GC samples, paired normal samples were available and the respective DNA-PET data was used to filter germline SVs from the SVs which were identified in the tumors. For this, extended mapping coordinates of the clusters of discordant paired-end tag (dPET) sequences which defined the SVs were searched for overlap with dPET clusters of the paired normal sample. In addition, and in particular for the tumors without paired normal samples (tumors 17, 26, 28 and 38) and TMK1, all SVs of the paired normal samples and of 16 unrelated non-cancer individuals were used for filtering. Further, simulations were performed in which paired sequence tags in a distance distribution of a representative library were randomly selected from the reference sequence and were mapped and processed by the pipeline. Resulting dPET clusters represented mapping artifacts and were used for SV filtering. Further, dPET clusters were compared with SVs in the database of genomic variants (http://dgv.tcag.ca/dgv/app/home), paired-end sequencing studies of non-cancer individuals when the larger SV overlapped by ≧80% with SVs identified in cancer genomes. The data processing by the standard pipeline resulted in a large number of small deletions for the blood sample of patient 82 due to the abnormal insert size distribution and all the deletions smaller than 12 kb were removed.
MCF-7 RNA Polymerase II ChIA-PET and GC DNA-PET Comparison
To investigate whether the two partner sites of germline and somatic SV of the study were enriched for loci which are in proximity of each other in the nucleus, overlap of SVs were tested with genome-wide chromatin interaction data sets derived from ChIA-PET sequencing of the breast cancer cell line MCF-7 with the rationale that some chromatin interactions might be conserved across different cell types.
Driver Fusion Gene Prediction
The potential driver fusion genes were predicted by in silico analysis as previously described. The in silico analysis is a network fusion centrality approach in which the position of a gene product within transcript networks is used to predict its importance for the network to function. The threshold value 0.37 was set for identifying the potential fusion drivers.
In-Frame Fusion Gene Confirmation and Screening by RT-PCR
One microgram of total RNA was reverse-transcribed to cDNA using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) according to the manufacturer's instruction. PCR was done with JumpStart™ REDAccuTaq LA DNA Polymerase (Sigma-Aldrich Inc.).
GC Fusion Gene Constructs and Retroviral Transfections
The GC fusion genes CLEC16A-EMP2, CLDN18-ARHGAP26, SNX2-PRDM6 and DUS2L-PSKH1 were amplified from tumor samples by PCR using 2× Phusion Mastermix with HF buffer (Thermo Scientific) and the following primers.
Open reading frame of the CLEC16A-EMP2 fusion was constructed with the FLAG peptide of pMXs-Puro in frame using forward primer
(BamHI, kozak sequence and start codon follow by the first coding nucleotides of CLEC16A) and reverse primer 5′-
CTCGAG
TTTGCGCTTCCTCAGTATCAG-3′
(NotI, stop codon, HA-tag and XhoI followed by the 3′ end of the coding sequence of EMP2).
Similarly, open reading frame of the CLDN18-ARHGAP26 fusion was constructed with forward primer 5′ GGCGCGGATCCGCCGCCACCATGGCCGTGACTGCCTGTCA-3′ (SEQ ID NO.: 13) (BamHI, kozak, start, CLDN18) and reverse primer
TCGAG
GAGGAACTCCACGTAATTCTCA-3′
(NotI, stop, HA-tag, XhoI, ARHGAP26).
Open reading frame of the SNX2-PRDM6 fusion was constructed using forward primer 5′-GGCGCTTAATTAAGCCGCCACCATGGCGGCCGAGAGGGAACC-3′ (SEQ ID NO.: 15) (PacI, kozak, start, SNX2) and reverse
CTCGAG
ATCCACTTCGATTGATTCTGG-3′
(NotI, stop, HA-tag, XhoI PRDM6).
Open reading frame of the DUS2L-PSKH1 fusion was constructed using forward primer 5′-GGCGCGGATCCGCCGCCACCATGATTTTGAATAGCCTCTC-3′ (SEQ ID NO.: 17) (BamHI, kozak, start, DUS2L) and reverse primer
CTCGAGGCCATTGTATTGCTGCTGGTAG-3′
(NotI, stop, HA-tag, XhoI, PSKH1).
MLL3-PRKAG2 was synthesized with the FLAG peptide of pMXs-Puro by the gBlock method (Integrated DNA Technologies, Inc). The PCR products or MLL3-PRKAG2 were cloned into pMXs-Puro retroviral vector (Cell biolabs, RTV-012). The pMXs-Puro retroviral vectors containing the fusion genes were co-transfected with pVSVG (pseudotyping construct) into GP2-293 cells using lipofectamine 2000 to produce virus. Both HGC27 and HeLa cells were then infected with the viral supernatant containing empty vector or the fusion genes. Stable transfectants were obtained and maintained under selection pressure by puromycin dihydrochloride (Sigma, P9620).
Construction of CLDN18 and ARHGAP26 Plasmids
Human CLDN18 cDNA was obtained from IMAGE consortium (http://www.imageconsortium.org/) and cloned with an N-terminal HA-tag into pcDNA3 vector. The last three amino acids (DYV) of CLDN18 which encodes PDZ-binding motif was mutated to alanines and referred to as CLDN18ΔP. The human ARHGAP26 (GRAF1 isoform 2) cDNA in pEGFP vector and pCMVmyc were kindly provided by Dr Richard Lundmark (Medical Biochemistry and Biophysics, Umeå University, 901 87 Umeå, Sweden).
Details of the ARHGAP26 isoform is as follows:
Transcript: ARHGAP26-008 ENST00000378004 (http://www.ensembl.org) (SEQ ID NO.: 135)
followed in frame by HA-tag followed by stop codon. The human influenza hemagglutinin (HA)-tag has one of the following nucleotide sequences: 5′ TAC CCA TAC GAT GTT CCA GAT TAC GCT 3′ or 5′ TAT CCA TAT GAT GTT CCA GAT TAT GCT 3′. It will also be understood that the stop codon can be selected from any one of the following: TAG, TAA, or TGA.
Fusion Gene Recurrence Significance Test
The statistical significance of the observed frequency of fusion genes was assessed using a randomization framework. SV profiles were defined that mimic the type, number and size distributions of SVs identified in the samples sequenced by DNA-PET. The SVs of a 15 GCs test data set were simulated using the SV profiles and the frequency of recurrent SVs on a simulated validation set of 85 GC samples was assessed. Letting N=10,000 be the number of random simulations and es the frequency in the validation data set of an SV s present in the test data set, P values (es) were defined as p/N, where p is the number of simulations where a SV k exists with a frequency ek≧es.
Cell Aggregation, Cell Adhesion and Wound Healing Assays
For cell aggregation assay, 20 μl of 1.2×106/ml cells were plated on tissue culture dishes as hanging drops and phase contrast images were obtained the next day using Nikon Eclipse TE2000-S.
For cell adhesion assay, 24-well plates were either non-treated or treated with 1 mg/ml of fibronectin and 10 μg/ml of rat collagen type 1 for 2 hrs and blocked with 0.1% BSA. 2.5×104/ml of cells were seeded and incubated at 37° C. for 2 hrs.
In detail, 24-well plates were treated with 1 mg/ml of fibronectin and 10 μg/ml of rat collagen type 1 for 2 hrs. The plates were subsequently washed and non-specific binding was prevented by treating the surfaces with 0.1% bovine serum albumin (BSA) for 20 mins. The surfaces were again washed with PBS and 2.5×104/ml of cells were seeded and incubated at 37° C. for 2 hrs. Cells were also seeded on untreated 24-well as control. Cells were imaged with phase contrast microscopy. For quantification of cells adhered to the surfaces, the cells were gently washed with PBS three times and fixed in PFA and counted.
For wound healing assay, 70 ul of 7×105 cells/ml were plated on culture insert in μ-Dish 35 mm (Ibidi). The following day, the insert was peeled off to create a wound and migration was imaged with Nikon Eclispe TE2000 until closure of the wound.
Cell Proliferation Assay
800 cells were seeded in quadruplicates for each condition in 24-well plates and readings were taken according to manufacturer's instructions (Cell Proliferation Reagent WST-1: Roche) for 7 days. Absorbance was measured using Infinite M200 Quad4 Monochromator (Tecan) at 450 nm using a reference wavelength of 650 nm.
Cell Invasion Migration Assay
0.5 ml of 1×105 stably transfected HeLa and MDCK cells in RPMI serum free media were plated into the Biocoat Matrigel invasion chamber according to manufacturer's instructions (Corning) with 5% FBS in media added as chemoattractant to the wells of the Matrigel invasion chamber for 24 hr. Specifically, 0.5 ml of 1×105 HeLa and MDCK cells stably transfected with CLDN18, ARHGAP26 and CLDN18-ARHGAP26 in RPMI serum free media were plated into the Biocoat Matrigel invasion chamber according to manufacturer's instructions (Corning). 5% FBS in media was added as chemoattractant to the wells of the Matrigel invasion chamber for 24 hr. The following day, the cells were fixed for 10 min in 3.7% PFA and the insert was washed with PBS. 0.1% of crystal violet was added to the insert for 10 min and washed twice with water. A cotton swap was used to remove any non-invading cells and washed again. The number invading cells were imaged using Nikon Eclipse TE2000-S and counted.
Transepithelial Epithelial Resistance (TER) Analysis
2×105 stably transfected MDCK cells were seeded on 12 mm Transwell inserts (Corning) to obtain a polarized monolayer. The next day, the inserts were placed in CellZcope (nanoAnalytics) for TER measurements.
Soft Agar Colony Formation Assay
5000 cells of HeLa and HGC27 stable cell lines were added to 2 ml soft agar (0.35% Noble agar and 2×FBS media) and plated onto solidified base layers (0.7% Nobel agar with 2×FBS media) with triplicates set up for each experiment. 2-4 weeks later, colonies were counted.
Fusion Genes
5 fusion genes were used in this study as detailed in Table 3 below.
Details on the five recurrent fusion genes are mentioned below.
All genomic coordinates are based on the February 2009 human reference sequence (GRCh37 or hg19; http://genome.ucsc.edu/). Transcript IDs are based on Ensembl genome database (http://www.ensembl.org/). Shaded in yellow are the coding parts of the 5′ fusion partner genes as discovered in the initial screen and shaded in green are the 3′ fusion partner genes.
Fusion Gene #1: CLEC16A-EMP2
CLEC16A
Genomic PCR confirmed breakpoint—chr16: 11073471
RT-PCR confirmed RNA fusion point in exon 9—chr16: 11073239
EMP2
Genomic PCR confirmed breakpoint—chr16: 10666428
RT-PCR confirmed RNA fusion point in exon 2 (5′ UTR)—chr16: 10641534
Transcript: CLEC16A-001 ENST00000409790
Transcript: CLEC16A-001 ENST00000409790
Transcript: EMP2-001 ENST00000359543
Transcript: EMP2-001 ENST00000359543
Transcript: EMP2-001 ENST00000359543
CLEC16A—EMP2 Fusion sequence exon 9 to exon 2 UTR
Protein Domain
Domains within the query sequence of 506 residues
CLEC16A—EMP2 Fusion sequence exon 4 to exon 2 UTR
Protein Domain
Domains within the query sequence of 351 residues
CLEC16A—EMP2 Fusion sequence exon 10 to exon 2 UTR
Protein Domain
Domains within the query sequence of 544 residues
Fusion Gene #2: CLDN18-ARHGAP26
CLDN18
Genomic PCR confirmed breakpoint in the discovery sample—chr3:137,752,065
RT-PCR confirmed RNA fusion point in exon 5—chr3: 137,749,947
ARHGAP26
Genomic PCR confirmed breakpoint in the discovery sample—chr5:142318274
RT-PCR confirmed RNA fusion point in exon 12—chr5: 142393645
Transcript: CLDN18-001 ENST00000343735
Transcript: CLDN18-001 ENST00000343735
Transcript: ARHGAP26-001 ENST00000274498
Transcript: ARHGAP26-001 ENST00000274498
CLDN18-ARHGAP26 Fusion sequence
Protein Domain
Domains within the query sequence of 695 residues
Fusion Gene #3: SNX2-PRDM6
Confirmed genomic breakpoint for SNX2 on chr5:122162808 located in intron 12-13 of Transcript: SNX2-001 (ENST00000379516)
Confirmed genomic breakpoint for PRDM6 on chr5:122437347 located at intron 3-4 of Transcript: PRDM6-001 (ENST00000407847)
Transcript: SNX2-001 ENST00000379516
Transcript: SNX2-001 ENST00000379516
Transcript: PRDM6-001 ENST00000407847
Transcript: PRDM6-001 ENST00000407847
SNX2-PRDM6 Fusion sequence exon 12 to exon 4
Protein Domains
No transmembrane domains.
SNX2-PRDM6 Fusion sequence exon 2 to exon 7
Protein Domains
No transmembrane domains.
Fusion Gene #4: MLL3-PRKAG2
Confirmed genomic breakpoint for MLL3 on chr7:151365906 (reference Transcript: MLL3-001 (ENST00000262189))
confirmed genomic breakpoint for PRKAG2 on chr7:151951997 (reference Transcript: PRKAG2-001 (ENST00000287878))
Transcript: MLL3-001 ENST00000262189
Transcript: MLL3-001 ENST00000262189
Transcript: PRKAG2-001 ENST00000287878
Transcript: PRKAG2-001 ENST00000287878
MLL3-PRKAG2 Fusion sequence exon 9 to exon 5
Protein Domain Exon 9 to Exon 5
Due to overlapping domains, there are 4 representations of the protein. No transmembrane domains.
MLL3-PRKAG2 Fusion sequence exon 6 to exon 7
Protein Domain Exon 6 to Exon 7
No transmembrane domains within the query sequence of 566 residues.
MLL3-PRKAG2 Fusion sequence exon 23 to exon 6
Stop
Protein Domain Exon 23 to Exon 6
Due to overlapping domains, there are 40 representation of the protein. No transmembrane domains.
Fusion Gene #5: DUS2L-PSKH1
Confirmed genomic breakpoints: DUS2L—chr16:67930935, PSKH1—chr16:68103638
Transcript: DUS2L-001 ENST00000565263
Transcript: DUS2L-001 ENST00000565263
Transcript: DUS2L-001 ENST00000565263
Transcript: PSKH1-001 ENST00000291041
Transcript: PSKH1-001 ENST00000291041
Transcript: PSKH1-001 ENST00000291041
DUS2L-PSKH1 Fusion sequence exon 10 to exon 2 UTR
DUS2L-PSKH1 Fusion sequence exon 10 to exon 2 UTR
Protein Domain
No transmembrane domain.
DUS2L-PSKH1 Fusion sequence exon 3 to exon 2 UTR
Protein Domain
No domains.
Genomic positions of the mRNA fusion points for each of the fusion genes in this study are presented in Table 4.
Genomic DNA was sequenced from 14 primary gastric tumors including ten paired normal samples and gastric cancer cell line TMK1 by DNA-PET. With approximately 2-fold by coverage and 200-fold physical coverage of the genome, 1,945 somatic SVs were identified (
Both germline and somatic breakpoints were enriched in repeat regions (P<10−5
It was observed that 2% of validated fusion points have a characteristic pattern where the inserted sequence originated from a locus near the fusion point (
The possibility that the rearrangement partner sites of somatic SVs tend to be in spatial proximity within the nucleus was tested by searching for overlap between SVs and chromatin interaction analysis by paired-end-tag (ChIA-PET) sequencing data. As a proof of concept, cell line-derived (MCF-7 and K562) chromatin interactions and tumor derived somatic SVs for breast cancer and chronic myeloid leukemia (CML), respectively, were compared and significant overlap was observed.
To investigate whether the two partner sites of germline and somatic SVs of the study were enriched for loci which are in proximity of each other in the nucleus, overlap of SVs were tested with genome-wide chromatin interaction data sets derived from ChIA-PET sequencing of the breast cancer cell line MCF-7 with the rationale that some chromatin interactions might be conserved across different cell types. (
Since ChIA-PET data of a gastric cell line was not available, data from breast cancer cell line MCF-7 was used, with the assumption that some chromatin interactions are stable across different tissues. 1,667 germline and 1,945 somatic SVs of the 15 GCs were overlapped with 87,253 chromatin interactions of MCF-7 and 61 (3.7%) germline and 19 (1%) somatic SV overlaps were found, more than expected by chance (P<0.001, permutation based,
14 recurrent somatic SVs were identified with stringent search criteria and an additional 173 were identified with relaxed search criteria. Recurrent rearrangements clustered in seven hotspots with FHIT, WWOX, MACROD2, PARK2, and PDE4D at known fragile sites and NAALADL2 and CCSER1 (FAM190A), at new hotspots. All recurrently rearranged genes were of relevance for cancer. Interestingly, tumor 17 and TMK1 which had the highest number of somatic SVs in the seven rearrangement hotspots (12 and 11, respectively), also ranged among the GCs with the largest number of somatic SVs (
Using the somatic SVs of the 15 GCs, 136 fusion genes were predicted, 97 of them were validated by genomic PCR and Sanger sequencing, and the expression of 44 was confirmed by reverse transcription polymerase chain reaction (RT-PCR) in the respective tumours. Fifteen expressed fusion genes were in-frame. Since constitutively active oncogenic fusion genes are usually in-frame fusions, focus was placed on this category to screen an additional set of 85 GC tumor/normal pairs by RT-PCRs and found SNX2-PRDM6 in one additional tumor, CLDN18-ARHGAP26 and DUS2L-PSKH1 in two additional tumors, MLL3-PRKAG2 in three additional tumors, and CLEC16A-EMP2 in four additional tumors, giving overall frequencies of 2-5% (
It was found that they were not expected by chance (P=0.00472), with higher levels of significance for two rediscoveries (P=9.98×10−5) and three rediscoveries (P=1.11×10−5). This suggests that these fusion genes are not randomly created but most likely by targeted rearrangement mechanisms and/or that the resulting fusion genes provide selective advantages,
To explore if the fusion genes provided selective advantages, bioinformatics and cell biological approaches were used. In silico, a network fusion centrality analysis was used to predict driver fusion genes. Among the 136 fusion genes of this study, 38 were classified as potential driver fusion genes, including CLDN18-ARHGAP26, SNX2-PRDM6 and MLL3-PRKAG2 (Table 5). Since MLL3-PRKAG2 and DUS2L-PSKH1 in TMK1 were identified, short interfering RNA (siRNA) experiments specific for the fusion points of the MLL3-PRKAG2 and DUS2L-PSKH1 transcripts was performed. Reduced cell proliferation by 63% was observed when silencing MLL3-PRKAG2 (
To investigate the function of CLDN18-ARHGAP26, CLEC16A-EMP2 and SNX2-PRDM6 in GC, stable overexpression was created in GC cell line HGC27, and showed increased cell proliferation rates for CLDN18-ARHGAP26 (85% increase, P=4.2×10−6, T-test
The high proliferation rate by overexpression of CLDN18-ARHGAP26 suggested an oncogenic role for this fusion gene, and further investigation of its function was performed. CLDN18-ARHGAP26 encodes a 75.6 kDa fusion protein containing all four transmembrane domains of CLDN18 and the RhoGAP domain of ARHGAP26, but lacking the C-terminal PDZ-binding motif of CLDN18 (
In all three tumors with CLDN18-ARHGAP26 fusions, the transcripts were joined by a cryptic splice site within the coding region of exon 5 of CLDN18 and the regular splice site of exon 12 of ARHGAP26 (
For immunofluorescence in tumor specimens, CLDN18 and ARHGAP26 antibodies were used which both were able to detect the CLDN18-ARHGAP26 fusion protein (
To understand the contribution of the fusion protein to the observed changes in epithelial integrity in the tumor sample, CLDN18, ARHGAP26 or CLDN18-ARHGAP26 were stably expressed in non-transformed epithelial MDCK cells. Viewed by phase contrast, control and MDCK-CLDN18 cell cultures showed the characteristic epithelial morphology (
To evaluate if the phenotypic changes induced by CLDN18-ARHGAP26 reflected an EMT, the expression of various EMT markers was investigated using quantitative PCR (qPCR). While E-cadherin mRNA levels were unchanged in ARHGAP26 and CLDN18-ARHGAP26 expressing cells, mRNA of the master EMT regulators SNAI1 (Snail) and SNAI2 (Slug) were decreased (
Interestingly, expression of CLDN18, but not the fusion protein, down-regulated N-cadherin and β-catenin expression was observed in transformed HeLa cells (
ARHGAP26 likely affects adhesion of cells to the ECM through its interaction with FAK and its regulation of RhoA, which in turn regulates focal adhesions. Adhesion assays showed that control and MDCK-CLDN18 cells attached and spread on either untreated or ECM-coated surfaces. Not only did ARHGAP26 and, even more so, CLDN18-ARHGAP26 expressing cells attach less efficiently to the surfaces (
Mirroring the changes in protein levels, a significant decrease in levels of PINCH1 and Paxillin transcripts was observed in MDCK-ARHGAP26 and MDCK-CLDN18-ARHGAP26 cells by qPCR (
In addition to the cytoplasmic components of focal adhesions, protein levels of integrin family members, which directly interact with the ECM components were analysed. Consistent with the poor attachment of MDCK-CLDN18-ARHGAP26 cells on collagen coated surfaces (
Claudins are critical components of the paracellular epithelial barrier, including the protection of the gastric tissue from the acidic milieu in the lumen. Alterations of this barrier function might cause chronic inflammation, a risk factor for the development of GC. Therefore, the role of CLDN18 and the fusion protein in barrier formation was investigated. Overexpression of CLDN18, which is not endogenously expressed in MDCK cells, resulted in a dramatic increase in the transepithelial electrical resistance (TER) of MDCK-CLDN18 monolayers. While ARHGAP26 had no significant effect on the TER, CLDN18-ARHGAP26 completely abolished the TER (
In GC cell line HGC27, CLDN18-ARHGAP26 induces a gain of proliferation (
RhoA regulates many actin events like actin polymerization, contraction and stress fiber formation upon growth factor receptor or integrin binding to their respective ligands. ARHGAP26 stimulates, via its GAP domain, the GTPase activities of CDC42 and RhoA, resulting in their inactivation. Since the CLDN18-ARHGAP26 fusion protein retains the GAP domain of ARHGAP26, it may still be able to inactivate RhoA. To test this, the effect of CLDN18-ARHGAP26 expression on stress fiber formation and the presence and subcellular localization of active RhoA (e.g. GTP-bound RhoA) were analysed. In HeLa cells, stable overexpression of ARHGAP26 or CLDN18-ARHGAP26 induced cytoskeletal changes, notably a reduction in stress fibers indicative of RhoA inactivation (
Changes in endocytosis can affect cell surface residence time and/or degradation of cell-ECM and cell-cell adhesion proteins as well as receptor tyrosine kinases (RTKs), thereby altering cell adhesion, migration and RTK signaling, which can drive carcinogenesis. In contrast to the other cell lines, HeLa cells expressing the CLDN18-ARHGAP26 fusion protein showed a significant reduction of endocytosis (
The fusion transcripts between DUS2L and PSKH1 were identified in the cancer cell line TMK1 and subsequently in two primary gastric tumors. However, in one tumor, the exon 3 of DUS2L was fused to the exon 2 (UTR region) of PSKH1 resulting in an out of frame fusion transcript (
Unpaired inversions created the fusion gene CLEC16A-EMP2 which were identified in five out of 100 GCs. Of CLEC16A, exon 4 (one tumor), exon 9 (two tumors) or exon 10 (two tumors) were fused to exon 2 of EMP2 (
SNX2-PRDM6 was found to be fused in frame in one gastric tumor (exon 12 of SNX2 fused to exon 4 of PRDM6) and out of frame in a second tumor (exon 2 of SNX2 fused to exon 7 of PRDM6,
ARHGAP26 is reported to be indispensable for clathrin independent endocytosis and many receptor tyrosine kinases (RTKs) can be internalized by both clathrin dependent and independent pathways. In order to evaluate the effect of the CLDN18-ARHGAP26 fusion protein on clathrin-independent endocytosis, fluorescein isothiocyanate (FITC) conjugated CTxB, a marker for clathrin-independent endocytosis, was incubated with live control HeLa cells or cells stably expressing CLDN18, ARHGAP26 or CLDN18-ARHAGP26 for 15 minutes. Cells were then fixed and internalized FITC-CTxB visualized by fluorescence microscopy. In contrast to the other cell lines, HeLa cells expressing the CLDN18-ARHGAP26 fusion protein showed a significant reduction in the amount of CTxB endocytosed (
Recurrent somatic SVs and recurrent fusion genes were observed in this study. The simulations show that the rate of recurrent fusion genes could not be explained by chance indicating that specific rearrangements are more likely to occur than others and/or that selective processes enrich for such rearrangements. By comparing the somatic SVs with a genome-wide view of chromatin interactions, significantly more overlaps of rearrangement sites with chromatin interactions were observed than expected by chance, suggesting that the chromatin structure contributes to recurrent fusions of distant loci in GC.
This is the first systematic correlation analysis between somatic SVs in cancer and chromatin interactions. Since the chromatin structure was profiled in a different cell type than GC, the actual rate of overlap between chromatin interactions and rearrangements may have been underestimated.
The validity, expression and reading frame characteristics of 136 fusion genes were evaluated, and five recurrent fusion genes were identified by an extended screen. CLDN18-ARHGAP26 was analysed in detail and functional properties promoting both, early cancer development and late disease progression were found. CLDN18 and ARHGAP26 are expressed in the gastric mucosa epithelium, where CLDN18 localizes to tight junctions (TJs) and ARHGAP26 to punctate tubular vesicular structures of epithelial cells. The CLDN18-ARHGAP26 fusion gene thus links functional protein domains of a regulator of RhoA to a TJ protein resulting in altered properties. These, as well as the aberrant localization of the GAP activity, result in changes to cellular functions that are associated with GC.
While CLDN18-ARHGAP26 was associated with increased proliferation, anchorage dependent growth and invasion in tumorigenic HeLa and HGC27 cells, such cellular processes were reduced (proliferation, wound closure) in non-transformed MDCK cells, suggesting that the degree of transformation influences some of the effects of the fusion protein, consistent with the multi-step model of carcinogenesis. In the relevant GC in situ as well as when over-expressed in MDCK cells, CLDN18-ARHGAP26 was linked to a loss of the epithelial phenotype.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10201400876T | Mar 2014 | SG | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/SG2015/050047 | 3/23/2015 | WO | 00 |
