CALCULATED INDEX OF GENOMIC EXPRESSION OF ESTROGEN RECEPTOR (ER) AND ER-RELATED GENES

Abstract

The present invention provides the identification and combination of genes that are expressed in tumors that are responsive to a given therapeutic agent and whose combined expression can be used as an index that correlates with responsiveness to that therapeutic agent. One or more of the genes of the present invention may be used as markers (or surrogate markers) to identify tumors that are likely to be successfully treated by that agent or class of agents such as hormonal or endocrine treatment.

Description

I. FIELD OF THE INVENTION

The present invention relates to the fields of medicine and molecular biology, particularly transcriptional profiling, molecular arrays and predictive tools for response to cancer treatment.

II. BACKGROUND

Endocrine treatments of breast cancer target the activity of estrogen receptor alpha (ER, gene name ESR1). The current challenges for treatment of patients with ER-positive breast cancer include the ability to predict benefit from endocrine (hormonal) therapy and/or chemotherapy, to select among endocrine agents, and to define the duration and sequence of endocrine treatments. These challenges are each conceptually related to the state of ER activity in a patient's breast cancer. Since ER acts principally at the level of transcriptional control, a genomic index to measure downstream ER-associated gene expression activity in a patient's tumor sample can help quantify ER pathway activity, and thus dependence on estrogen, and intrinsic sensitivity to endocrine therapy. Treatment-specific predictors can enable available multiplex genomic technology to provide a way to specifically address a distinct clinical decision or treatment choice.

SUMMARY OF THE INVENTION

Embodiments of the invention include methods of calculating an index, e.g., an estrogen receptor (ER) reporter index or a sensitivity to endocrine treatment (SET) index, for assessing the hormonal sensitivity of a tumor comprising one or more of the steps of: (a) obtaining gene expression data from samples obtained from a plurality of patients; (b) calculating one or more reference gene expression profiles from a plurality of patients with a specific diagnosis, e.g., cancer diagnosis; (c) normalizing the expression data of additional samples to the reference gene expression profile; (d) measuring and reporting estrogen receptor (ER) gene expression from the profile as a method for defining ER status of a cancer; (e) identifying the genes to define a profile to measure ER-related transcriptional activity in any cancer sample; (f) defining one or more reference ER-related gene expression profiles; (g) calculating a weighted index or index (e.g., a SET index) based on ER-related gene expression in any patient sample(s) and the ER-related reference profile; and/or (h) combining the measurements of ER gene expression and the index (e.g., weighted index or SET index) for ER-related gene expression to measure and report the gene expression of ER and ER-related transcriptional profile as a continuous or categorical result. In certain aspects assessing the likely sensitivity of any cancer to treatment by measuring ER and ER-related gene expression singly or as a combined result. In certain embodiments, the cancer is suspected of being a hormone-sensitive cancer, preferably an estrogen-sensitive cancer. In certain aspects, the suspected estrogen-sensitive cancer is breast cancer. The ER-related genes may include one or more genes selected from two-hundred ER related genes or gene probes. In certain aspects of the invention, ER related genes or gene probes include 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 ER related genes or gene probes. In particular embodiments one or more genes are selected from Table 1 or Table 2. The weighted or calculated index may be based on similarity with the reference ER-related gene expression profile(s). In a further aspect of the invention similarity is calculated based on: (a) an algorithm to calculate a distance metric, such as one or a combination of Euclidian, Mahalanobis, or general Miknowski norms; and/or (b) calculation of a correlation coefficient for the sample based on expression levels or ranks of expression levels. The calculation of the weighted or reporter index may include various parameters (e.g., patient covariates) related to the disease condition including, but not limited to the parameters or characteristics of tumor size, nodal status, grade, age, and/or evaluation of prognosis based on distant relapse-free survival (DRFS) or overall survival (OS) of patients.

Embodiments of the invention include patients that are ER-positive and receiving hormonal therapy. In certain aspects the hormonal therapy includes, but is not limited to tamoxifen therapy and may include other known hormonal therapies used to treat cancers, particularly breast cancer. The treatment administered is typically a hormonal therapy, chemotherapy or a combination of the two. Additional aspects of the invention include evaluation of risk stratification of noncancerous cells and may be used to mitigate or prevent future disease. Still further aspects of the invention include normalization by a single digital standard. The method may further comprise normalizing expression data of the one or more samples to the ER-related gene expression profile. The expression data can be normalized to a digital standard. The digital standard can be a gene expression profile from a reference sample.

Further embodiments of the invention include methods of assessing patient sensitivity to treatment comprising one or more steps of: (a) determining expression levels of the ER gene and/or one or more additional ER-related genes; (b) calculating the value of the ER reporter index (e.g., a SET index); (c) assessing or predicting the response to hormonal therapy based on the value of the index; (d) assessing or predicting the response to an administered treatment (e.g., chemotherapy) based on the value of the index, and/or (e) selecting a treatment(s) for a patient based on consideration of the predicted responsiveness to hormonal therapy and/or chemotherapy.

In yet still further embodiments of the invention include a calculated index for predicting response (e.g., a response to treatment) produced by the method comprising the steps of: (a) obtaining gene expression data from samples obtained from a plurality of cancer patients; (b) normalizing the gene expression data; and (c) calculating an index (e.g., a weighted or SET index) based on the ER gene and one or more additional ER-related gene expression levels in the patient sample. In certain aspects the ER-related genes are selected as described supra. Parameters (e.g., patient covariates) used in conjunction with the calculation of the index includes, but is not limited to tumor size, nodal status, grade, age, evaluation of distant relapse-free survival (DRFS) or of overall survival (OS) of the patients and various combinations thereof. Typically, the patients are ER-positive and receiving hormonal therapy, preferably tamoxifen therapy. The methods of the invention may also include treatment administered as a combination of one or more cancer drugs. In particular aspects, the treatment administered is a hormonal therapy, a chemotherapy, or a combination of hormonal therapy and chemotherapy.

In yet still further embodiments of the invention include a calculated index for predicting response to therapy for late-stage (recurrent) cancer as performed by the method comprising the steps of: (a) obtaining gene expression data from samples obtained from a plurality of stage IV cancer patients; (b) normalizing the expression data; (c) calculating an index based on the ER gene and/or one or more additional ER-related gene expression levels in the patient sample; and (d) predicting response to therapy. Typically, the patients are ER-positive and have previously received, or are currently receiving hormonal therapy. The methods of the invention may also include treatment administered as a combination of one or more cancer drugs. In particular aspects, the treatment administered is a hormonal therapy, a chemotherapy, or a combination of hormonal therapy and chemotherapy.

Other embodiments of the invention include methods of assessing, e.g., assessing quantitatively, the estrogen receptor (ER) status of a cancer sample by measuring transcriptional activity comprising two or more of the steps of: (a) obtaining a sample of cancerous tissue from a patient; (b) determining mRNA gene expression levels of the ER gene in the sample; (c) establishing a cut-off ER mRNA value from the distribution of ER transcripts in a plurality of cancer samples, and/or (d) assessing ER status based on the mRNA level of the ER gene in the sample relative to the pre-determined cut-off level of mRNA transcript. The sample may be a biopsy sample, a surgically excised sample, a sample of bodily fluids, a fine needle aspiration biopsy, core needle biopsy, tissue sample, or exfoliative cytology sample. In certain aspects, the patient is a cancer patient, a patient suspected of having hormone-sensitive cancer, a patient suspected of having an estrogen or progesterone sensitive cancer, and/or a patient having or suspected of having breast cancer. In further aspects of the invention, the expression levels of the genes are determined by hybridization, nucleic amplification, or array hybridization, such as nucleic acid array hybridization. In certain aspects the nucleic acid array is a microarray. In still further embodiments, nucleic acid amplification is by polymerase chain reaction (PCR).

Embodiments of the invention may also include kits for the determination of ER status of cancer comprising: (a) reagents for determining expression levels of the ER gene and/or one or more additional ER-related genes in a sample; and/or (b) algorithm and software encoding the algorithm for calculating an ER reporter index from expression of ER and ER-related genes in a sample to determine the sensitivity of a patient to hormonal therapy.

Other embodiments of the invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the invention applies to other aspects of the invention as well and vice versa. The embodiments in the Example section are understood to be embodiments of the invention that are applicable to all aspects of the invention.

The terms “inhibiting,” “reducing,” or “prevention,” or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.

The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

Throughout this application, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

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

FIG. 1. Selection probabilities P_g(50), P_g(100), P_g(200) for the 200 top-ranking probe sets in terms of their Spearman's rank correlation with the ESR1 transcript (probe set 205225_at) plotted as a function of the probe set's rank in the original dataset. Probabilities were estimated from 1000 bootstrap samples of the original dataset.

FIG. 2. Distribution of ranks of the top 200 genes estimated from 1000 bootstrap replications of the original dataset as a function of the magnitude of the Spearman's rank correlation with the ESR1 transcript.

FIGS. 3A-3D. Distribution of the index of expression of the 200 ER-related genes by ER status for (FIG. 3A) 277 tamoxifen-treated patients and (FIG. 3B) 286 node-negative untreated patients. (FIGS. 3C and 3D) Dependence of ER gene expression index on ESR1 mRNA expression for patient populations corresponding to panels (FIG. 3A) and (FIG. 3B).

FIG. 4. Replicate measurements of ESR1 expression, PGR expression, ER reporter index and sensitivity to endocrine treatment (SET) index in 35 sample pairs of experimental replicates using residual RNA. Also shown is the 45° line through the origin. FIG. 4A (ESR1), FIG. 4B (PGR), FIG. 4C (ER Reporter Index), and FIG. 4D (SET Index).

FIGS. 5A-5C. Predicted marginal risk of distant relapse at 10 years in ER-positive breast cancer patients treated with adjuvant tamoxifen as a continuous function of genomic covariates: (FIG. 5A) ESR1 (ER) expression level, (FIG. 5B) log-transformed PGR expression level, and (FIG. 5C) genomic sensitivity to endocrine therapy (SET) index. The dashed lines show the 95% confidence interval of the predicted risk rates.

FIGS. 6A-6D. Kaplan-Meier estimates of relapse-free survival in ER-positive patients treated with adjuvant tamoxifen (FIG. 6A, FIG. 6C) or in patients not receiving systemic therapy after surgery (FIG. 6B, FIG. 6D). Groups were defined by the SET index (FIG. 6A, FIG. 6B) or the median-dichotomized log-transformed PGR expression (FIG. 6C, FIG. 6D). P-values are from the log-rank test.

FIGS. 7A-7B. Kaplan-Meier estimates of relapse-free survival in ER-positive patients treated with adjuvant tamoxifen grouped by nodal status: (FIG. 7A) node-negative group; (FIG. 7B) node-positive group. P-values are from the log-rank test.

FIG. 8A-8D. Box plots demonstrate genomic measurements in 351 ER-positive samples categorized by AJCC Stage (58 stage I, 123 stage IIA, 107 stage IIB, 44 stage III, and 18 stage IV). Each box indicates the median and interquartile range, and the whisker lines extend 1.5× the interquartile range above the 75th percentile and below the 25th percentile. FIG. 8A=SET index; FIG. 8B=ESR1; FIG. 8C=Log PGR; FIG. 8D=GAPDH.

DETAILED DESCRIPTION OF THE INVENTION

It has already been established that the overall transcriptional profile in breast cancers is dependent on ER status, being largely determined in ER-positive breast cancer by the genomic activity of ER on the transcription of numerous genes (Perou et al., 2000; van't Veer et al., 2002; Gruvberger et al., 2001; Pusztai et al., 2003). The inventors contemplate that the amount of ER-associated reporter gene expression is an indicator of ER transcriptional activity, likely dependence on ER activity, and sensitivity to hormonal therapy. Differences in expression of ER mRNA (the receptor) and ER reporter genes (the transcriptional output) might contribute to variable response of patients with ER-positive breast cancers to hormonal therapy (Buzdar, 2001; Howell and Dowsett, 2004; Hess et al, 2003). Herein, a set of genes are defined that are co-expressed with ER from an independent public database of Affymetrix U133A gene profiles from 286 lymph node-negative breast cancers and calculated an index score for their expression (Wang et al., 2005). Another goal was to determine whether the expression level of ESR1 gene, and value of this index for expression of ER reporter (associated) genes, is associated with distant relapse-free survival (DRFS) in other patients following adjuvant hormonal therapy with tamoxifen.

There are four main approaches to improving the ability to predict responsiveness to endocrine therapies. One approach is a standard predictive or chemopredictive study focused on treatment, in which a sufficiently powered discovery population of subjects is used to define a predictive test that must then be proven to be accurate in a similarly sized validation population (Ransohoff, 2005; Ransohoff 2004). Several studies have used this approach to define predictive genes for adjuvant tamoxifen therapy (Ma et al., 2004; Jansen et al., 2005; Loi et al., 2005). There are advantages to this approach, particularly when samples are available from mature studies for retrospective analysis. But two disadvantages are that the study design is empirical and that adjuvant treatment introduces surgery as a confounding variable, because it is impossible to ever know which patients were cured by their surgery and would never relapse, irrespective of their sensitivity to systemic therapy. Neoadjuvant chemotherapy trials enable a direct comparison of tumor characteristics with pathologic response (Ayers et al., 2004). While an empirical study design is needed for chemopredictive studies of cytotoxic chemotherapy regimens because multiple cellular pathways are likely to be disrupted, endocrine therapy of breast cancer specifically targets ER-mediated tumor growth and survival. The compositions and methods of the present invention may define and measure this ER-mediated effect supplanting the need for a limited empirical study design.

A second approach is to identify genes that are downregulated in vivo after treatment with an endocrine agent. This involves a small sample size of patients who undergo repeat biopsies, but is complicated by the selection of agent and dose used, variable timing of downregulation of different genes after therapy, and variable treatment effect in different tumors.

A third approach is to quantify receptor expression as accurately as possible. Semiquantitative scoring of ER immunoflourescent/immunohistochemical (IFIC) staining is related to disease-free survival following adjuvant tamoxifen (Harvey et al., 1999). For example, measurement of 16 selected genes (mostly related to ER, proliferation, and HER-2) using RT-PCR in a central reference laboratory predicts survival of women with tamoxifen-treated node-negative breast cancer (Paik et al., 2004). In a recent report, measurement of ER mRNA using RT-PCR diagnoses ER IHC status with 93% overall accuracy (Esteva et al., 2005). It was also recently reported that ER mRNA measurements from the same RT-PCR assay predict survival after adjuvant tamoxifen (Paik et al., 2005). So, if gene expression microarrays can reliably measure ER mRNA in a way that can be standardized in different laboratories, those measurements should predict response to endocrine treatment. Certain aspects of the invention described herein demonstrate that measurements of ER mRNA expression levels from microarrays also predict distant relapse-free survival following adjuvant tamoxifen therapy (Tables 4 and 5, and FIG. 6). However, other gene expression measurements from the microarray are informative as well.

A fourth approach, selected by the inventors, measures ER gene expression and the transcriptional output from ER activity, taking advantage of the high-throughput microarray platform. This approach theoretically applies to all endocrine treatments and does not require the empirical discovery and validation study populations. If a continuous scale of endocrine responsiveness exists, then specific endocrine treatments could be matched to likely response. Some patients would have an excellent response from tamoxifen, but others may need more potent endocrine treatment to respond to the same extent. A challenge with this approach is to accurately define the number and correct ER reporter genes to measure. The approach was to define ER reporter genes from a large, independent data set of 286 breast cancer profiles from Affymetrix U133A arrays. It is not necessary that these patients receive endocrine treatment, or to know their immunohistochemical ER status or survival, in order to define the genes most correlated with ER gene expression. Even with the relatively large sample size of 286 cases, the inventors calculated that 200 genes should be included as reporter genes in order to contain the 50 most ER-related genes with 98.5% confidence and the 100 most related genes with about 90% confidence (FIG. 1). This demonstrates the importance of a sufficiently large reporter gene set to capture a reliable transcriptional signature for ER activity in breast cancers (Perou et al., 2000; Van't Veer et al., 2002; Gruvberger et al., 2001; Pusztai et al., 2003).

If quantitative measurements of the ER-related expression, expression of ER mRNA, and/or ER activity (represented by a calculated index of ER reporter gene expression) accurately predict benefit from hormonal therapy, it is possible to develop a continuous genomic scale of measurement for ER expression and activity. This scale could be used to identify subsets of patients with ER-positive breast cancer that: (1) are expected to benefit from tamoxifen alone, (2) require more potent endocrine therapy, (3) may require chemotherapy along with endocrine therapy, or (4) are unlikely to benefit from any endocrine therapy.

To assess expression of at least 5, 25, 50, 100 or 200 reporter (ER-related) genes in a sample, the inventors first developed a gene-expression-based ER associated index. ER-positive and ER-negative reference signatures, or centroids, were then described as the median log-transformed expression value of each of the 200 reporter genes in the 209 ER-positive and 77 ER-negative subjects, respectively. For new samples, the similarity between the log-transformed 200-gene ER associated gene expression signature with the reference centroids was determined based on Hoeffding's D statistic (Hollander and Wolfe, 1999). D takes into account the joint rankings of the two variables and thus provides a robust measure of association that, unlike correlation-based statistics, will detect nonmonotonic associations (in statistical terms, it detects a much broader class of alternatives to independence than correlation-based statistics). The ER reporter index (RI) was defined as the difference between the similarities with the ER+ and ER− reference centroids: RI=D⁺−D⁻.

The 200-gene signature of a tumor with high ER-dependent transcriptional activity will resemble more closely the ER-positive centroid and therefore D⁺ will be greater than D⁻ and RI will be positive. The opposite will be the case for tumors with low ER-related activity and thus RI will be small or negative. Subtraction of D⁻ normalizes the reporter index relative to the basal levels of expression of the ER-related genes in ER negative tumors. Because of this and since D is a distribution-free statistic, RI is relatively insensitive to the method used to normalize the microarray data and therefore can be computed across datasets. From the RI, a genomic index of sensitivity to endocrine therapy (SET) was calculated as follows: SET=100(RI+0.2)³. The offset translated RI to mostly positive values and was then transformed to normality using an unconditional Box-Cox power transformation. Finally, the maximum likelihood estimate of the exponent was rounded to the closest integer and the index was scaled to a maximum value of 10.

Embodiments of the present invention also provide a clinically relevant measurement of estrogen receptor (ER) activity within cells by accurately quantifying the transcriptional output due to estrogen receptor activity. This measure or index of the ER pathway or ER activity is an index or measure of the dependence on this growth pathway, and therefore, likely susceptibility to an anti-estrogen receptor hormonal therapy. There are a growing number of hormonal therapies that are used for patients with cancer or to protect from cancer and that vary in their efficacy, cost, and side effects. Aspects of the invention will assist doctors to make improved recommendations about whether and how long to use hormonal therapy for patients with breast cancer or ER-positive breast cancer, particularly those with ER-positive status as established by the existing immunochemical assay, and which hormonal therapy to prescribe for a patient based on the amount of ER-related transcriptional activity measured from a patient's biopsy that indicates the likely sensitivity to hormonal therapy and so matches the treatment selected to the predicted sensitivity to treatment.

Embodiments of the invention are pathway-specific, are applicable to any sample cohort, and are not dependent on inherent biostatistical bias that can limit the accuracy of predictive profiles derived empirically from discovery and validation trial designs linking genes to observed clinical or pathological responses. One advantage of the assay, in addition to its ability to link genomic activity to clinical or pathological response, is that it is quantitative, accurate, and directly comparable using results from different laboratories.

In one aspect of the invention, a calculated index is used to measure the expression of many genes that represent activity of the estrogen receptor pathway within the cells that provides independently predictive information about likely response to hormonal therapy, and that improves the response prediction otherwise obtained by measuring expression of the estrogen receptor alone. The invention includes the methods for standardizing the expression values of future samples to a normalization standard that will allow direct comparison of the results to past samples, such as from a clinical trial. The invention also includes the biostatistical methods to calculate and report the results.

In certain aspects of the invention, measurements of ER and ER-related genes from microarrays have demonstrated to be comparable in standardized datasets from two different laboratories that analyzed two different types of clinical samples (fine needle aspiration cytology samples and surgical tissue samples) and that these accurately diagnose ER status as defined by existing immunochemical assays. In further aspects of the invention, measurements of ER and ER-related genes using this technique have been demonstrated to independently predict distant relapse-free survival in patients who were treated with local therapy (surgery/radiation) followed by post-operative hormonal therapy with tamoxifen. In still further aspects, these gene expression measurements were demonstrated to outperform existing measurements of ER for prediction of survival with this hormonal therapy. In yet still further aspects, measurement of ER-related genes were demonstrated to add to the predictive accuracy of measurements of ER gene expression in the survival analysis of tamoxifen-treated women.

Further embodiments of the invention include kits for the measurement, analysis, and reporting of ER expression and transcriptional output. A kit may include, but is not limited to microarray, quantitative RT-PCR, or other genomic platform reagents and materials, as well as hardware and/or software for performing at least a portion of the methods described. For example, custom microarrays or analysis methods for existing microarrays are contemplated. Also, methods of the invention include methods of accessing and using a reporting system that compares a single result to a scale of clinical trial results. In yet still further aspects of the invention, a digital standard for data normalization is contemplated so that the assay result values from future samples would be able to be directly compared with the assay value results from past samples, such as from specific clinical trials.

The clinical relevance for measurements of ER mRNA and ER related genes from microarrays is also demonstrated herein. Some exemplary advantages to the current composition and methods include, but are not limited to: (1) standardized, quantitative reporting of ER mRNA expression that is comparable in different sample types and laboratories, (2) use of different methods for defining genomic profiles to predict response to adjuvant endocrine treatments, and (3) combining ER-related reporter genes expression to develop a measurable scale or index of estrogen dependence and likely sensitivity to endocrine therapy.

The performance of certain embodiments of a microarray-based ER determination is presented in relation to the current immunohistochemical “gold” standard for evaluation of ER. It is important to remember that IHC assays for ER in routine clinical use are imperfect. The existing IHC assay for ER has only modest positive predictive value (30-60%) for response to various single agent hormonal therapies (Bonneterre et al, 2000; Mouridsen et al, 2001). There are also occasional false negative results. Much of the recognized inter-laboratory differences that affect the IHC results for ER are caused in part by problems associated with tissue fixation methods and antigen retrieval in paraffin tissue sections (Rhodes et al., 2000; Rudiger et al., 2002; Rhodes, 2003; Taylor et al., 1994; Regitnig et al., 2002). Finally, IHC is at least a qualitative assay (reported as positive or negative) and at most a semiquantitative assay (reported as a score). There is still a need to further improve the accuracy with which pathologic assays for ER can predict response to endocrine therapies.

The microarrays provide a suitable method to measure ER expression from clinical samples. ER mRNA levels measured by microarrays, such as Affymetrix U133A gene chips, in fine needle aspirates (FNA), core needle biopsy, and/or frozen tumor tissue samples of breast cancer correlated closely with protein expression by enzyme immunoassay and by routine immunohistochemistry. This is consistent with the previously observed correlation between ER mRNA expression using Northern blot and ER protein expression (Lacroix et al., 2001). An expression level of ER mRNA (ESR1 probe set 205225_)≧500 correctly identified ER-positive tumors (IHC≧10%) with overall accuracy of 96% (95% CI, 90%-99%) in the original set of 82 FNAs and this threshold was validated with 95% overall accuracy (95% CI, 88%-98%) in an independent set of 94 tissue samples (see Table 3). If any ER staining is considered to be ER-positive, the overall accuracy was 98% for FNAs and 99% for tissues. These results indicate that ER status can be reliably determined from gene expression microarray data, with the advantage of providing comparable results from cytologic and surgical samples, and from different laboratories. With appropriately standardized methods for analysis of data, a microarray platform may also provide robust clinical information of ER status.

ER-positive breast cancer includes a continuum of ER expression that might reflect a continuum of biologic behavior and endocrine sensitivity. Others have reported that some breast cancers are difficult to predict as ER-positive based on transcriptional profile and described non-estrogenic growth effects, such as HER-2, more frequently in this small subset of tumors with aggressive natural history (Kun et al., 2003). Indeed, ER mRNA levels are lower in breast cancers that are positive for both ER and HER2 (Konecny et al., 2003). Another group defined a gene expression signature from cDNA arrays that could predict ER protein levels (enzyme immunoassay) and another signature that predicted flow cytometric S-phase measurements (Gruvberger et al., 2004). Their finding of a reciprocal relationship supports the concept that less ER-positive breast cancers are more proliferative. This relationship is also factored into the calculation of the Recurrence Score that adds the values for proliferation and HER-2 gene groups and subtracts the values for the ER gene group (Paik et al., 2004; Paik et al., 2005). Molecular classification from unsupervised cluster analysis shows the same thing by identifying subtypes of luminal-type (ER-positive) breast cancer (Sorlie et al., 2001). The inverse relationship between ER expression and genes associated with proliferation and other growth pathways is best explained by viewing differentiation as a continuum in which cells become increasingly less proliferative and more dependent on ER stimulation as they differentiate. It follows that there would be an inverse relationship between greater sensitivity to endocrine therapy in differentiated tumors and greater sensitivity to chemotherapy in less differentiated tumors. Measurements along this scale could be valuable for treatment selection.

Randomized clinical trials have demonstrated a survival benefit for some patients who receive additional endocrine therapy with an aromatase inhibitor (compared to placebo) after 5 years of adjuvant tamoxifen (Goss et al., 2003; Bryant and Wolmark, 2003). Although there was a 24% relative reduction in deaths after 2.4 years of letrozole, the absolute difference in recurrence or new primaries was only 2.2% at 2.4 years (Goss et al., 2003, Burnstein, 2003). Without a test to identify patients who actually benefit from prolonged adjuvant endocrine therapy, the resulting decision to provide routine extension of adjuvant endocrine treatment (possibly for an indefinite period) in all women with ER-positive cancer could be a costly and potentially avoidable practice for the healthcare community that would benefit an unidentified minority (Buzdar, 2001). It is therefore helpful to consider that this genomic SET index of ER-associated gene expression might identify patients with intermediate endocrine sensitivity as candidates for extended adjuvant endocrine therapy.

A genomic scale of intrinsic endocrine sensitivity might also provide an improved scientific basis for selection of the most appropriate subjects for inclusion in clinical trials. The ATAC and BIG 1-98 trials enrolled 9,366 and 8,010 postmenopausal women, respectively, and both demonstrated 3% absolute improvement in disease-free survival (DFS) at 5 years from adjuvant aromatase inhibition, compared to tamoxifen (Howell et al., 2005; Thurlimann et al., 2005). Aromatase inhibition as first-line endocrine treatment for all postmenopausal women with ER-positive breast cancer would achieve this survival benefit in 3% of patients at significant cost, and might relegate an effective and less expensive treatment (tamoxifen) to relative obscurity. It is also likely that identification of potentially informative subjects, based on predicted partial endocrine sensitivity from indicators such as the SET index, could reduce the size and cost of adjuvant trials, demonstrate larger absolute survival benefit from improved treatment, and establish who should receive each treatment in routine practice after a positive trial result.

As the cost and complexity of endocrine therapy increase, diagnostic tools are needed not merely for prognosis, but, using strong biological rationale, to demonstrate clinical benefit when they are used to guide the selection and duration of endocrine agents therapy. Indicators such as the SET index can predict response to tamoxifen rather than intrinsic prognosis, and should be independent of stage, grade, and the expression levels of ESR1 and PGR. Continuing validation of the SET index with samples from trials of other hormonal agents would help continual refinement of this clinical interpretation.

TABLE 1
Reporter genes for ER-related genomic activity
and use in calculating index
		Unigene	Gene
Rank	Probe Set ID	ID	Symbol	Rs	Pg (200)
1	209603_at	169946	GATA3	0.783	1.000
2	215304_at	159264		0.779	1.000
3	218195_at	15929	C6orf211	0.774	1.000
4	212956_at	411317	KIAA0882	0.771	1.000
5	209604_s_at	169946	GATA3	0.764	1.000
6	202088_at	79136	SLC39A6	0.757	1.000
7	209602_s_at	169946	GATA3	0.749	1.000
8	212496_s_at	301011	JMJD2B	0.733	1.000
9	212960_at	411317	KIAA0882	0.724	1.000
10	215867_x_at	5344	AP1G1	0.724	1.000
11	214164_x_at	512620	CA12	0.721	1.000
12	203963_at	512620	CA12	0.719	1.000
13	41660_at	252387	CELSR1	0.709	1.000
14	218259_at	151076	MRTF-B	0.695	1.000
15	204667_at	163484	FOXA1	0.689	1.000
16	211712_s_at	430324	ANXA9	0.684	1.000
17	218532_s_at	82273	FLJ201S2	0.677	1.000
18	212970_at	15740	FLJ14001	0.677	1.000
19	209459_s_at	1588	ABAT	0.676	0.999
20	204508_s_at	512620	CA12	0.675	1.000
21	218976_at	260720	DNAJC12	0.673	0.998
22	217838_s_at	241471	EVL	0.673	1.000
23	218211_s_at	297405	MLPH	0.669	1.000
24	222275_at	124165	MRPS30	0.666	1.000
25	218471_s_at	129213	BBS1	0.666	0.999
26	214053_at	7888		0.666	0.999
27	203438_at	155223	STC2	0.664	1.000
28	213234_at	6189	KIAA1467	0.664	0.999
29	219197_s_at	435861	SCUBE2	0.657	0.999
30	212692_s_at	209846	LRBA	0.657	0.999
31	200711_s_at	171626	SKP1A	0.654	1.000
32	205074_at	15813	SLC22A5	0.653	1.000
33	203685_at	501181	BCL2	0.653	1.000
34	209460_at	1588	ABAT	0.653	0.999
35	222125_s_at	271224	PH-4	0.651	1.000
36	204798_at	407830	MYB	0.651	0.999
37	212985_at	15740	FLJ14001	0.648	1.000
38	203929_s_at	101174	MAPT	0.647	0.998
39	202089_s_at	79136	SLC39A6	0.642	0.997
40	205696_s_at	444372	GFRA1	0.639	0.997
41	209681_at	30246	SLC19A2	0.637	0.999
42	212495_at	301011	JMJD2B	0.637	0.999
43	218510_x_at	82273	FLJ20152	0.634	0.995
44	208682_s_at	376719	MAGED2	0.632	0.994
45	212195_at	529772		0.630	0.997
46	51192_at	29173	SSH-3	0.630	0.999
47	40016_g_at	212787	KIAA0303	0.628	0.997
48	212638_s_at	450060	WWP1	0.627	0.994
49	218692_at	354793	FLJ20366	0.624	0.991
50	213077_at	283283	FLJ21940	0.623	0.985
51	203439_s_at	155223	STC2	0.623	0.995
52	212441_at	79276	KIAA0232	0.622	0.988
53	210652_s_at	112949	C1orf34	0.621	0.990
54	219981_x_at	288995	ZNF587	0.620	0.984
55	205186_at	406050	DNALI1	0.620	0.990
56	213627_at	376719	MAGED2	0.620	0.987
57	200670_at	437638	XBP1	0.617	0.985
58	218437_s_at	30824	LZTFL1	0.617	0.987
59	206754_s_at	1360	CYP2B6	0.616	0.985
60	209696_at	360509	FBP1	0.616	0.987
61	201826_s_at	238126	CGI-49	0.615	0.984
62	219833_s_at	446047	EFHC1	0.610	0.975
63	203928_x_at	101174	MAPT	0.610	0.976
64	216092_s_at	22891	SLC7A8	0.609	0.985
65	200810_s_at	437351	CIRBP	0.609	0.977
66	204811_s_at	389415	CACNA2D2	0.609	0.968
67	44654_at	294005	G6PC3	0.609	0.974
68	202371_at	194329	FLJ21174	0.608	0.970
69	209173_at	226391	AGR2	0.607	0.971
70	212196_at	529772		0.606	0.953
71	210720_s_at	324104	APBA2BP	0.606	0.965
72	204497_at	20196	ADCY9	0.605	0.965
73	214440_at	155956	NAT1	0.604	0.960
74	205009_at	350470	TFF1	0.603	0.964
75	204862_s_at	81687	NME3	0.601	0.971
76	219562_at	3797	RAB26	0.600	0.949
77	50965_at	3797	RAB26	0.599	0.951
78	218966_at	111782	MYO5C	0.598	0.961
79	217979_at	364544	TM4SF13	0.596	0.972
80	209759_s_at	403436	DCI	0.596	0.938
81	212637_s_at	450060	WWP1	0.594	0.951
82	218094_s_at	256086	C20orf35	0.592	0.954
83	219222_at	11916	RBKS	0.592	0.941
84	202121_s_at	12107	BC-2	0.591	0.940
85	215001_s_at	442669	GLUL	0.591	0.940
86	210085_s_at	430324	ANXA9	0.590	0.934
87	210958_s_at	212787	KIAA0303	0.589	0.940
88	201596_x_at	406013	KRT18	0.588	0.928
89	212209_at	435249	THRAP2	0.587	0.923
90	221139_s_at	279815	CSAD	0.586	0.924
91	201384_s_at	458271	M17S2	0.586	0.910
92	213283_s_at	416358	SALL2	0.586	0.927
93	202908 at	26077	WFS1	0.585	0.917
94	219786_at	121378	MTL5	0.585	0.918
95	214109_at	209846	LRBA	0.584	0.930
96	203791_at	181042	DMXL1	0.583	0.914
97	205012_s_at	155482	HAGH	0.583	0.903
98	212492_s_at	301011	JMJD2B	0.582	0.902
99	218026_at	16059	HSPC009	0.579	0.905
100	210272_at	1360	CYP2B6	0.579	0.897
101	204199_at	432842	RALGPS1	0.577	0.892
102	202752_x_at	22891	SLC7A8	0.577	0.886
103	217645_at	531103		0.576	0.882
104	213419_at	324125	APBB2	0.576	0.888
105	219919_s_at	29173	SSH-3	0.575	0.861
106	213365_at	248437	MGC16943	0.574	0.861
107	219206_x_at	126372	CGI-119	0.574	0.883
108	221751_at	388400	PANK3	0.573	0.875
109	211596_s_at	528353	LRIG1	0.572	0.863
110	221963_x_at	356530		0.572	0.867
111	202641_at	182215	ARL3	0.572	0.850
112	201754_at	351875	COX6C	0.571	0.857
113	219741_x_at	515644	ZNF552	0.569	0.848
114	209224_s_at		NDUFA2	0.568	0.862
115	212099_at	406064	RHOB	0.568	0.836
116	205794_s_at	292511	NOVA1	0.568	0.836
117	219913_s_at	171342	CRNKL1	0.568	0.816
118	204934_s_at	432750	HPN	0.567	0.830
119	209341_s_at	413513	IKBKB	0.567	0.816
120	204231_s_at	528334	FAAH	0.567	0.817
121	203571_s_at	511763	C10orf116	0.567	0.807
122	204045_at	95243	TCEAL1	0.566	0.833
123	202636_at	147159	RNF103	0.566	0.788
124	202962_at	15711	KIF13B	0.565	0.798
125	208865_at	318381	CSNK1A1	0.563	0.801
126	201825_s_at	238126	CGI-49	0.563	0.806
127	219686_at	58241	STK32B	0.562	0.806
128	57540_at	11916	RBKS	0.560	0.782
129	212416_at	31218	SCAMP1	0.559	0.801
130	201170_s_at	171825	BHLHB2	0.559	0.758
131	40093_at	155048	LU	0.558	0.773
132	219414_at	12079	CLSTN2	0.557	0.761
133	209623_at	167531	MCCC2	0.556	0.758
134	202772_at	444925	HMGCL	0.555	0.752
135	208517_x_at	446567	BTF3	0.553	0.734
136	213018_at	21145	ODAG	0.552	0.764
137	204703_at	251328	TTC10	0.551	0.731
138	203801_at	247324	MRPS14	0.551	0.730
139	203246_s_at	437083	TUSC4	0.550	0.733
140	218769_s_at	239154	ANKRA2	0.549	0.740
141	203476_at	82128	TPBG	0.549	0.706
142	217770_at	437388	PIGT	0.548	0.736
143	35666_at	32981	SEMA3F	0.547	0.694
144	212508_at	24719	MOAP1	0.546	0.686
145	208712_at	371468	CCND1	0.545	0.703
146	204863_s_at	71968	IL6ST	0.544	0.710
147	204284_at	303090	PPP1R3C	0.544	0.672
148	203628_at	239176	IGF1R	0.544	0.674
149	200719_at	171626	SKP1A	0.544	0.668
150	214919_s_at		MASK-BP3	0.544	0.669
151	205376_at	153687	INPP4B	0.544	0.691
152	202263_at	334832	CYB5R1	0.543	0.674
153	218450_at	294133	HEBP1	0.543	0.660
154	213285_at	146180	LOC161291	0.543	0.666
155	209740_s_at	264	DXS1283E	0.543	0.653
156	205380_at	15456	PDZK1	0.543	0.661
157	203144_s_at	368916	KIAA0040	0.543	0.656
158	214552_s_at	390163	RABEP1	0.542	0.660
159	202814_s_at	15299	HIS1	0.540	0.629
160	205776_at	396595	FMO5	0.539	0.633
161	217906_at	415236	KLHDC2	0.539	0.640
162	212148_at	408222	PBX1	0.539	0.620
163	220581_at	287738	C6orf97	0.538	0.643
164	200811_at	437351	CIRBP	0.538	0.574
165	217894_at	239155	KCTD3	0.538	0.580
166	206197_at	72050	NME5	0.537	0.610
167	202454_s_at	306251	ERBB3	0.537	0.614
168	218394_at	22795	FLJ22386	0.535	0.601
169	201413_at	356894	HSD17B4	0.535	0.593
170	40569_at	458361	ZNF42	0.535	0.574
171	221856_s_at	3346	FLJ11280	0.535	0.576
172	210336_x_at	458361	ZNF42	0.534	0.584
173	211621_at	99915	AR	0.533	0.573
174	204623_at	82961	TFF3	0.533	0.533
175	40148_at	324125	APBB2	0.533	0.581
176	212446_s_at	387400	LASS6	0.532	0.543
177	210735_s_at	279916	CA12	0.531	0.540
178	214924_s_at	457063	OIP106	0.531	0.561
179	203071_at	82222	SEMA3B	0.531	0.522
180	213527_s_at	301463	LOC146542	0.530	0.531
181	208617_s_at	82911	PTP4A2	0.530	0.517
182	213249_at	76798	FBXL7	0.529	0.552
183	205645_at	334168	REPS2	0.529	0.520
184	208788_at	343667	ELOVL5	0.529	0.543
185	205769_at	11729	SLC27A2	0.528	0.501
186	213712_at	246107	ELOVL2	0.528	0.510
187	212697_at	432850	LOC162427	0.528	0.503
188	219900_s_at	435303	FLJ20626	0.528	0.485
189	213832_at	23729		0.527	0.490
190	213049_at	167031	GARNL1	0.527	0.474
191	59437_at	414028	C9orf116	0.527	0.504
192	204072_s_at	390874	13CDNA73	0.526	0.451
193	210108_at	399966	CACNA1D	0.526	0.489
194	214855_s_at	167031	GARNL1	0.525	0.459
195	209662_at	528302	CETN3	0.525	0.441
196	219687_at	58650	MART2	0.525	0.470
197	217191_x_at		COX6CP1	0.524	0.440
198	203538_at	13572	CAMLG	0.524	0.442
199	213702_x_at	324808	ASAH1	0.522	0.456
200	212744_at	26471	BBS4	0.522	0.458

In some aspects, although not intending to bound to any single theory, the ER reporter index can be of importance for tumors with high ER mRNA expression. If ER mRNA and the reporter index are high, this can describe a highly endocrine-dependent state for which tamoxifen alone seems to be sufficient for prolonged survival benefit. Patients with high ER mRNA expression but low reporter index appear to derive initial benefit from tamoxifen, but that is not sustained over the long term. Those patients' tumors are likely to be partially endocrine-dependent and might benefit from more potent endocrine therapy in the adjuvant setting. Some women might also benefit from more potent endocrine therapy. A measurable scale of ER gene expression and genomic activity might be applicable to any endocrine therapy that targets ER or other hormonal receptor activity. The relation of an index to efficacy of different endocrine therapies could be used to guide the selection of first-line treatment (e.g., chemotherapy versus endocrine therapy), influence the selection of endocrine agent based on likely endocrine sensitivity, and possibly to re-evaluate endocrine sensitivity if ER-positive breast cancer recurs.

Typically for clinical utility one would define the optimal probe set for ESR1 (ERα gene) on the Affymetrix U133A GeneChip™ to measure ER gene expression. The ESR1 205225_probe set produces the highest median and greatest range of expression and the strongest correlation with ER status because this probe set recognizes the most 3′ end of ESR1 (NetAffx search tool at www.affymetrix.com). The initial reverse transcription (RT) of mRNA sequences in each sample begins at the unique poly-A tail at the 3′ end of mRNA. Therefore, the 3′ end is likely to be the most represented part of any mRNA sequence, and probes that target the 3′ end generally produce the strongest hybridization signal.

In other aspects of the invention it is preferred that biostatistical methods be used that allow standardization of microarray data from any contributing laboratory. At present, direct comparison of IHC results for ER from multiple centers is difficult because technical staining methods differ, positive and negative tissue controls are laboratory-dependent, and interpretation of staining is subjective to the interpretation of the individual pathologist or the threshold setting of the image analysis system being used (Rhodes et al., 2000; Rhodes, 2003; Regitnig et al., 2002). Even in quantitative RT-PCR assays, the expression of genes of interest are calculated relative to only one or several intrinsic housekeeper genes in each assay. The techniques for RNA extraction from fresh samples and preparation for hybridization to Affymetrix microarrays are available from standardized laboratory protocols. However, it should not be overlooked that uniform normalization of microarray data from every breast cancer sample to a digital standard (e.g., U133A dCHIP dataset) will consistently calculate the expression of all genes of interest relative to the expression of thousands of intrinsic control genes. This availability of multiple controls to standardize expression levels of all genes on the microarray is a robust mathematical control that can explain the comparable results from measurements of ER mRNA expression levels in different sample types and in different laboratories. Adoption of an agreed dCHIP standard for data normalization of breast cancer samples using the Affymetrix U133A array could lead to a digital standard available to laboratories for clinical trials and for routine diagnostics.

The implications of establishing standard analysis tools for development of a useful clinical assay are clear. When diagnostic microarrays are introduced into the clinic through a central reference laboratory, then uniform data normalization and standardized experimental procedure require internal quality control procedures by the central laboratory. However, in a decentralized system where each center performs its own profiling following a standard procedure using the same microarray platform, a single digital standard should be available for data normalization. This allows different laboratories to generate data that is directly comparable to a common standard.

TABLE 2
Genes indicative of the responsiveness of a cancer cell to therapy
Probe.Set	Accession	Name	T-stat	P-val
203930_s_at	NM_016835.1	Microtubule-associated protein	−6.42	5.25 × 10-08
212745_s_at	A1813772	Bardet-Biedl syndrome 4	−6.25	9.40 × 10-08
203928_x_at	NM_016835.1	Microtubule-associated protein	−5.99	2.70 × 10-07
206401_s_at	J03778.1	Microtubule-associated protein	−5.73	7.02 × 10-07
203929_s_at	NM_016835.1	Microtubule-associated protein	−5.52	1.26 × 10-06
212207_at	AK023837.1	KIAA1025 protein	−5.37	2.21 × 10-06
212046_x_at	X60188.1	Mitogen-activated protein kinase	−5.33	3.43 × 10-06
210469_at	BC002915.1	Discs, large (Drosophila) homol	−5.28	3.53 × 10-06
205074_at	NM_003060.1	Solute carrier family 22 (organ	−5.13	5.45 × 10-06
204509_at	NM_017689.1	Hypothetical protein FLJ20151	−5.02	6.15 × 10-06
205696_s_at	NM_005264.1	GDNF family receptor alpha 1	−5.00	1.06 × 10-05
219741_x_at	NM_024762.1	Hypothetical protein FLJ21603	−4.94	1.00 × 10-05
215616_s_at	AB020683.1	KIAA0876 protein	−4.86	1.43 × 10-05
208945_s_at	NM_003766.1	Beclin 1 (coiled-coil, myosin-l	−4.86	1.48 × 10-05
217542_at	BE930512	ESTs	−4.80	1.84 × 10-05
202204_s_at	AF124145.1	Autocrine motility factor recep	−4.74	2.05 × 10-05
204916_at	NM_005855.1	Receptor (calcitonin) activity	−4.70	2.92 × 10-05
218769_s_at	NM_023039.1	Ankyrin repeat, family A (RFXAN	−4.70	2.58 × 10-05
219981_x_at	NM_017961.1	Hypothetical protein FLJ20813	−4.66	4.44 × 10-05
222131_x_at	BC004327.1	Hypothetical protein BC014942	−4.64	3.26 × 10-05
213234_at	AB040900.1	KIAA1467 protein	−4.60	3.73 × 10-05
219197_s_at	AI424243	CEGP1 protein	−4.57	3.45 × 10-05
205425_at	NM_005338.3	Huntington interacting protein	−4.51	8.86 × 10-05
213504_at	W63732	COP9 subunit 6 (MOV34 homolog,	−4.50	4.98 × 10-05
201413_at	NM_000414.1	Hydroxysteroid (17-beta) dehydr	−4.46	5.71 × 10-05
203050_at	NM_005657.1	Tumor protein p53 binding prote	−4.45	7.53 × 10-05
212494_at	AB028998.1	KIAA1075 protein	−4.43	9.46 × 10-05
209173_at	AF088867.1	Anterior gradient 2 homolog (Xe	−4.41	6.36 × 10-05
201124_at	AL048423	Integrin, beta 5	−4.41	7.76 × 10-O5
205354_at	NM_000156.3	Guanidinoacetate N-methyltransf	−4.39	8.11 × 10-05
212444_at	AA156240	Homo sapiens cDNA: FLJ22182 fis	−4.37	7.71 × 10-05
205225_at	NM_000125.1	Estrogen receptor 1	−4.37	8.12 × 10-05
211000_s_at	AB015706.1	Interleukin 6 signal transducer	−4.36	9.16 × 10-05
204012_s_at	AL529189	KIAA0547 gene product	−4.36	8.63 × 10-05
203682_s_at	NM_002225.2	Isovaleryl Coenzyme A dehydroge	−4.35	7.60 × 10-05
220357_s_at	NM_016276.1	Serum/glucocorticoid regulated	−4.35	5.94 × 10-05
216173_at	AK025360.1	Homo sapiens cDNA: FLJ21707 fis	−4.32	7.65 × 10-05
210230_at	BC003629.1	RNA, U2 small nuclear	−4.26	9.95 × 10-05
219044_at	NM_018271.1	Hypothetical protein FLJ10916	−4.25	1.75 × 10-04
218761_at	NM_017610.1	Likely ortholog of mouse Arkadi	−4.23	1.35 × 10-04
210826_x_at	AF098533.1	RAD17 homolog (S. pombe)	−4.22	1.44 × 10-04
210831_s_at	L27489.1	Prostaglandin E receptor 3 (sub	−4.22	1.07 × 10-04
211233_x_at	M12674.1	Estrogen receptor 1	−4.21	1.20 × 10-04
218807_at	NM_006113.2	Vav 3 oncogene	−4.20	1.46 × 10-04
210129_s_at	AF078842.1	DKFZP434B103 protein	−4.19	1.09 × 10-04
39313_at	AB002342	Protein kinase, lysine deficien	−4.19	1.23 × 10-04
213245_at	AL120173	Homosapiens cDNA FLJ30781 fis,	−4.18	1.43 × 10-04
214053_at	AW772192	Homo sapiens clone 23736 mRNA s	−4.18	1.51 × 10-04
205352_at	NM_005025.1	Serine (or cysteine) proteinase	−4.17	1.47 × 10-04
213623_at	NM_007054.1	Kinesin family member 3A	−4.15	1.88 × 10-04
215304_at	U79293.1	Human clone 23948 mRNA sequence	−4.13	1.40 × 10-04
203009_at	NM_005581.1	Lutheran blood group (Auberger	−4.13	1.80 × 10-04
218692_at	NM_017786.1	Hypothetical protein FLJ20366	−4.13	1.76 × 10-04
218976_at	NM_021800.1	J domain containing protein 1	−4.12	1.76 × 10-04
201405_s_at	NM_006833.1	COP9 subunit 6 (MOV34 homolog,	−4.11	1.63 × 10-04
202168_at	NM_003187.1	TAF9 RNA polymerase II, TATA bo	−4.11	2.01 × 10-04
216109_at	AK025348.1	Homo sapiens cDNA: FLJ21695 fis	−4.11	1.77 × 10-04
219051_x_at	NM_024042.1	Hypothetical protein MGC2601	−4.10	2.34 × 10-04
210908_s_at	AB055804.1	Prefoldin 5	−4.09	1.71 × 10-04
221728_x_at	AK025198.1	Homo sapiens cDNA FLJ30298 fis,	−4.07	2.11 × 10-04
203187_at	NM_001380.1	Dedicator of cyto-kinesis 1	−4.06	2.22 × 10-04
212660_at	AI735639	KIAA0239 protein	−4.04	2.56 × 10-04
212956_at	AB020689.1	KIAA0882 protein	−4.01	2.27 × 10-04
217838_s_at	NM_016337.1	RNB6	−4.01	2.14 × 10-04
218621_at	NM_016173.1	HEMK homolog 7 kb	−4.01	1.92 × 10-04
201681_s_at	AB0111855.1	Discs, large (Drosophila) homol	−4.01	2.49 × 10-04
209884_s_at	AF047033.1	Solute carrier family 4, sodium	−4.00	2.98 × 10-04
201557_at	NM_014232.1	Vesicle-associated membrane pro	−3.99	2.23 × 10-04
219338_s_at	NM_017691.1	Hypothetical protein FLJ20156	−3.99	2.94 × 10-04
217828_at	NM_024755.1	Hypothetical protein FLJ13213	−3.98	2.42 × 10-04
209339_at	U76248.1	Seven in absentia homolog 2 (Dr	−3.98	2.26 × 10-04
214218_s_at	AV699347	Homo sapiens cDNA FLJ30298 fis,	−3.97	2.82 × 10-04
221643_s_at	AF016005.1	Arginine-glutamic acid dipeptid	−3.96	2.57 × 10-04
218211_s_at	NM_024101.1	Melanophilin	−3.95	3.05 × 10-04
221483_sat	AF084555.1	Cyclic AMP phosphoprotein, 19 k	−3.95	2.83 × 10-04
211864_s_at	AF207990.1	Fer-1-like 3, myoferlin (C. ele	−3.92	3.29 × 10-04
202392_s_at	NM_014338.1	Phosphatidylserine decarboxylas	−3.92	4.33 × 10-04
214164_x_at	BF752277	Adaptor-related protein complex	−3.91	3.52 × 10-04
204862_s_at	NM_002513.1	Non-metastatic cells 3, protein	−3.91	3.55 × 10-04
215552_s_at	AI073549	Estrogen receptor 1	−3.91	3.33 × 10-04
211235_s_at	AF258450.1	Estrogen receptor 1	−3.90	3.13 × 10-04
210833_at	AL031429	Prostaglandin E receptor 3 (sub	−3.89	3.06 × 10-04
204660_at	NM_005262.1	Growth factor, augmenter of liv	−3.89	2.79 × 10-04
211234_x_at	AF258449.1	Estrogen receptor 1	−3.89	3.10 × 10-04
201508_at	NM_001552.1	Insulin-like growth factor bind	−3.88	4.04 × 10-04
213527_s_at	AI350500	Similar to hypothetical protein	−3.85	4.33 × 10-04
202048_s_at	NM_014292.1	Chromobox homolog 6	−3.84	4.15 × 10-04
206794_at	NM_005235.1	v-erb-a erythroblastic leukemia	−3.84	3.87 × 10-04
201798_s_at	NM_013451.1	Fer-1-like 3, myoferlin (C. ele	−3.83	4.44 × 10-04
213523_at	AI671049	Cyclin E1	3.81	4.14 × 10-04
209050_s_at	AI421559	Ral guanine nucleotide dissocia	3.83	4.07 × 10-04
217294_s_at	U88968.1	Enolase 1, (alpha)	3.84	4.48 × 10-04
201555_at	NM_002388.2	MCM3 minichromosome maintenance	3.84	4.41 × 10-04
201030_x at	NM_002300.1	Lactate dehydrogenase B	3.85	3.85 × 10-04
202912_at	NM_001124.1	Adrenomedullin	3.86	3.59 × 10-04
204050_s_at	NM_001833.1	Clathrin, light polypeptide (Lc	3.88	3.97 × 10-04
202342_s_at	NM_015271.1	Tripartite motif-containing 2	3.88	4.43 × 10-04
209393_s_at	AF047695.1	Eukaryotic translation initiati	3.89	4.21 × 10-04
219774_at	NM_019044.1	Hypothetical protein FLJ10996	3.93	3.86 × 10-04
204162_at	NM_006101.1	Highly expressed in cancer, nc	3.93	2.94 × 10-04
216237_s_at	AA807529	MCM5 minichromosome maintenance	3.96	2.84 × 10-04
214581_x_at	BE568134	Tumor necrosis factor receptor	3.99	3.07 × 10-04
209408_at	U63743.1	Kinesin-like 6 (mitotic centrom	3.99	2.23 × 10-04
208370_s_at	NM_004414.2	Down syndrome critical region g	4.02	2.94 × 10-04
203744_at	NM_005342.1	High-mobility group box 3	4.02	2.02 × 10-04
209575_at	BC001903.1	Interleukin 10 receptor, beta	4.03	2.84 × 10-04
200934_at	NM_003472.1	DEK oncogene (DNA binding)	4.05	2.54 × 10-04
202341_s_at	AA149745	Tripartite motif-containing 2	4.06	2.87 × 10-04
200996_at	NM005721.2	ARP3 actin-related protein 3 ho	4.06	2.42 × 10-04
206392_s_at	NM_002888.1	Retinoic acid receptor responde	4.06	2.28 × 10-04
206391_at	NM_002888.1	Retinoic acid receptor responde	4.07	2.52 × 10-04
201797_s_at	NM_006295.1	Valyl-tRNA synthetase 2	4.07	2.17 × 10-04
209358_at	AF118094.1	TAF11 RNA polymerase II, TATA b	4.07	2.34 × 10-04
209201_x_at	L01639.1	Chemokine (C-X-C motif) recepto	4.09	2.80 × 10-04
209016_s_at	BC002700.1	Keratin 7	4.14	1.69 × 10-04
221957_at	BF939522	Pyruvate dehydrogenase kinase,	4.15	2.22 × 10-04
218350_s_at	NM_015895.1	Geminin, DNA replication inhibi	4.16	1.64 × 10-04
201897_s_at	NM_001826.1	p53-regulated DDA3	4.21	1.36 × 10-04
209642_at	AF043294.2	BUB1 budding uninhibited by ben	4.22	1.22 × 10-04
201930_at	NM_005915.2	MCM6 minichromosome maintenance	4.23	1.16 × 10-04
202870_s_at	NM_001255.1	CDC20 cell division cycle 20 ho	4.23	1.07 × 10-04
221485_at	NM_004776.1	UDP-Gal:betaGlcNAc beta 1,4- ga	4.26	1.08 × 10-04
211919_s_at	AF348491.1	Chemokine (C-X-C motif) recepto	4.27	1.61 × 10-04
218887_at	NM_015950.1	Mitochondrial ribosomal protein	4.27	8.93 × 10-05
216295_s_at	X81636.1	H.sapiens clathrin light chain	4.28	1.17 × 10-04
218726_at	NM_018410.1	Hypothetical protein DKFZp762E1	4.28	1.19 × 10-04
204989_s_at	BF305661	Integrin, beta 4	4.30	1.01 × 10-04
221872_at	AI669229	Retinoic acid receptor responde	4.31	1.12 × 10-04
206746_at	NM_001195.2	Beaded filament structural prot	4.32	9.33 × 10-05
201231_s_at	NM_001428.1	Enolase 1, (alpha)	4.42	5.76 × 10-05
204203_at	NM_001806.1	CCAAT/enhancer binding protein	4.42	6.44 × 10-05
211555_s_at	AF020340.1	Guanylate cyclase 1, soluble, b	4.47	5.11 × 10-05
202200_s_at	NM_003137.1	SFRS protein kinase 1	4.47	5.17 × 10-05
213101_s_at	Z78330	Homo sapiens mRNA; cDNA DKFZp68	4.49	7.76 × 10-05
204600_at	NM_004443.1	EphB3	4.51	5.81 × 10-05
212689_s_at	AA524505	Zinc finger protein	4.52	5.10 × 10-05
209773_s_at	BC001886.1	Ribonucleotide reductase M2 po1	4.55	3.18 × 10-05
204962_s_at	NM_001809.2	Centromere protein A, l7kDa	4.62	3.00 × 10-05
211519_s_at	AY026505.1	Kinesin-like 6 (mitotic centrom	4.62	2.41 × 10-05
204825_at	NM_014791.1	Maternal embryonic leucine zipp	4.73	2.45 × 10-05
203287_at	NM_005558.1	Ladinin 1	4.74	2.06 × 10-05
204913_s_at	AI360875	SRY (sex determining region Y)-	4.77	2.44 × 10-05
217028_at	AJ224869		4.82	2.56 × 10-05
204750_s_at	BF196457	Desmocollin 2	4.84	1.78 × 10-05
216222_s_at	AI561354	Myosin X	4.84	1.93 × 10-05
1438_at	X75208	EphB3	5.02	9.02 × 10-06
203693_s_at	NM_001949.2	E2F transcription factor 3	5.17	4.83 × 10-06
205548_s_at	NM_006806.1	BIG family, member 3	5.64	1.96 × 10-06
201976_s_at	NM_012334.1	Myosin X	5.68	8.74 × 10-07
213134_x_at	AI765445	BlG family, member 3	5.76	1.31 × 10-06
40016g_at	AB002301	KIAA0303 protein	4.26	1.071 × 10-04
206352_s_at	AB013818	peroxisome biogenesis factor 10	4.28	5.79 × 10-05
205074_at	AB015050	solute carrier family 22 member 5	4.64	2.24 × 10-05
213527_s_at	AC002310	similar to hypothetical protein	4.62	3.16 × 10-05
		MGC13138
216835_s_at	AF035299	docking protein 1,62 kDa	4.44	3.32 × 10-05
209617_s_at	AF035302	catenin (cadherin-associated protein),	5.16	1.7 × 10-06
		delta 2 (neural plakophilin-related arm-
		repeat protein)
208945_s_at	AF139131	beclin 1 (coiled-coil, myosin-like BCL2	5.61	5.0 × 10-07
		interacting protein)
222275_at	AI039469	mitochondrial ribosomal protein S30	4.51	2.16 × 10-05
203929_s_at	AI056359	microtubule-associated protein tau	6.60	0.0 × 10-04
215552_s_at	AI073549	Estrogen receptor 1	4.51	2.51 × 10-05
212956_at	AI348094	KIAA0882 protein	4.40	7.0 × 10-05
204913_s_at	AI360875	SRY (sex determining region Y)-box 11	−4.45	9.92 × 10-05
213855_s_at	AI500366	lipase, hormone-sensitive	4.17	1.08 × 10-04
212239_at	AI680192	phosphoinositide-3-kinase, regulatory	4.36	4.71 × 10-05
		subunit, polypeptide 1 (p85 alpha)
203928_x_at	AI870749	microtubule-associated protein tau	5.91	8 × 10-08
214124_x at	AL043487	FGFR1 oncogene partner	5.18	3.1 × 10-06
212195_at	AL049265	MRNA; cDNA DKFZp564F053	4.25	1.11 × 10-04
210222_s_at	BC000314	reticulon 1	4.08	1.07 × 10-04
210958_s_at	BC003646	KIAA0303 protein	4.43	4.26 × 10-05
204863_s_at	BE856546	interleukin 6 signal transducer (gp130,	4.28	8.20 × 10-05
		oncostatin M receptor)
213911_s_at	BF718636	H2A histone family, member Z	−4.16	1.10 × 10-04
212207_at	BG426689	thyroid hormone receptor associated	6.06	1.0 × 10-07
		protein 2
209696_at	D26054	fructose-1,6-bisphosphatase 1	4.29	9.21 × 10-05
209443_at	J02639	serine (or cysteine) proteinase inhibitor,	4.21	6.95 × 10-05
		clade A (alpha-1 antiproteinase,
		antitrypsin), member 5
202862_at	NM_000137	fumarylacetoacetate hydrolase	4.34	5.59 × 10-05
		(fumarylacetoacetase)
214440_at	NM_000662	N-acetyltransferase 1 (arylamine N-	4.24	6.75 × 10-05
		acetyltransferase)
208305_at	NM_000926	progesterone receptor	4.15	8.19 × 10-05
202204_s_at	NM_001144	autocrine motility factor receptor	5.28	1.29 × 10-06
204862_s_at	NM_002S13	non-metastatic cells 3, protein expressed	4.30	8.95 × 10-05
		in
202641_at	NM_004311	ADP-ribosylation factor-like 3	4.24	9.46 × 10-05
200896_x_at	NM_004494	hepatoma-derived growth factor (high-	−4.87	1.38 × 10-05
		mobility group protein 1-like)
203071_at	NM_004636	sema domain, immunoglobulin domain	4.65	1.63 × 10-05
		(Ig), short basic domain, secreted,
		(semaphorin) 3B
205012_s_at	NM_005326	hydroxyacylglutathione hydrolase	4.60	3.62 × 10-05
204916_at	NM_005855	receptor (calcitonin) activity modifying	5.47	5.10 × 10-07
		protein 1
204792_s_at	NM_014714	KIAA0590 gene product	4.14	1.12 × 10-04
208202_s_at	NM_015288	PHD finger protein 15	4.18	1.08 × 10-04
217770_at	NM_015937	phosphatidylinositol glycan, class T	4.33	5.43 × 10-05
218671_s_at	NM_016311	ATPase inhibitory factor 1	4.18	9.04 × 10-05
219872_at	NM_016613	hypothetical protein DKFZp434L142	4.10	1.03 × 10-04
219197_s_at	NM_020974	signal peptide, CUB domain, EGF-like 2	5.43	6.8 × 10-07
203485_at	NM_021136	reticulon 1	4.18	7.56 × 10-05
206936_x_at	NM_022335	NADH dehydrogenase (ubiquinone) 1,	4.28	6.46 × 10-05
		subcomplex unknown, 2, 14.5kDa
220540_at	NM_022358	potassium channel, subfamily K,	4.68	1.32 × 10-05
		member 15
219438_at	NM_024522	hypothetical protein FLJ12650	4.82	6.68 × 10-06
205696_s_at	2674 U97144	GDNF family receptor alpha 1	4.89	7.15 × 10-06

In addition to other know methods of cancer therapy, hormone therapies may be employed in the treatment of patients identified as having hormone sensitive cancers. Hormones, or other compounds that stimulate or inhibit these pathways, can bind to hormone receptors, blocking a cancer's ability to get the hormones it needs for growth. By altering the hormone supply, hormone therapy can inhibit growth of a tumor or shrink the tumor. Typically, these cancer treatments only work for hormone-sensitive cancers. If a cancer is hormone sensitive, a patient might benefit from hormone therapy as part of cancer treatment. Sensitive to hormones is usually determined by taking a sample of a tumor (biopsy) and conducting analysis in a laboratory.

Cancers that are most likely to be hormone-receptive include: Breast cancer, Prostate cancer, Ovarian cancer, and Endometrial cancer. Not every cancer of these types is hormone-sensitive, however. That is why the cancer must be analyzed to determine if hormone therapy is appropriate.

Hormone therapy may be used in combination with other types of cancer treatments, including surgery, radiation and chemotherapy. A hormone therapy can be used before a primary cancer treatment, such as before surgery to remove a tumor. This is called neoadjuvant therapy. Hormone therapy can sometimes shrink a tumor to a more manageable size so that it's easier to remove during surgery.

Hormone therapy is sometimes given in addition to the primary treatment—usually after—in an effort to prevent the cancer from recurring (adjuvant therapy). In some cases of advanced (metastatic) cancers, such as in advanced prostate cancer and advanced breast cancer, hormone therapy is sometimes used as a primary treatment.

Hormone therapy can be given in several forms, including: (A) Surgery—Surgery can reduce the levels of hormones in your body by removing the parts of your body that produce the hormones, including: Testicles (orchiectomy or castration), Ovaries (oophorectomy) in premenopausal women, Adrenal gland (adrenalectomy) in postmenopausal women, Pituitary gland (hypophysectomy) in women. Because certain drugs can duplicate the hormone-suppressive effects of surgery in many situations, drugs are used more often than surgery for hormone therapy. And because removal of the testicles or ovaries will limit an individual's options when it comes to having children, younger people are more likely to choose drugs over surgery. (B) Radiation—Radiation is used to suppress the production of hormones. Just as is true of surgery, it's used most commonly to stop hormone production in the testicles, ovaries, and adrenal and pituitary glands. (C) Pharmaceuticals—Various drugs can alter the production of estrogen and testosterone. These can be taken in pill form or by means of injection. The most common types of drugs for hormone-receptive cancers include: (1) Anti-hormones that block the cancer cell's ability to interact with the hormones that stimulate or support cancer growth. Though these drugs do not reduce the production of hormones, anti-hormones block the ability to use these hormones. Anti-hormones include the anti-estrogens tamoxifen (Nolvadex) and toremifene (Fareston) for breast cancer, and the anti-androgens flutamide (Eulexin) and bicalutamide (Casodex) for prostate cancer. (2) Aromatase inhibitors—Aromatase inhibitors (AIs) target enzymes that produce estrogen in postmenopausal women, thus reducing the amount of estrogen available to fuel tumors. AIs are only used in postmenopausal women because the drugs can't prevent the production of estrogen in women who haven't yet been through menopause. Approved AIs include letrozole (Femara), anastrozole (Arimidex) and exemestane (Aromasin). It has yet to be determined if AIs are helpful for men with cancer. (3) Luteinizing hormone-releasing hormone (LH-RH) agonists and antagonists—LH-RH agonists—sometimes called analogs—and LH-RH antagonists reduce the level of hormones by altering the mechanisms in the brain that tell the body to produce hormones. LH-RH agonists are essentially a chemical alternative to surgery for removal of the ovaries for women, or of the testicles for men. Depending on the cancer type, one might choose this route if they hope to have children in the future and want to avoid surgical castration. In most cases the effects of these drugs are reversible. Examples of LH-RH agonists include: Leuprolide (Lupron, Viadur, Eligard) for prostate cancer, Goserelin (Zoladex) for breast and prostate cancers, Triptorelin (Trelstar) for ovarian and prostate cancers and abarelix (Plenaxis).

One class of pharmaceuticals are the Selective Estrogen Receptor Modulators or SERMs. SERMs block the action of estrogen in the breast and certain other tissues by occupying estrogen receptors inside cells. SERMs include, but are not limited to tamoxifen (the brand name is Nolvadex, generic tamoxifen citrate); Raloxifene (brand name: Evista), and toremifene (brand name: Fareston).

EXAMPLES

The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1

Material and Methods

Patients and Samples. Studies were conducted using different cohorts of samples: 132 patients (82 were ER-positive) from UT M.D. Anderson Cancer Center (MDACC) prior to pre-operative adjuvant chemotherapy, 18 patients from MDACC with metastatic (AJCC Stage IV) ER-positive breast cancer, 277 patients from three different institutions (109 from Oxford, UK; 87 from Guy's Hospital, London UK; 81 from Uppsala, Sweden) who were uniformly treated with adjuvant tamoxifen, and 286 patients (209 were ER-positive) with node-negative disease from a single institution who did not receive any systemic chemotherapy treatment. At MDACC, pre-treatment fine needle aspiration (FNA) samples of primary breast cancer were obtained using a 23-gauge needle and the cells from 1-2 passes were collected into a vial containing 1 ml of RNAlater™ solution (Ambion, Austin Tex.) and stored at −80° C. until use, whereas archival frozen samples were evaluated from resected, metastatic, ER-positive breast cancer. All patients signed an informed consent for voluntary participation to collect samples for research. At other institutions, fresh tissue samples of surgically resected primary breast cancer were frozen in OCT compound and stored at −80° C.

Patients in this study had invasive breast carcinoma and were characterized for estrogen receptor (ER) expression using immunohistochemistry (IHC) and/or enzyme immunoassay (EIA). Immunohistochemical (IHC) assay for ER was performed on formalin-fixed paraffin-embedded (FFPE) tissue sections or Camoy's-fixed FNA smears using the following methods: FFPE slides were first deparaffinized, then slides (FFPE or FNA) were passed through decreasing alcohol concentrations, rehydrated, treated with hydrogen peroxide (5 minutes), exposed to antigen retrieval by steaming the slides in tris-EDTA buffer at 95° C. for 45 minutes, cooled to room temperature (RT) for 20 minutes, and incubated with primary mouse monoclonal antibody 6F11 (Novacastra/Vector Laboratories, Burlingame, Calif.) at a dilution of 1:50 for 30 minutes at RT (Gong et al., 2004). The Envision method was employed on a Dako Autostainer instrument for the rest of the procedure according to the manufacturer's instructions (Dako Corporation, Carpenteria, Calif.). The slides were then counterstained with hematoxylin, cleared, and mounted. Appropriate negative and positive controls were included. The 96 breast cancers from OXF were ER-positive by enzyme immunoassay as previously described, containing>10 femtomoles of ER/mg protein (Blankenstein et al., 1987).

Estrogen receptor (ER) expression was characterized using immunohistochemistry (IHC) and/or enzyme immunoassay (EIA). IHC staining of ER was interpreted at MDACC as positive (P) if ≧10% of the tumor cells demonstrated nuclear staining, low expression (L) if <10% of the tumor cell nuclei stained, and negative (N) if there was no nuclear staining. Low expression (<10%) is reported in routine patient care as negative, but some of those patients potentially benefit from hormonal therapy (Harvey et al., 1999).

RNA extraction and gene expression profiling. RNA was extracted from the MDACC FNA samples using the RNAeasy Kit™ (Qiagen, Valencia Calif.). The amount and quality of RNA was assessed with DU-640 U.V. Spectrophotometer (Beckman Coulter, Fullerton, Calif.) and it was considered adequate for further analysis if the OD260/280 ratio was ≧1.8 and the total RNA yield was ≧1.0 μg. RNA was extracted from the tissue samples using Trizol (InVitrogen, Carlsbad, Calif.) according to the manufacturer's instructions. The quality of the RNA was assessed based on the RNA profile generated by the Bioanalyzer (Agilent Technologies, Palo Alto, Calif.). Differences in the cellular composition of the FNA and tissue samples have been reported previously (Symmans et al., 2003). In brief, FNA samples on average contain 80% neoplastic cells, 15% leukocytes, and very few (<5%) non-lymphoid stromal cells (endothelial cells, fibroblasts, myofibroblasts, and adipocytes), whereas tissue samples on average contain 50% neoplastic cells, 30% non-lymphoid stromal cells, and 20% leukocytes (Symmans et al., 2003). A standard T7 amplification protocol was used to generate cRNA for hybridization to the microarray. No second round amplification was performed. Briefly, mRNA sequences in the total RNA from each sample were reverse-transcribed with SuperScript II in the presence of T7-(dT)24 primer to produce cDNA. Second-strand cDNA synthesis was performed in the presence of DNA Polymerase I, DNA ligase, and Rnase H. The double-stranded cDNA was blunt-ended using T4 DNA polymerase and purified by phenol/chloroform extraction. Transcription of double-stranded cDNA into cRNA was performed in the presence of biotin-ribonucleotides using the BioArray High Yield RNA transcript labeling kit (Enzo Laboratories). Biotin-labeled cRNA was purified using Qiagen RNAeasy columns (Qiagen Inc.), quantified and fragmented at 94° C. for 35 minutes in the presence of 1× fragmentation buffer. Fragmented cRNA from each sample was hybridized to each Affymetrix U133A gene chip, overnight at 42° C. The U133A chip contains 22,215 different probe sets that correspond to 13,739 human UniGene clusters (genes). Hybridization cocktail was prepared as described in the Affymetrix technical manual. dCHIP Vi.3 (available via the internet at dchip.org) software was used to generate probe level intensities and quality measures including median intensity, % of probe set outliers and % of single probe outliers for each chip.

Microarray Data Analysis. The raw intensity files (CEL) from each microarray were normalized using dChip V1.3 software (dchip.org). After normalization, the 75th percentile of pixel level was used as the intensity level for each feature on a microarray (see mdanderson.org/pdf/biostats_utmdabtr00503.pdf via the world wide web). Multiple features representing each probe set were aggregated using the perfect match model to form a single measure of intensity.

Definition of ER Reporter Genes. ER reporter genes were defined from an independent public dataset of Affymetrix U133A transcriptional profiles from 286 node-negative breast cancer samples (Wang et al., 2005). Expression data had been normalized to an average probe set intensity of 600 per array (Wang et al., 2005). The dataset was filtered to include 9789 probe sets with most variable expression, where P₀≧5, P₇₅−P₂₅≧100, and P₉₅/P₅≧3 (P_q is the q^th percentile of intensity for each probe set). Those were ranked by Spearman's rho (Kendall and Gibbons, 1990) with ER mRNA (ESR1 probe set 205225_at) expression, of which 2217 probe sets were significantly and positively associated with ESR1 (t-test of correlation coefficients with one-sided significance level of 99.9% and estimated false discovery rate (FDR) of 0.45%). The size of the reporter gene set was then determined by a bootstrap-based method that accounts for sampling variability in the correlation coefficient and in the resulting probe sets rankings (Pepe et al., 2003). The entire dataset was re-sampled 1000 times with replacement at the subject level (i.e., when one of the 286 subjects was selected in the bootstrap sample, the 2217 candidate probe sets from that subject were included in the dataset). Each probe set was ranked according to its correlation with ESR1 in each bootstrap dataset. The probability (P) of selection for each probe set (g) in a reporter gene set of defined length (k) was calculated as P[Rank(g)≦k]. A similar computation provided estimates of the power to detect the truly co-expressed genes from a study of a given size (Pepe et al., 2003).

Genes that are truly co-expressed with ESR1 have selection probabilities close to 1, but the selection probability diminishes quickly for lower order probe sets (FIG. 1). The probability of selecting the top 50 ER-associated probes would be 98.5% if the ER reporter gene list included 200 probes, 87.0% if 100 probes, and 41.3% if 50 probes (FIG. 1). An ER reporter list with 200 top-ranking probes would include the top 50 probes with 98.5% probability and the top 100 probes with about 93% probability (FIG. 1). The distribution of ranks is very tight for genes that are strongly correlated with ESR1 having median ranks close to 1 (FIG. 2). However, both the median rank and the variance of the distribution of ranks increase for genes that are moderately correlated with ESR1. The gene ranks for genes with Spearman's rho>0.65 are less than 200 with the exception of a few outliers (FIG. 2). Therefore as opposed to selecting the reporter genes by choosing an arbitrary cutoff on the correlation coefficient, this approach identifies the 100 genes that are most-strongly correlated with ESR1 with high power (>93%). The size of the reporter gene set was selected to be 200 probe sets, based on the bootstrap-estimated selection probabilities (FIG. 1) and the requirement to detect the top 100 truly co-expressed genes with >90% power. The original dataset was re-sampled with replacement at the subject level (i.e., when one of the 286 subjects was selected in the bootstrap sample, the 2217 candidate probe sets from that subject were included in the dataset to generate 1000 different bootstrap datasets. Each candidate probe set was ranked according to its correlation with ESR1 within each bootstrap dataset and the degree of confidence in the ranking of each probe set was quantified in terms of the selection probability, Pg(k). The probability (P) of selection for each probe set (g) in a reporter gene set of defined length (k) was calculated as P[Rank(g)]≦k.

Calculation of Expression Index (Sensitivity to Endocrine Treatment Index). To quantify the expression of the 200 reporter genes in new samples, the inventors first developed a gene-expression-based ER associated index. ER-positive and ER-negative reference signatures, or centroids, were then described as the median log-transformed expression value of each of the 200 reporter genes in the 209 ER-positive and 77 ER-negative subjects, respectively. For new samples, the similarity between the log-transformed 200-gene ER associated gene expression signature with the reference centroids was determined based on Hoeffding's D statistic (Hollander and Wolfe, 1999). D takes into account the joint rankings of the two variables and thus provides a robust measure of association that, unlike correlation-based statistics, will detect nonmonotonic associations (in statistical terms, it detects a much broader class of alternatives to independence than correlation-based statistics). The ER reporter index (RI) was defined as the difference between the similarities with the ER+ and ER− reference centroids: RI=D⁺−D⁻.

The 200-gene signature of a tumor with high ER-dependent transcriptional activity resembles more closely the ER-positive centroid and therefore D⁺ will be greater than D⁻ and RI will be positive. The opposite will be the case for tumors with low ER-related activity and thus RI will be small or negative. Subtraction of D normalizes the reporter index relative to the basal levels of expression of the ER-related genes in ER negative tumors. Because of this and since D is a distribution-free statistic, RI is relatively insensitive to the method used to normalize the microarray data and therefore can be computed across datasets. From the RI, a genomic index of sensitivity to endocrine therapy (SET) was calculated as follows: SET=100(RI+0.2)³. The offset translated RI to mostly positive values and was then transformed to normality using an unconditional Box-Cox power transformation. Finally, the maximum likelihood estimate of the exponent was rounded to the closest integer and the index was scaled to a maximum value of 10.

Statistical Analysis of Distant relapse-free survival (DRFS). Distant relapse-free survival (DRFS) was defined as the interval from breast surgery until diagnosis of distant metastasis. Covariate effects on distant relapse risk after tamoxifen treatment were evaluated using log-rank test in multivariate Cox proportional hazards models stratified by institution. The covariates we included were genomic measurement of likely sensitivity to endocrine therapy (SET index), gene expression levels of estrogen receptor (ESR1, probe set 205225) and progesterone receptor (PGR, probe set 208305), age at diagnosis, tumor histologic grade and tumor stage (revised American Joint Committee on Cancer (AJCC) staging system). ESR1 was normally distributed, but PGR levels were log-transformed to normality. To determine the continuous relation between the SET index and 10-year DRFS, the data were fitted by Cox proportional hazards models having a smoothing spline approximation with 2 degrees of freedom of the SET index as the only covariate (Therneau and Grambsch, 2000). The baseline cumulative hazard rate was estimated from the Cox model based on the Nelson-Aalen estimator and the predicted rate of distant relapse was then obtained from the Breslow-type estimator of the survival function. Confidence intervals of the survival estimate were calculated based on the Tsiatis variance estimates of the cumulative log-hazards (Therneau and Grambsch, 2000). A similar approach was used to determine the continuous relation between ESR1 and PGR expression and DRFS.

Likely sensitivity to endocrine therapy was classified as low, intermediate, or high using cutoff points of the SET index values determined by fitting on the entire dataset (n=277) a stratified multivariate Cox model to predict DRFS in relation to age, histologic grade, stage, median-dichotomized ESR1, median-dichotomized PGR, and the trichotomous SET indicator variable using different thresholds. Thresholds that resulted in maximum or near maximum log-profile likelihood for this model were selected as most informative cut points for predicting DRFS (Tableman and Kim, 2004). The same thresholds were maintained for subsequent analyses of the untreated patients. All statistical computations were performed in R(R Development Core Team, 2005).

Example 2

Correlation Between ER mRNA Expression Levels and ER Status

Intensity values of ESR1 (ER) gene expression from microarray experiments were compared to the results from standard IHC and enzyme immunoassays in 82 FNA samples (MDACC). The Affymetrix U133A GeneChip™ has six probe sets that recognize ESR1 mRNA at different sequence locations. A comparison of the different probe sets using the 82 FNA dataset is presented in Table 3. All the ESR1 probe sets showed high correlation with ER status determined by immunohistochemistry (Kruskal-Wallis test, p<0.0001). The probe set 205225_had the highest mean, median, and range of expression and was most correlated with ER status (Spearman's correlation, R=0.85, Table 3).

TABLE 3
The mean, median, and range of expression of the six probe sets that
identify ERα gene (ESR1) are compared using the results from 82 FNA
samples. Expression of each ESR1 probe set is correlated to ER status
(positive, low, or negative) and to the expression of the ESR1 205225—
probe set (R values, Spearman‘s rank correlation test).
Probe Set		Spearman
ER	Signal Intensity	Correlation With
ESR1	Mean	Median	Range	ER Status	205225—
205225—	1633	912	6802	0.85	1.00
215552—	192	136	671	0.81	0.86
217190—	152	122	429	0.72	0.84
211233—	234	178	663	0.71	0.88
211235—	189	139	674	0.69	0.88
211234—	236	209	462	0.64	0.83

Example 3

ER Reporter Genes

The consistency of identifying top-ranking genes depends on factors that affect the sampling variability in the correlation coefficient, such as the size of the dataset and the strength of the underlying true association between the candidate genes and ESR1. The inventors evaluated the consistency in the ranking of the candidate ER reporter genes in terms of the selection probability estimated from 1000 bootstrapped datasets. FIG. 1 shows that the selection probability was high for the top-ranking probes, i.e., the top-ranking probes rank consistently at the top of the list, but it diminished quickly with increasing rank. Furthermore, the selection probability of a candidate gene of a given rank showed a strong dependence on the number of candidate probes selected. For example, the probability of consistently selecting the truly top 50 ER-associated probes was 98.5% if the top 200 candidate probes are selected, 87.0% if the top 100 probes are selected, and only 41.3% if the top 50 probes are selected (FIG. 1). Based on these considerations, the inventors defined the ER reporter list to include the 200 top-ranking probes to ensure that the 100 most-strongly associated probes with ESR1, which are expected to be biologically relevant, would be among the reporter genes with about 90% probability. The entire list included 200 probe sets (excluding those that detect ESR1) representing 163 different genes and 7 uncharacterized transcripts (Table 1).

Example 4

ER Reporter Index is Independent of ESR1 Expression

The ER reporter index (RI) was calculated for the tamoxifen-treated group and the node-negative untreated group. The RI was predominantly positive in ER-positive subjects and predominately negative in ER-negative subjects with the two ER-conditional distributions being distinct and well separated (FIGS. 3A and 3B), which supports ER RI as an indicator of ER-associated activity. To evaluate whether the levels of ER RI are correlated with ESR1 mRNA expression levels, the RI was plotted vs. ESR1 expression for both groups (FIGS. 3C and 3D). Although both ESR1 mRNA and RI were lower in ER-negative subjects, there was no apparent trend in ER-positive subjects. This suggests that, even though the estrogen reporter genes were identified as being co-expressed with ESR1, the overall expression pattern of this group of genes as captured by the ER reporter index conveys information on ER-signaling that is not captured by ESR1.

Example 5

Reproducibility of Reporter Genes and SET Index

The in vivo transcription and microarray hybridization steps were repeated using residual sample RNA from 35 FNA samples. The 35 original and replicate sample pairs demonstrated excellent reproducibility of the gene expression measurements and calculated indices (FIG. 4). The concordance correlation coefficients were (Lin, 1989; 2000): 0.979 (95% CI 0.958-0.989) for the pairs of ESR1 expression measurements, 0.953 (95% CI 0.909-0.976) for PGR expression, 0.985 (95% CI 0.972-0.992) for ER reporter index values, and 0.972 (95% CI 0.945-0.986) for the pairs of SET index measurements exhibiting excellent accuracy (minimal deviation of the best fit line from the 45° line) and good precision in all cases.

Example 6

Characterization of ER Reporter Genes

The 200 ER reporter probe sets represent 163 unique genes and 7 uncharacterized transcripts (Table 1). These contain twenty-seven probe sets that represent 23 genes on chromosome 5, and 20 probe sets that represent 18 genes on chromosome 1. Mapping the 163 genes to the KEGG pathway database indicated representation of several signaling pathways including focal adhesion, Wnt, Jak-STAT, and MAPK signaling pathways. Furthermore, mapping to gene ontology (GO) categories indicated that the biological processes “fatty acid metabolism,” “pyrimidine ribonucleotide biosynthesis,” and “apoptosis” are over-represented in this set relative to chance based on the hypergeometric test (p-values<0.03). The distributions of reporter genes for ER-positive and ER-negative breast cancers were distinct and well separated, consistent with an indicator of ER-associated activity (FIGS. 3A and 3B). Both ESR1 and reporter genes were lower in ER-negative subjects, but there was no apparent correlation in ER-positive subjects (FIGS. 3C and 3D). Therefore, although the ER reporter genes were identified by their co-expression with ESR1, the overall expression pattern of this group of genes (as captured by the index) conveys information on ER-signaling that is independent of ER gene expression level alone.

Example 7

Distant Relapse after Adjuvant Tamoxifen Therapy

Univariate Cox proportional hazards models were employed to evaluate the risk of distant relapse at 10 years after adjuvant tamoxifen treatment as continuous functions of expression levels of the estrogen receptor gene (ESR1), progesterone receptor gene (PGR), and the 200-gene index of reporter genes for sensitivity to endocrine therapy (SET index) (FIG. 5). ER gene expression (ESR1, FIG. 5A) was not a significant predictor of 10-year relapse rate (LRT p=0.16), but higher progesterone receptor gene expression (PGR, FIG. 5B) was significantly associated with lower relapse rates at 10 years (HR 0.62; 95% CI 0.44-0.88; LRT p=0.005). Higher SET index levels (FIG. 5C) were also significantly associated with lower 10-year relapse rates (HR 0.70; 95% CI 0.56-0.86; LRT p<0.001). The mean relapse-free survival at 10 years for subjects with SET index<2 was 57.1% (95% CI 41.1-80.3) whereas for those with SET index>5 was 90.0% (95% CI 82.5-97.7) (FIG. 5C).

Example 8

Distant Relapse in Untreated Patients—SET Index is Independent of Prognosis

To address the possibility that observed differences in DRFS could be due to indolent prognosis, rather than benefit from adjuvant tamoxifen, the same covariates were evaluated as potential prognostic factors of DRFS in 209 ER-positive patients who did not receive adjuvant systemic therapy. Consistent with the effects in the tamoxifen treated group, ER expression level (ESR1, FIG. 6A) was not significantly associated with the 5-year relapse rate in untreated patients (LRT p=0.75), and higher progesterone receptor (PGR, FIG. 6B) was significantly associated with lower relapse rates at 5 years (HR 0.78, 95% CI 0.67-0.90; LRT p<0.001). However, the effect of the SET index (FIG. 6C) on the 5-year relapse rate in untreated patients was small and marginally significant (HR 0.90, 95% CI 0.82-1.00; LRT p=0.043).

Example 9

Independence of Genomic Predictors in Multivariate Survival Analyses

The continuous gene-expression-based predictors (ESR1, PGR, and SET index) were evaluated in a multivariate Cox model in relation to patient's age, tumor histologic grade and tumor AJCC stage for ER-positive patients treated with adjuvant tamoxifen. SET index was a significant predictor of relapse after adjuvant tamoxifen treatment (HR 0.72; 95% CI 0.54-0.95), whereas the effect of PGR expression was not statistically significant (Table 4, Treated Patients). Conversely, when patients with ER-positive breast cancer who did not receive adjuvant treatment were evaluated with the same multivariate model, it was found that PGR expression was independently prognostic (HR 0.72; 95% CI 0.58-0.89), whereas the effect of SET index was not statistically significant (Table 4, Untreated Patients). Therefore the SET index was independently predictive of benefit from adjuvant tamoxifen therapy, but not prognostic in patients with ER-positive breast cancer who did not receive adjuvant treatment.

TABLE 4
Multivariate Cox analysis of continuous gene-expression-based covariates of
DRFS in patients with ER-positive breast cancer. Treated patients (left column) received
adjuvant tamoxifen, whereas untreated patients (right column) had node-negative disease
and did not receive adjuvant treatment. ‡PGR expression values were log-transformed.
	Treated Patients (n=211)	Untreated Patients (n=142)
Effect	HR (95% CI)	P-value	HR (95% CI)	P-value
Age	1.09 (0.30-3.90)	0.89	0.59 (0.31-1.11)	0.10
>50 vs. ≦ 50
Histologic Grade	1.09 (0.54-2.22)	0.81	1.93 (0.92-4.04)	0.08
3 vs. 1 or 2
AJCC Stage	1.96 (0.80-4.78)	0.14	1.13 (0.64-1.97)	0.68
II or III vs. I
ER Expression	1.00 (1.00-1.00)	0.72	1.00 (1.00-1.00)	0.13
PGR Expression	0.93 (0.61-1.40)	0.72	0.72 (0.58-0.89)	0.002
Sensitivity to Endocrine	0.72 (0.54-0.95)	0.022	0.99 (0.86-1.14)	0.86
Therapy Index

The SET index was developed to measure ER-related gene expression in breast cancer samples with a hypothesis that this would represent intrinsic endocrine sensitivity. The inventors found that SET index had a steep and linear association with improved 10-year relapse-free survival in women who received tamoxifen as their only adjuvant therapy (FIG. 2), and was the only significant factor in multivariate analysis of DRFS that included grade, stage, age, and expression levels of ESR1 and PGR (Table 4). The information from SET index is mostly predictive of benefit from endocrine treatment, rather than prognosis (FIG. 6, Table 4).

Example 10

Classes of Endocrine Sensitivity Defined By Set Index

The almost linear functional dependence of the likelihood of distant relapse on the genomic endocrine sensitivity (SET) index (FIG. 5C) makes it possible to define three classes by specifying two cut points. Optimal thresholds were chosen to maximize the predictability of the trichotomous SET index in a multivariate Cox model, and occurred at the 50^th and 65^th percentiles of SET distribution corresponding to index values 3.71 and 4.23, respectively. The three classes of predicted sensitivity to endocrine therapy (low, intermediate, and high sensitivity) were evaluated in a multivariate Cox model stratified by institution that included dichotomized age, histologic grade, AJCC stage, and the median-dichotomized gene expression of ESR1 and PGR. The likelihood of distant relapse after tamoxifen therapy was significantly lower in those in the high SET group, compared with the low SET group (HR=0.24, 95% CI 0.09-0.59, p=0.002). There was no significant difference between intermediate and low SET groups (HR=0.67; 95% CI 0.30-1.49; p=0.33).

Example 11

SET Index and Classes Correlate with Distant Relapse-Free Survival

Kaplan-Meier estimators of relapse-free survival were compared for the three classes of SET index in the patients with ER-positive breast cancer who received adjuvant tamoxifen (FIG. 7A) with those who did not receive adjuvant therapy (FIG. 7B). The 35% of subjects with high SET had improved and sustained survival benefit from adjuvant tamoxifen, whereas the 50% of subjects with low SET did not obtain as much benefit from adjuvant tamoxifen (FIG. 7A). Most interesting were the 15% of subjects with intermediate SET. In the untreated cohort (FIG. 7B), subjects with intermediate SET had similar prognosis to those with low SET. However, in the tamoxifen treated cohort (FIG. 7A), subjects with intermediate SET had similar prognosis to those with high SET for the first 6 to 7 years of follow up. Furthermore, within 2 years after the completion of endocrine therapy these patients with intermediate SET began to experience distant relapse at a rate that was similar to the low SET group during the first 3 to 4 years of follow up (FIGS. 7A and 7B). Finally, the Kaplan-Meier estimators of relapse-free survival based on PGR expression (FIGS. 3C and 3D) confirm the combined prognostic and predictive effects of PGR (also shown in FIGS. 5B and 6B) and demonstrate less pronounced separation of the survival curves than SET in tamoxifen treated subjects (FIGS. 7A and 7C).

The inventors observed the same effects of SET class on DRFS of patients treated with adjuvant tamoxifen when the inventors stratified this cohort by known nodal status and separately evaluated the three classes of SET index in 115 node-negative patients (FIG. 8A) and 140 node-positive patients (FIG. 8B). These three classes of SET appear to identify approximately 35% of patients who have sustained benefit from adjuvant tamoxifen alone, approximately 50% who have minimal benefit from tamoxifen, and approximately 15% of patients whose benefit from tamoxifen continues during their adjuvant treatment, but is not sustained after endocrine therapy is completed.

Patients with high endocrine sensitivity (SET index values in upper 35%) had sustained benefit from adjuvant tamoxifen, compared to untreated patients (FIG. 7). This effect was evident when comparing untreated prognosis with tamoxifen treatment in node-negative patients (FIGS. 7B and 8A). Rare relapse events during tamoxifen treatment might still occur because of individual differences in compliance, metabolism due to variant genotype of cytochrome p450 2D6, or interaction from selective serotonin reuptake inhibitors used as antidepressants or to treat hot flashes. These can limit metabolism of tamoxifen to more active metabolites, thereby decreasing treatment efficacy, and are obviously unrelated to the activity of ER in the breast cancer cells (Stearns et al., 2003; Jin et al., 2005). Patients with low SET index values (lower 50%) derived minimal benefit from adjuvant tamoxifen, irrespective of nodal status (FIGS. 11 and 12). The effect of adjuvant tamoxifen (compared to untreated prognosis) is particularly revealing for patients with intermediate SET index (FIG. 7). These patients derived benefit from tamoxifen during their adjuvant treatment, but relinquished this survival benefit after cessation of treatment. Subjects with intermediate SET index started to accrue distant relapse events within 2 years of discontinuing adjuvant tamoxifen, and at a rate that was similar to the subjects with low SET index (treatment or prognosis) in the early period of follow up. This suggests that intermediate SET index values identified patients who might benefit from prolonged and/or more effective endocrine therapy used in current crossover treatment strategies (Goss et al., 2003).

Example 12

SET Index and Chemotherapy Response in ER-Positive Breast Cancer

Groups with low, intermediate, and high SET index were compared with pathologic response outcome in the 82 patients with ER-positive breast cancer who received neoadjuvant chemotherapy with paclitaxel (12 weekly cycles) followed by fluorouracil, doxorubicin, and cyclophosphamide (4 cycles q3 weeks) (Ayers et al., 2004). The same SET classes were as for the survival analyses after adjuvant tamoxifen. There were 8 patients with ER-positive cancer who achieved pathologic complete response (pCR) in the breast and axilla, of which 7 had low SET and one had intermediate SET (Table 5). Conversely, none of the 11 patients with ER-positive breast cancer and high SET, and only one of 11 patients with intermediate SET, achieved pCR from neoadjuvant T/FAC chemotherapy (Table 5).

TABLE 5
Pathologic response to neoadjuvant T/FAC chemotherapy in ER-positive
patients compared with predicted sensitivity to endocrine therapy
(SET risk groups).
	Chemotherapy Response (ER + patients)
Sensitivity to
Endocrine Therapy	Compete Pathologic
(SET) Group	Response	Residual Disease
Low	7	53
Intermediate	1	10
High	0	11

Example 13

SET Index and Stage of ER-Positive Cancer

There was a progressive decline in the values for the sensitivity to endocrine therapy (SET) index with increasing AJCC stage of ER-positive breast cancers (FIG. 8A, p<0.001). The decrease is only marginally significant for the transcriptional levels of ESR1 (FIG. 8B, p=0.04) and PGR (FIG. 8C, p=0.05), whereas the transcriptional level of a housekeeper gene (GAPDH) does not vary with stage (FIG. 8D, p=0.77). This analysis was done for 351 breast cancers that were ER-positive by IHC and had known stage of disease at the time of sample (58 stage I, 123 stage IIA, 107 stage IIB, 44 stage III, and 18 stage IV). The significance of stage-related trends was evaluated by treating tumor stage as an ordinal covariate in ordinary least squares regression with orthogonal polynomial contrasts. The p-values correspond to the significance of the linear term (based on the t-test). All samples from Stage I to III breast cancer were collected prior to any treatment. The 18 samples of Stage IV ER-positive breast cancer were from relapsed disease in 17 patients and at the time of initial presentation in one, and these included 14 patients who had received previous hormonal treatment with tamoxifen and/or aromatase inhibition. There was no obvious difference in the genomic expression levels of ESR1 or SET index in the 14 patients with Stage IV breast cancer who had received prior hormonal therapy, compared to the 4 who had not (ANOVA p=0.9).

Stage-dependent differences in biomarker measurements have obvious clinical importance, particularly for biomarkers of critical targeted cellular pathways. SET index values successively declined with advancing stage, whereas changes in ESR1 and PGR were less distinct (FIG. 8). One explanation is that tumors with less intrinsic dependence on estrogen are more biologically aggressive, and hence more likely to present with larger size and nodal metastasis. Additionally, biological progression of ER-positive breast cancer probably includes progressive dissociation from estrogen dependence through recruitment of other growth and survival pathways. The SET index captures these important differences in tumor biology with greater acuity than measurements of ER and PR. If significant decrease in genomic SET index values between matched primary tumors and subsequent distant metastases were demonstrated, then SET index could be used to monitor changes in the ER genomic pathway (and endocrine sensitivity) during the course of disease.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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Claims

1. A method of assessing cancer patient sensitivity to treatment comprising the step of preparing a sensitivity to endocrine therapy (SET) index based on expression in a patient sample of one or more ER-related genes selected from Table 1.

2. The method of claim 1, further comprising selecting a treatment based on the SET index.

3. The method of claim 1, wherein the ER-related genes comprise 25 or more ER related genes of Table 1.

4. The method of claim 3, wherein the ER-related genes comprise 50 or more ER related genes of Table 1.

5. The method of claim 4, wherein the ER-related genes comprise 100 or more ER related genes of Table 1.

6. The method of claim 4, wherein the ER-related genes comprise 200 ER related genes of Table 1.

7. The method of claim 1, wherein the SET index includes covariates of tumor size, nodal status, grade, and age.

8. The method of claim 1, wherein the SET index includes evaluation of overall survival (OS).

9. The method of claim 8, wherein the SET index includes evaluation of distant relapse-free survival (DRFS).

10. The method of claim 1, wherein the treatment is a combination of one or more cancer therapy.

11. The method of claim 1, wherein the treatment is hormonal therapy.

12. The method of claim 11, wherein the hormonal therapy is tamoxifen therapy, aromatase inhibitor therapy, or SERM therapy.

13. The method of claim 11, wherein the treatment is chemotherapy.

14. The method of claim 11, wherein the treatment is a combination of hormonal therapy and chemotherapy.

15. The method of claim 1, wherein the patients are diagnosed with early or late-stage cancer.

16. A method of calculating a sensitivity to endocrine treatment (SET) index comprising the steps of: (a) identifying a gene set of one or more estrogen receptor (ER)-related genes indicative of ER transcriptional activity by assessing gene expression in a reference population of tumor samples from cancer patients, defining a reference ER-related gene set; and (b) preparing a calculated index using an assessment of ER-related gene expression in one or more samples relative to the reference ER-relate gene expression.

17. The method of claim 16, further comprising assessing sensitivity of a cancer to therapy using the calculated index.

18. The method of claim 17, wherein the therapy is hormonal therapy or chemotherapy.

19. The method of claim 18, wherein the therapy comprises both hormonal therapy and chemotherapy.

20. The method of claim 19, further comprising selecting a class or individual hormonal therapy.

21. The method of claim 20, wherein the hormonal therapy is tamoxifen therapy, aromatase inhibitor therapy, or SERM therapy.

22. The method of claim 17, further comprising identifying a patient that will benefit from an extended duration of therapy.

23. The method of claim 16, wherein all or part of the reference tumor samples are from patients diagnosed with a hormone sensitive cancer.

24. The method of claim 23, wherein the hormone sensitive cancer is an estrogen sensitive cancer.

25. The method of claim 24, wherein the estrogen-sensitive cancer is breast cancer.

26. The method of claim 16, wherein the gene set comprises 25 to 200 ER related genes.

27. The method of claim 26, wherein the gene set comprises 50 to 200 ER related genes.

28. The method of claim 27, wherein the gene set comprises 200 ER related genes.

29. The method of claim 16, wherein the calculated index includes a metric indicative of ER status of all or part of the reference tumor samples.

30. The method of claim 16, wherein the calculated index includes covariates of tumor size, nodal status, grade, and age.

31. The method of claim 16, wherein the calculated index includes evaluation of survival of the patient population sampled for all or part of the reference population of tumor samples.

32. The method of claim 31, wherein calculation of the index includes evaluation of distant relapse-free survival (DRFS) of the patient population.

33. The method of claim 16, wherein the patient population include ER-positive or both ER positive and ER negative samples.

34. The method of claim 16, further comprising normalizing expression data of the one or more samples to the ER-related gene expression profile.

35. The method of claim 34, wherein the expression data is normalized to a digital standard.

36. The method of claim 35, wherein the digital standard is a gene expression profile from a reference sample.

37.-42. (canceled)