Diagnosis, Prognosis and Prediction of Recurrence of Breat Cancer

Abstract

The present invention relates to methods and compositions for the diagnosis, prognosis, and prediction of breast cancer. More specifically, the invention relates to classification of breast cancer tissue samples based on measuring the expression of a set of marker genes. The set is useful for the identification of clinically important breast cancer subtypes. Methods are disclosed for prediction, diagnosis and prognosis of breast cancer.

Description

TECHNICAL FIELD OF THE INVENTION

The present invention relates to methods and compositions for the diagnosis, prognosis, and prediction of breast cancer. More specifically, the invention relates to classification of breast cancer tissue samples based on measuring the expression of a set of marker genes. The set is useful for the identification of clinically important breast cancer subtypes. Methods are disclosed for prediction, diagnosis and prognosis of breast cancer.

BACKGROUND OF THE INVENTION AND PRIOR ART

Breast cancer is one of the leading causes of cancer death in women in western countries. More specifically breast cancer claims the lives of approximately 40,000 women and is diagnosed in approximately 200,000 women annually in the United States alone. Over the last few decades, adjuvant systemic therapy has led to markedly improved survival in early breast cancer (EBCTCG, 1998 a+b). This clinical experience has led to consensus recommendations offering adjuvant systemic therapy for the vast majority of breast cancer patients (Goldhirsch et al., 2003). In breast cancer a multitude of treatment options are available which can be applied in addition to the routinely performed surgical removal of the tumor and subsequent radiation of the tumor bed. Three main and conceptually different strategies are endocrine treatment, chemotherapy and treatment with targeted therapies. Prerequisite for treatment with endocrine agents is expression of hormone receptors in the tumor tissue i.e. either estrogen, progesterone or both. Several endocrine agents with different mode of action and differences in disease outcome when tested in large patient cohorts are available. Tamoxifen is one of the oldest endocrine drugs that significantly reduced the risk of tumor recurrence. Apparently, even more effective are aromatase inhibitors which belong to a new endocrine drug class. In contrast to tamoxifen which is a competitive inhibitor of estrogen binding aromatase inhibitors block the production of estrogen itself thereby reducing the growth stimulus for estrogen receptor positive tumor cells. Recent clinical trials have demonstrated an even better disease outcome for patients treated with these agents compared to patients treated with tamoxifen. Still, some patients experience a relapse despite endocrine treatment and in particular these patients might benefit from additional therapeutic drugs. Chemotherapy with anthracyclines, taxanes and other agents have been shown to be efficient in reducing disease recurrence in estrogen receptor positive as well as estrogen receptor negative patients. The NSABP-20 study compared tamoxifen alone against tamoxifen plus chemotherapy in node negative estrogen receptor positive patients and showed that the combined treatment was more effective than tamoxifen alone. Recently, a systemically administered antibody directed against the Her2neu antigen on the surface of tumor cells have been shown to reduce the risk of recurrence several fold in a patients with Her2neu over expressing tumors.

Yet, most if not all of the different drug treatments have numerous potential adverse effects which can severely impair patients' quality of life (Shapiro and Recht, 2001; Ganz et al., 2002). This makes it mandatory to select the treatment strategy on the basis of a careful risk assessment for the individual patient to avoid over- as well as under treatment.

Arguably, the most important histopathological factor for risk stratification in primary breast cancer is the nodal status (Chia et al., 2004; Fisher et al., 1993; Jatoli et al., 1999). Patients with node-negative breast cancer have a favourable long-term prognosis with 10-years survival rates between 67% and 76% even without adjuvant systemic therapies (Fisher et al., 1993; Chia et al., 2004). To further elucidate the prognosis of this substantial subgroup of patients, several other factors such as the age of the patients, tumor size, estrogen receptor status and histological grade are commonly applied to identify those patients with only a minimal risk of recurrence (Chia et al., 2004). Only in these carefully selected patients can adjuvant systemic therapy be omitted without risk of under treatment (Goldhirsch et al., 2003). However, this group with a minimal risk comprises only very few of all node-negative breast cancer patients. An abundance of potential prognostic factors have been analysed in recent years often in studies with varying quality and sometimes conflicting results (Altman and Lyman, 1998).

More recently, gene expression profiling studies with DNA microarray technologies were able to show distinct subtypes of breast cancer (Perou et al., 2000). Five major subtypes described as luminal type A, luminal type B, basal like, Her2neu like and normal like tumors were identified by two dimensional hierarchical clustering. Luminal type A and B tumors were mainly estrogen receptor positive and basal like tumors estrogen receptor negative. Importantly, in survival analysis the subtypes showed significantly differences in outcome with the basal like and Her2neu tumors having the worst outcome and with luminal like A patients having the best outcome (Sorlie et al, 2001, 2003). However, this “class discovery” approach based on unsupervised two dimensional hierarchical cluster analysis appeared not to be effective for class prediction. First, by this technique tumor samples are ordered in a row according to the calculated similarity and slight variations of the algorithm or distance metrics can result in large differences of sample orders. In addition, inclusion of a few additional samples can have tremendous influence on sample order so that a robust and reproducible classification is difficult. Furthermore, cluster of genes related to putative clinical relevant tumor subclasses have been identified by visual inspection instead of appropriate statistical evaluation. Consequently, neither discovered classes nor genes selected to characterize them allow reproducible and robust classification.

Expression profiles could be linked to prognosis by several investigators using supervised analysis methods that are assumed to be more appropriate for class prediction studies. Van't Veer et al. identified a prognostic signature consisting of 70 respectively 231 genes in a finding cohort of 78 sporadic breast cancers of node negative women younger than 53 years of age (Van't Veer et al., 2002; Van de Vijver et al., 2002). They used a case versus control statistics, with development of metastasis within five years defined as case and disease free survival of more than five years as control, and found that the expression values of at least 70 genes could be used to calculate an average “good prognosis” profile. Unknown tumor samples were classified by correlation of the gene expression of these 70 genes to the good prognosis signature. In a subsequent validation study the significance as a predictor of survival was confirmed (Van de Vijver et al., 2002) although a multicenter external validation study showed that the predictor performed less well as previously published (Piccart et al., SABC presentation 2004). Huang et al., 2003 described gene expression predictors of lymph node status and recurrence. They used k-means clustering of 7030 genes with a target of 500 clusters. For all resulting 496 clusters the dominant singular factor was obtained and used as “metagene” in a tree model analysis. They noted that poor outlook with respect to survival is related to the vigorous proliferative ability of the tumor. Aggregates of distinct groups of genes were capable of predicting lymph node status and patient outcome at least in the small cohort which was used in the analysis. Distinct gene expression alterations were found to be associated with different tumor grades (Ma et al., 2003). Grade I and grade III breast tumors exhibit reciprocal gene expression patterns, whereas grade II tumors exhibit a hybrid pattern of grade I and grade III signatures. Similarly, a gene expression signature differentiating grade I versus grade II tumors was found by another group using a high density single colour gene expression platform. Using this signature, which they called “Genomic Grade Index (GGI)” they showed that the GGI could stratify histological grade II tumors into tumors resembling either more genomic grade I or genomic grade III tumors (Sotiriou et al., 2005). ER-alpha (ER) status is an essential determinant of clinical and biological behaviour of human breast cancers. Generally, patients with ESR1-negative tumors tend to have a worse prognosis than patients with ESR1-positive tumors. The underlying reason for this phenomenon is probably the large genetic difference between these two distinct tumor subtypes. Several gene expression studies found that numerous genes are tightly co-regulated with the estrogen receptor and that the estrogen receptor status might be more reliably determined by measuring ESR1 mRNA than the protein by immunohistochemistry (Dressman et al., 2001). In a previous study two prognostic gene expression profiles have been identified for ER-positive and ER-negative tumors, respectively (Wang et al. 2005). The ER status had been determined by ligand binding assay or immuno-histochemistry. Expression values of 60 probe sets measured by Affymetrix HG U133A oligonucleotide gene chips for ER-positive samples and 16 probe sets for ER-negative samples were used to classify separately both tumor types into a high and low risk prognostic class.

Gene expression profiling not only has been utilized for identification of prognostic genes but also for development of classification algorithms capable of predicting response of a tumor toward a given drug treatment. Gene signatures and corresponding algorithms have been identified for predicting tumor response toward docetaxel based on a 92 gene predictor (Chang et al. 2003), paclitaxel followed by fluorouracil, doxorubicin and cyclophosphamide using a model based on expression values of 74 genes (Ayers et al. 2004) or tamoxifen using a 44 gene signature (Jansen et al. 2005) and a 62 probe set signature (Loi et al., 2005) respectively. In another study, gene expression profiles of tumors of tamoxifen treated patients were used to define a two-gene ratio supposed to be predictive of disease free survival (Ma et al., 2004). However, neither the 44 gene signature nor the two-gene ratio proposed to predict response to tamoxifen could be validated in a subsequent study (Loi et al., 2005). A multigene assay comprising the measurement of 21 genes (16 breast cancer related genes and 5 housekeeping genes) was shown to predict recurrence of tamoxifen-treated breast cancer (Paik et al. 2004). The genes were selected from a limited list of genes derived from the literature and tested for prognostic and predictive power by expression profiling in patient samples. However, since the genes tested comprise only a minor subset of all genes expressed in breast tumour tissue and the panel of 16 breast cancer related genes is strongly biased in that it predominantly measures the degree of proliferation, it is highly likely, that a more comprehensive gene expression profiling approach will yield a better predictor.

Most gene identification methods use per-gene (univariate) statistics such as t-test (Chang et al. 2003), signal to noise ratio (Golub et al. 1999), significance analysis in microarrays SAM (Tusher et al., 2001) or univariate Cox regression (Wang et al. 2005). In recent years, multivariate models have become increasingly popular (Shrunken Centroids (Tibshirani et al., 2001, 2002), KNN (Khan et al. 2002), SVM (Lee 2000, 2001), Artificial Neural Networks (Burke et al., 1995), multivariate Cox Regression (Pawitan et al., 2004; van de Vijver et al., 2002; Li et al., 2003)). The goals remain the same as in the univariate context: to distinguish between two or more different classes and to produce a predictor that can assign a class to a given previously unknown sample while using a minimal set of genes only. Since multivariate models usually allow for geometrically more complex separations, the issue of overfitting the data arises. This is especially a problem if the model has a lot of parameters to be estimated from the training data. Selection of the minimal number of genes needed to successfully capture the nature of the subclasses is also somewhat arbitrary (up to the point of over-fitting the training data) since higher testset accuracy can possibly be achieved by allowing the use of a larger number of genes in the predictor. A disadvantage of most studies using the standard strategy of supervised gene identification is the fact that the corresponding algorithms utilize a high number of genes that are potentially unstable as predictors in the general population. The main reason for this problem can be ascribed to the way how the genes of the classifier are selected. In most cases the number of expression levels measured (p) will exceed the number of patient samples (n) by orders of magnitude (n<<p) so that the selected genes and algorithms are highly prone to over estimating the quality of predictor performance, because the molecular signatures strongly depended on the selection of patients in the gene finding cohort, which may not adequately represent the patient population the classifier is intended for. For instance, with data from the study by van't Veer and colleagues and a gene finding set of the same size as in the original publication (n=78), only 14 of 70 genes from the published signature were included in more than half of 500 signatures generated after multiple randomisation of the training set, although virtually the same gene finding algorithm was used, namely Pearson correlation with binary patient status (Michiels et al. 2005). Furthermore, samples apparently belonging to a different clinical class, e.g. a sample from a patient with an early distant metastasis and another sample from a patient with no metastasis for many years after diagnosis, still might be very similar with regard to their gene expression pattern. The underlying reasons for the different behaviour of tumors with very similar expression profiles might be subtle and difficult to correlate to gene expression. In any case, all these aspects make it very difficult to extract the most informative genes and to build a high performance classifier.

SUMMARY OF THE INVENTION

The present invention is based on the unexpected finding that robust classification of breast tumor tissue samples into clinically relevant subgroups can be achieved by predictors that use a small set of specific marker genes. The idea of the invention is to predict the class of a previously unknown tissue sample (i.e. its gene expression profile) hierarchically by separating a number of mutually disjoint groups of classes at a time (FIG. 1). In each node in this tree (where a partial classification is done), only a very small number of genes is used to reliably distinguish the classes or groups of classes until the sample can uniquely be assigned to a single class (the leaves of the tree structure). One embodiment of the method uses a hierarchical binary classification technique (n=2) involving the computation of in-class-probability for each sample point to each class. In another embodiment, the approach is able to cope with an arbitrary number of classes (n>2) at the same time. The whole set of partial classifiers builds the global classifier. The number of genes used in each partial classifier can be as low as 2, but also larger numbers of genes may be used.

It is an unexpected finding that the overall predictor is robust in the sense that in a random permutation of the sample-to-class mapping for each partial classifier, the best possible classifier on the original data is significantly better than the best one on randomized data.

Compared to the supervised methods mentioned in the previous section, the classification method described in the invention is capable to distinguish between tumours that are genetically very different yet behave very similar with regard to a particular clinical parameter. Furthermore, it uses a much smaller set of genes for class separations and achieves a significantly higher accuracy on test data. In that respect, it out-performs prior classifiers. Special gene sets are provided for the classification of a breast tumor sample into clinically relevant subclasses.

The method comprises:

a) Measuring the expression of genes in a collection of breast tumor specimens.

b) Normalising the raw signal intensities of the gene measurements of each individual array using either signal intensities of housekeeping genes measured on the same array or a global scaling approach, in which all signal intensities of an array multiplied with a factor so that the signal intensities of all arrays of the experiment have the same median (or mean).

c) Filtering for those genes that first, are technically well measurable, e.g. with a median signal intensity higher than background signal+3 standard deviations of repeated background measurements and secondly, variable expressed within said specimen collection, e.g. having a coefficient of variation of larger than 5% for log transformed expression values.

d) Performing an unsupervised principle component analysis (PCA) on conditions (samples) using the selected genes with appropriate computer programs like GeneSpring® (Silicon Genetics, Redwood City, Calif., USA).

e) Displaying the PCA outcome in a two or preferentially three dimensional condition scatter graph using preferentially principal components 1, 2 and 3 (FIG. 1a).

f) Visualising categorical clinical information, e.g. estrogen receptor status, presence and absence of metastasis, clinical grade, or histological tumor type, or numerical clinical information, e.g. time to metastasis, time to local recurrence, or age, in the graphical display, e.g. by colouring the respective classes by discrete or continuous colouring, respectively (FIG. 1b).

g) Identifying clinically relevant subclasses by I) similar clinical characteristics only, II) by similar clinical characteristics and mutual proximity within the PCA. In accordance to f), similarity in clinical characteristics is visualised by similar colours, so it is easy to extract from the visualisation (FIG. 1c).

h) Labelling of the samples according to the identified subclasses. Clinically relevant breast cancer subclasses that have been identified include:

• • Estrogen receptor positive breast tumours with a
  • i. very low likelihood for disease recurrence (FHL++)
  • ii. low likelihood for disease recurrence (FHL+, FHL++, ESR1++)
  • iii. high likelihood for disease recurrence (ESR1 LM, ESR1 EM, ESR1 ER)
  • iv. high likelihood for early disease recurrence (ESR1 ER, ESR1 EM)
  • v. high likelihood for late disease recurrence (ESR1 LM)
  • vi. high likelihood for early distant metastasis (ESR1EM), (FIG. 1d)
  • vii. high likelihood for early local recurrence (ESR1 ER)
  • Estrogen receptor negative breast tumors with a
  • viii. low likelihood for disease recurrence (ESR-A)
  • ix. high likelihood for disease recurrence (ESR-B)
  • x. intermediate likelihood for disease recurrence (ESR-C, ESR-D)

i) Identifying genes suitable for classification of said breast cancer subclasses using t-statistics, signal to noise ratio, fishers exact test, support vector machines or any other method previously described to derive separating genes. Special preference is put on genes whose median expression level across all samples in the collection is above the lower quartile of the medians of all genes measured.

j) In particular, said subclasses may be characterized on the gene expression level by fitting multivariate normal distributions to each subclass, either with distinctly, partial commonly or commonly chosen or estimated distribution parameters, and selecting a prediction class for a previously unknown sample based on the probability distributions and/or pointwise probability of the gene expression values of the sample under investigation used in the distributions of the training clusters (including, but not limited to e.g. the likeliest cluster).

k) Said algorithm may use 2 or more genes or means or medians of gene sets derived prior to classifier training by a grouping procedure such as but not limited to unsupervised clustering or correlation graph analysis.

l) Said algorithm may in parts use univariate gene expression distributions and/or values of single genes, medians or means of gene sets previously derived for partial classification. “Estrogen receptor positive” and “estrogen receptor negative”, within the meaning of the invention, relates to the classification of tumors to one of the classes based on methods like immunohistochemistry (IHC), ligand binding assay (DCC) or ESR1 mRNA measurement of preferentially micro-dissected or macro-dissected tumor tissue.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a depicts the result of an unsupervised principle component analysis of 212 breast tumour samples using variable expressed genes.

FIG. 1 b depicts the result of an unsupervised principle component analysis of 212 breast tumor samples using variable expressed genes coloured according to ESR1 status (1 if signal intensity>1000, 0 if signal intensity ≦1000).

FIG. 1 c depicts the results of an unsupervised principle component analysis of 212 breast tumor samples using variable expressed genes coloured according to time to metastasis (TTM). Samples without metastasis are set to 180 regardless of follow up time.

FIG. 1 d depicts the results of an unsupervised principle component analysis of 212 breast tumor samples using variable expressed genes. A subgroup of estrogen receptor positive tumors with a high likelihood of early metastasis has been labelled (ESR+ EM) based on information provided in FIGS. 1b and 1c.

FIG. 2 depicts an example of a hierarchical classification tree.

FIG. 3 depicts the separation scheme used for an embodiment of the invention.

FIG. 4 depicts the separation scheme used for an embodiment of the invention with reference numerals.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of building a classificator for the classification of breast cancer samples into clinically relevant sub-classes, said method comprising

(a) collecting data on the expression level of a plurality of genes in a plurality of breast tumor samples,

(b) performing an unsupervised principle component analysis on data derived from said data collected under (a),

(c) visualizing the outcome of said principle component analysis under (b),

(d) visualizing categorical clinical information for individual samples in said visualization of step (c),

(e) identifying clinically relevant sub-classes as regions in said visualization of step (d),

(f) identifying marker genes and threshold values for expression levels of said marker genes, suitable for classification of said breast cancer samples into said clinically relevant breast cancer classes.

The present invention further relates to methods of building a classificator for the classification of breast cancer samples into clinically relevant sub-classes, wherein said classification of said breast cancer samples is in a hierarchical classification tree.

Methods of the invention are preferably built exclusively from binary classification steps.

According to another aspect of the invention, said data derived from said data collected under step (a) is obtained by normalization of said collected data.

According to another aspect of the invention, the method further comprises filtering for genes that are technically well measurable and/or variably expressed in said plurality of breast tumor samples.

According to another aspect of the invention said visualization is a visualization of a three-dimensional space, spanned by the first three principle components of said principle component. analysis.

Preferably, said visualization of said categorical clinical information is by using a color code, a symbol code and/or a size code. Different categories are assigned different colors, different shapes (i.e. different symbols), or different sizes of the symbols used for visualization of the PCA results.

The present invention also relates to a system for building a classificator for the classification breast cancer samples into clinically relevant sub-classes, said system being adapted to perform methods of the invention as described above.

Such systems advantageously comprise

(a) means for performing an unsupervised principle component analysis on data derived from gene expression data,

(b) means for visualizing the outcome of said principle component analysis under (a) in a multidimensional space,

(c) means for visualizing categorical clinical information of individual samples in said visualization of (b).

Another aspect of the invention relates to a method for the classification of a breast cancer from a sample of said tumor, said method comprising

(a) assigning the sample to a first aggregate breast cancer class (2) if the sample is ESR(+), or to a second aggregate breast cancer class (3) if the sample is ESR(−),

(b) if said sample is in the first aggregate breast cancer class (2), then

• • (i) assigning the sample to a 3rd (4) or a 4th (5) aggregate breast cancer class, based on marker gene expression;
  • (ii) if said sample is in the 3rd aggregate breast cancer class (4), then assigning the sample to a first (8) or a second (9) elementary breast cancer class, based on marker gene expression;.
  • (iii) if said sample is in the 4th aggregate breast cancer class (5), then assigning the sample to a third (10) or a fourth (11) elementary breast cancer class, based on marker gene expression;

(c) if said sample is in the second aggregate breast cancer class (3), then

• • (i) assigning the sample to a fifth (6) or a 6th (7) aggregate breast cancer class, based on marker gene expression,
  • (ii) if said sample is in the fifth aggregate breast cancer class (6), then assigning the sample to a fifth elementary breast cancer class (12) or a 7th aggregate breast cancer class (13), based on marker gene expression,
  • (iii) if said sample is in said 7th aggregate breast cancer class (13), then assigning the sample to a 6th (16) or 7th (17) elementary breast cancer class
  • (iv) if said sample is in said 6th aggregate breast cancer class, then assigning said sample to an 8th aggregate breast cancer class (14) or to a 10th elementary breast cancer class (15),
  • (v) if said sample is in said 8th aggregate breast cancer class (14), then assigning said sample to an 8th (18) or 9th (19) elementary breast cancer class.

Another aspect of the invention relates to the method described above, wherein

(a) said assigning said sample to a 3rd (4) or 4th (5) aggregate breast cancer class is based on a bivariate classifier using the expression level of two genes selected from Table 1,

(b) said assigning said sample to a first (8) or second (9) elementary breast cancer class is based on a bivariate classifier using the expression level of two genes selected from Table 2,

(c) said assigning said sample to a 3rd (10) or 4th (11) elementary breast cancer class is based on a bivariate classifier using the expression level of two genes selected from Table 3,

(d) said assigning said sample to a 5th (6) or 6th (7) aggregate breast cancer class is based on a bivariate classifier using the expression level of two genes selected from Table 4,

(e) said assigning said sample to a 5th elementary breast cancer class (12) or a 7th aggregate breast cancer class (13) is based on a bivariate classifier using the expression level of two genes selected from Table 5,

(f) said assigning said sample to a 6th (16) or 7th (17) elementary breast cancer class is based on a bivariate classifier using the expression level of two genes selected from Table 6,

(g) said assigning said sample to an 8th aggregate breast cancer class (14) or a 10th elementary breast cancer class (15) is based on a bivariate classifier using the expression level of two genes selected from Table 7,

(h) said assigning said sample to an 8th (18) or 9th (19) elementary breast cancer class is based on a bivariate classifier using the expression level of two genes selected from Table 8.

Another aspect of the invention relates to the above methods, wherein

(a) said assigning said sample to a 3rd (4) or 4th (5) aggregate breast cancer class is based on a bivariate classifier using the expression level of two genes selected from the group consisting of 21821_s_at, 213441_x_at, 214404_x_at and 220192_x_at and 208190_s_at, or selected from the group consisting of 219572_at, 204641_at, 207828_s_at and 219918_s_at, or selected from the group consisting of 202580_x_at, 221436 s_at, 202035_s_at, 202036_s_at and 202037_s_at;

(b) said assigning said sample to a first (8) or second (9) elementary breast cancer class is based on a bivariate classifier using the expression level of 206978_at and 203960_s_at or the absolute expression level of 204502_at and 214433_s_at, or the absolute expression level of 209374_s_at or 206133_at;

(c) said assigning said sample to a 3rd (10) or 4th (11) elementary breast cancer class is based on a bivariate classifier using the expression level of two genes selected from the group consisting of 209392_at, 210839_at, 209135_at and 210896_s_at, or selected from the group consisting of 219777_at and 213508_at, or selected from the group consisting of 218806_s_at, 218807_at and 208370_s_at;

(d) said assigning said sample to a 5th (6) or 6th (7) aggregate breast cancer class is based on a bivariate classifier using the absolute expression level of 208747_s_at and 38158s_at, or 216401_x_at and 204222_s_at, or 214768_x_at and 202238_s_at;

(e) said assigning said sample to a 5th elementary breast cancer class (12) or a 7th aggregate breast cancer class (13) is based on a bivariate classifier using the expression level of 213288_at and 204897_at, or the expression level of two genes selected from the group consisting of 203868_s_at, 203438_at and 203439_s_at, or the expression level of 209374_s_at and 203895_at;

(f) said assigning said sample to a 6th (16) or 7th (17) elementary breast cancer class is based on a bivariate classifier using the absolute expression level of two genes selected from the group consisting of 218468_s_at, 218469_at, 203438_at and 203439_s_at, or selected from the group consisting of 201656_at, 215177_s_at and 201627_s_at, or selected from 219197_s_at and 209291_at;

(g) said assigning said sample to an 8th aggregate breast cancer class (14) or a 10th elementary breast cancer class (15) is based on a bivariate classifier using the absolute expression level of two genes selected from the group consisting of 205479_s_at, 211668_s_at, 203797_at, or selected from the group consisting of 212935_at and 212494_at, or selected from the group consisting of 221530_s_at and 202177_at;

(h) said assigning said sample to an 8th (18) or 9th (19) elementary breast cancer class is based on a bivariate classifier using the absolute expression level of two genes selected from the group consisting of 209714_s_at and 204259_at, or selected from 209200_at and 204041_at, or selected from the group consisting of 202954_at, 208079_s_at, 204092_s_at and 218644_at.

Further aspects of the invention are shown in by way of the following examples.

EXAMPLES

Example 1

Isolation of RNA From Tumor Tissue

RNA Isolation From Frozen Tumour Tissue Sections

Frozen sections were taken for histology and the presence of breast cancer was confirmed in samples from 212 patients. Tumor cell content exceeded 30% in all cases and was above 50% in most cases. Approximately 50 mg of snap frozen breast tumour tissue was crushed in liquid nitrogen. RLT-Buffer (QIAGEN, Hilden, Germany) was added and the homogenate spun through a QIAshredder column (QIAGEN, Hilden, Germany). From the eluate total RNA was isolated by the RNeasy Kit (QIAGEN, Hilden, Germany) according to the manufacturers instruction. RNA yield was determined by UV absorbance and RNA quality was assessed by analysis of ribosomal RNA band integrity on the Agilent Bioanalyzer (Palo Alto, Calif., USA).

Example 2

Determination of Expression Levels

Gene Expression Measurement Utilizing HG-U133A Microarrays of Affymetrix

Starting from 5 μg total RNA labelled cRNA was prepared for all 212 tumour samples using the Roche Microarray cDNA Synthesis, Microarray RNA Target Synthesis (T7) and Microarray Target Purification Kit according to the manufacturer's instruction. In brief, synthesis of first strand cDNA was done by a T7-linked oligo-dT primer, followed by second strand synthesis. Double-stranded cDNA product was purified and then used as template for an in vitro transcription reaction (IVT) in the presence of biotinylated UTP. Labelled cRNA was hybridized to HG-U133A arrays (Santa Clara, Calif., USA) at 45° C. for 16 h in a hybridization oven at a constant rotation (60 r.p.m.) and then washed and stained with a streptavidin-phycoerythrin conjugate using the GeneChip fluidic station. We scanned the arrays at 560 nm using the GeneArray Scanner G2500A from Hewlett Packard. The readings from the quantitative scanning were analysed using the Microarray Analysis Suit 5.0 (MAS 5.0) from Affymetrix. In the analysis settings the global scaling procedure was chosen which multiplied the output signal intensities of each array to a mean target intensity of 500. Array images were visually inspected for defects and quality controlled using the Refiner Software from GeneData. Routinely we obtained over 50 percent present calls per chip as calculated by MAS 5.0.

Example 3

Labelling of Breast Cancer Samples into Subclasses After Principle Component Analysis

All 212*.chp files generated by MAS 5.0 were converted to *.txt Files and loaded into GeneSpring® software (Silicon Genetics, Redwood City, Calif., USA). An experiment group was created using the following normalisation settings. Values below 0.01 were set to 0.01. Each measurement was divided by the 50th percentile of all measurements in that sample. Each gene was divided by the median of its measurements in all samples. If the median of the raw values was below 10 then each measurement for that gene was divided by 10 if the numerator was above 10, otherwise the measurement was thrown out. Next, genes were filtered for quality with regard to the technical measurement. In a first step genes from the default list “all genes”. whose flags in the experiment group were “Present” in at least 10 of the 212 samples were selected for further analysis. Secondly, remaining genes were filtered for variable expression within the experiment group. For that purpose only genes were considered eligible for further analysis when the normalized signal intensity was above 3 or below 0.3 in at least 10 of the 212 samples. Several other cut off values used for filtering of variable genes as well as choosing genes on the basis of coefficient of variation calculations (e.g. >5% for log 2 transformed signal intensities) yielded gene list of similar usefulness for subsequent principal component analysis (PCA).

Example 4

Classification of Breast Cancer Samples Into Subclasses From Expression Levels of Marker Genes

1. The overall classifier on the breast cancer data (n=212 samples (tissue samples) with p˜22k gene expression levels each) was derived in the following steps:

• • a) A separation of the samples was carried out by distinguishing estrogen receptor negative and estrogen receptor positive samples by comparing the absolute, relative or standardized expression level of an estrogen related gene with a thresholding value. In an embodiment of the algorithm, the gene ESR1 was used with a threshold of 1000, yielding estrogen receptor state negative (called ESR− from now on) for ESR1 expressions smaller than 1000 and estrogen receptor state positive (called ESR+ from now on) for ESR1 expressions greater or equal to 1000.
  • b) For the both groups (ESR+ and ESR−) separately, genes with advantageous properties were identified in an unsupervised manner including general quality measures like present calls, minimum expression, minimum median expression, minimum mean expression, standardized variance, normal variance, signal-to-noise ratio and by other means on the raw or processed data (e.g. logarithmized data). In an embodiment of the method, genes were selected to be present in at least 5 samples, to have a minimum mean expression of 250 and a standardized standard deviation exceeding 8% for logarithmised data.
  • c) For each partial predictor, genes may be used single or in groups, where groups of genes are replaced by one or more quantity derived from the group member genes by linear or nonlinear functions of the member genes, including (but not limited to) means, medians, minimum and maximum values or principal components. In an embodiment of the method, genes sets were “pooled” to increase overall stability and take advantage of redundancy of the underlying genetic network. Clusters of co-expressed genes that had a complete correlation graph in terms of Pearson correlation to a minimum threshold of 0.8 were identified. Each “pool” of genes was replaced by a single value (for each tissue sample) by taking the arithmetic average expression of all genes in the pool.
  • d) A separation strategy was chosen by grouping sample labels (e.g. ESR− A,B as one group and ESR− C,D as another). The separation may use a strictly hierarchical approach, direct classification or majority decisions using sets of multiple partial classifiers. In an embodiment of the method, a strictly hierarchical separation strategy was chosen as illustrated in FIG. 3.
  • e) Each partial separation inside ESR− and ESR+ uses a multivariate per-class normal distribution to assign a class to an unknown tissue sample as described in items i), j), k) in the Summary of the Invention chapter. In an embodiment of the method, bivariate normal distributions were used to estimate pointwise in-class probabilities of an unknown sample.
  • f) The parameters of the multivariate distributions can be estimated from the all of the data or a subset thereof using standard statistic methods such as (but not limited to) arithmetic mean (over samples) and covariance (over samples). The parameters of the distribution may be estimated simultaneously (i.e. the value under consideration is expected to be constant over two or more classes) or separately (i.e. the value under consideration is estimated in each class separately). In an embodiment of the method, the mean and the covariance of the distribution were estimated for each class separately.
  • g) Parameters for the distributions may be selected by exhaustive search, steepest descent or other optimization techniques known to a scientist skilled in the art of mathematics with respect to one or more objectives measuring the performance (quality) of each possible classifier. Parameters include linear and nonlinear mappings of one or more gene expression levels. In an embodiment of the method, exhaustive search with respect to the selection of two different gene pools in the meaning of item c) was performed with the objective of minimizing the arithmetic mean of 100 ten-fold cross validation test set misclassification rates. If this objective did not yield a unique (partial) classifier, cross entropy (misclassification error) was computed for the predicted and true classes of the test set samples, and the predictor with the lowest cross entropy was chosen.
  • h) With the optimal set of genes determined by g), parameters of the final partial classifier distribution may be estimated in a way described in f) using either the full or a partial set of available samples. In an embodiment of the method, mean and covariance of the bivariate normal distribution was estimated for each class separately by using all samples bearing the labels under discussion in the partial classifier.

For the separation of (ESR1− A, ESR1− B) against (ESR1− C, ESR1− D), the following partial classifier is used:

• • i) With g₁ being the mean of the binary logarithm of the absolute expression levels of genes 218211_s_at, 213441_x_at, 214404_x_at, and 220192_x_at, and g₂ being the binary logarithm of the absolute expression level of gene 208190_s_at, evaluate

$\begin{array}{c}p_1:=\frac{1}{\sqrt{{\left(2·\pi \right)}^2·\det {\Sigma }_1}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_1\right)}^t{\Sigma }_1^{-1}g-{\mu }_1\right)\\ p_2:=\frac{1}{\sqrt{{\left(2·\pi \right)}^2·\det {\Sigma }_2}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_2\right)}^t{\Sigma }_2^{-1}\left(g-{\mu }_2\right)\right)\\ \mathrm{with}\\ g:=\left(\begin{array}{c}{g}_1\\ g_2\end{array}\right),{\mu }_1:=\left(\begin{array}{c}7.69\\ 10.39\end{array}\right),{\mu }_2:=\left(\begin{array}{c}10.53\\ 9.96\end{array}\right),\\ {\Sigma }_1:=\left(\begin{array}{cc}0.80& -0.073\\ -0.073& 0.32\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}1.37& 0.71\\ 0.71& 0.92\end{array}\right)\end{array}$

• • If p₁>p₂, we assign the unknown sample to the first group of clusters, ESR1− A, ESR1− B, and if not, to the second group of clusters, ESR1− C, ESR1− D.
  • ii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: binary logarithm of raw expression values of 219572_at, g₂: mean of binary logarithms of raw expression values of 204641_at, 207828_s_at, and 219918_s_at, and

${\mu }_1:=\left(\begin{array}{c}8.06\\ 9.78\end{array}\right),{\mu }_2:=\left(\begin{array}{c}9.57\\ 8.48\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.48& 0.0078\\ 0.0078& 0.41\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.44& 0.17\\ 0.17& 0.99\end{array}\right)$

• • iii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: mean of binary logarithms of raw expression values of 202580_x_at and 221436_s_at, g₂: mean of binary logarithms of raw expression values of 202035_s_at, 202036_s_at and 202037_s_at, and

${\mu }_1:=\left(\begin{array}{c}9.49\\ 10.76\end{array}\right),{\mu }_2:=\left(\begin{array}{c}8.12\\ 8.18\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.37& 10.76\\ 0.37& -0.33\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.66& -0.28\\ -0.28& 2.33\end{array}\right)$

• • For the separation of (ESR1− A) against (ESR1− B), the following partial classifier is used:
  • i) With g₁ being the binary logarithm of the absolute expression level of 206978_at and g₂ being the binary logarithm of the absolute expression level of 203960_s_at evaluate

$\begin{array}{c}p_1:=\frac{1}{\sqrt{{\left(2·\pi \right)}^2·\det {\Sigma }_1}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_1\right)}^t{\Sigma }_1^{-1}\left(g-{\mu }_1\right)\right)\\ p_2:=\frac{1}{\sqrt{{\left(2·\pi \right)}^2·\det {\Sigma }_2}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_2\right)}^t{\Sigma }_2^{-1}\left(g-{\mu }_2\right)\right)\\ \mathrm{with}\\ g:=\left(\begin{array}{c}{g}_1\\ g_2\end{array}\right),{\mu }_1:=\left(\begin{array}{c}8.68\\ 8.61\end{array}\right),{\mu }_2:=\left(\begin{array}{c}7.48\\ 8.29\end{array}\right),\\ {\Sigma }_1:=\left(\begin{array}{cc}0.56& -0.20\\ -0.20& 0.55\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.23& -0.034\\ -0.034& 0.18\end{array}\right)\end{array}$

• • If p₁>p₂, we assign the unknown sample to the first cluster, ESR1− A, and if not, to the second cluster, ESR1− B.
  • ii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: binary logarithm of raw expression value of 204502_at, g₂: binary logarithm of raw expression value of 214433_s_at, and

${\mu }_1:=\left(\begin{array}{c}9.36\\ 9.92\end{array}\right),{\mu }_2:=\left(\begin{array}{c}8.58\\ 9.06\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.25& -0.32\\ -0.32& 1.47\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.22& -0.26\\ -0.26& 0.87\end{array}\right)$

• • iii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: binary logarithm of raw expression value of 209374_s_at, g₂: binary logarithm of raw expression value of 206133_at, and

${\mu }_1:=\left(\begin{array}{c}12.48\\ 8.90\end{array}\right),{\mu }_2:=\left(\begin{array}{c}9.90\\ 7.71\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}2.11& -0.075\\ -0.075& 0.67\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}2.97& -0.44\\ -0.44& 0.40\end{array}\right)$

• • For the separation of (ESR1− C) against (ESR1− D), the following partial classifier is used:
  • i) With g₁ being the mean of the binary logarithms of the absolute expression levels of 209392_at and 210839_s_at and g₂ being the mean of the binary logarithms of the absolute expression level of209135_at and 210896_s_at, evaluate

$\begin{array}{c}p_1:=\frac{1}{\sqrt{{\left(2·\pi \right)}^2·\det {\Sigma }_1}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_1\right)}^t{\Sigma }_1^{-1}\left(g-{\mu }_1\right)\right)\\ p_2:=\frac{1}{\sqrt{{\left(2·\pi \right)}^2·\det {\Sigma }_2}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_2\right)}^t{\Sigma }_2^{-1}\left(g-{\mu }_2\right)\right)\\ \mathrm{with}\\ g:=\left(\begin{array}{c}{g}_1\\ g_2\end{array}\right),{\mu }_1:=\left(\begin{array}{c}11.25\\ 8.84\end{array}\right),{\mu }_2:=\left(\begin{array}{c}8.85\\ 10.10\end{array}\right),\\ {\Sigma }_1:=\left(\begin{array}{cc}0.18& 0.26\\ 0.26& 0.64\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.97& -0.052\\ -0.052& 0.85\end{array}\right)\end{array}$

• • If p₁>p₂, we assign the unknown sample to the first cluster, ESR1− C, and if not, to the second cluster, ESR1− D.
  • ii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: binary logarithm of raw expression value of 219777_at, g₂: binary logarithm of raw expression value of 213508_at, and

${\mu }_1:=\left(\begin{array}{c}9.89\\ 9.06\end{array}\right),{\mu }_2:=\left(\begin{array}{c}8.10\\ 10.10\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.13& 0.11\\ 0.11& 0.13\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}1.03& 0.065\\ 0.065& 0.75\end{array}\right)$

• • iii) Another choice for genes, μ₁, μ₂, Σ₁ and Σ₂ is g₁: mean of binary logarithms of raw expression values of 218806_s_at and 218807_at, g₂: binary logarithm of raw expression value of 208370_s_at, and

${\mu }_1:=\left(\begin{array}{c}8.03\\ 10.00\end{array}\right),{\mu }_2:=\left(\begin{array}{c}9.47\\ 9.20\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.13& 0.15\\ 0.15& 0.23\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.62& 0.022\\ 0.022& 0.41\end{array}\right)$

• • For the separation of (ESR1++, ESR1+ ER, ESR1+ EM) against (ESR1+ FHL+, ESR1+ FHL++, ESR1+ LM), the following partial classifier is used:
  • i) With g₁ being the binary logarithm of the absolute expression level of 208747_s_at and g₂ being the binary logarithm of the absolute expression level of 38158_at, evaluate

$p_1:=\frac{1}{\sqrt{{\left(2-\pi \right)}^2-{\det \Sigma }_1}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_1\right)}^t{\Sigma }_1^{-1}\left(g-{\mu }_1\right)\right)$ $p_2:=\frac{1}{\sqrt{{\left(2-\pi \right)}^2-{\det \Sigma }_2}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_2\right)}^t{\Sigma }_2^{-1}\left(g-{\mu }_2\right)\right)$ $\mathrm{with}$ $g:=\left(\begin{array}{c}{g}_1\\ g_2\end{array}\right),{\mu }_1:=\left(\begin{array}{c}10.82\\ 8.28\end{array}\right),{\mu }_2:=\left(\begin{array}{c}12.37\\ 7.54\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}1.13& -0.10\\ -0.10& 0.37\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.23& 0.072\\ 0.072& 0.33\end{array}\right)$

• • If p₁>p₂, we assign the unknown sample to the first group of clusters, ESR1++, ESR1+ ER, ESR1+ EM, and if not, to the second group of clusters, ESR1+ FHL+, ESR1+ FHL++, ESR1+ LM.
  • ii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: binary logarithm of raw expression values of 216401_x_at, g₂: binary logarithm of raw expression values of 204222_s_at, and

${\mu }_1:=\left(\begin{array}{c}6.27\\ 7.41\end{array}\right),{\mu }_2:=\left(\begin{array}{c}9.73\\ 8.43\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}3.79& 0.050\\ 0.050& 0.28\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}1.43& 0.13\\ 0.13& 0.23\end{array}\right)$

• • iii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: binary logarithm of raw expression values of 214768_x_at, g₂: binary logarithm of raw expression values of 202238_s_at, and

${\mu }_1:=\left(\begin{array}{c}7.88\\ 9.73\end{array}\right),{\mu }_2:=\left(\begin{array}{c}10.05\\ 10.91\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}1.36& -0.15\\ -0.15& 0.97\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}1.18& -0.14\\ -0.14& 0.34\end{array}\right)$

• • For the separation of (ESR1++) against (ESR1+ ER, ESR1+ EM), the following partial classifier is used:
  • i) With g₁ being the binary logarithm of the absolute expression level of 213288_at and g₂ being the binary logarithm of the absolute expression level of 204897_at, evaluate

$p_1:=\frac{1}{\sqrt{{\left(2-\pi \right)}^2-{\det \Sigma }_1}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_1\right)}^t{\Sigma }_1^{-1}\left(g-{\mu }_1\right)\right)$ $p_2:=\frac{1}{\sqrt{{\left(2-\pi \right)}^2-{\det \Sigma }_2}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_2\right)}^t{\Sigma }_2^{-1}\left(g-{\mu }_2\right)\right)$ $\mathrm{with}$ $g:=\left(\begin{array}{c}{g}_1\\ g_2\end{array}\right),{\mu }_1:=\left(\begin{array}{c}8.89\\ 7.73\end{array}\right),{\mu }_2:=\left(\begin{array}{c}9.24\\ 8.51\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.15& 0.025\\ 0.025& 0.32\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.85& -0.29\\ -0.29& 0.49\end{array}\right)$

• • If p₁>₂, we assign the unknown sample to the first cluster, ESR1++, and if not, to the second group of clusters, ESR1+ ER, ESR1+ EM.
  • ii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: binary logarithm of raw expression value of 203868_s_at, g₂: mean of binary logarithms of raw expression values of 203438_at and 203439_s_at, and

${\mu }_1:=\left(\begin{array}{c}7.70\\ 11.04\end{array}\right),{\mu }_2:=\left(\begin{array}{c}8.68\\ 10.18\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.24& 0.00063\\ 0.00063& 1.24\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.28& 0.067\\ 0.067& 2.46\end{array}\right)$

• • iii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: binary logarithm of raw expression value of 209374_s_at, g₂: binary logarithm of raw expression value of 203895_at, and

${\mu }_1:=\left(\begin{array}{c}7.47\\ 6.55\end{array}\right),{\mu }_2:=\left(\begin{array}{c}8.96\\ 7.90\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}1.32& 0.30\\ 0.30& 1.04\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}2.25& -0.46\\ -0.46& 1.70\end{array}\right)$

• • For the separation of (ESR1+ ER) against (ESR1+ EM), the following partial classifier is used:
  • i) With g₁ being the mean of the binary logarithms of the absolute expression level of 218468_s_at and 218469_at and g₂ being the mean of the binary logarithms of the absolute expression level of 203438_at and 203439_s_at, evaluate

$p_1:=\frac{1}{\sqrt{{\left(2-\pi \right)}^2-{\det \Sigma }_1}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_1\right)}^t{\Sigma }_1^{-1}\left(g-{\mu }_1\right)\right)$ $p_2:=\frac{1}{\sqrt{{\left(2-\pi \right)}^2-{\det \Sigma }_2}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_2\right)}^t{\Sigma }_2^{-1}\left(g-{\mu }_2\right)\right)$ $\mathrm{with}$ $g:=\left(\begin{array}{c}{g}_1\\ g_2\end{array}\right),{\mu }_1:=\left(\begin{array}{c}7.40\\ 11.08\end{array}\right),{\mu }_2:=\left(\begin{array}{c}8.66\\ 9.06\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}1.24& 0.41\\ 0.41& 1.73\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.77& 0.48\\ 0.48& 1.09\end{array}\right)$

• • If p₁>p₂, we assign the unknown sample to the first cluster, ESR1+ ER, and if not, to the second cluster, ESR1+ EM.
  • ii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: mean of binary logarithms of raw expression values of 201656_at and 215177_s_at, g₂: binary logarithm of raw expression value of 201627_s_at, and

${\mu }_1:=\left(\begin{array}{c}8.94\\ 8.77\end{array}\right),{\mu }_2:=\left(\begin{array}{c}8.17\\ 9.78\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.32& -0.031\\ -0.031& 0.38\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.66& 0.14\\ 0.14& 0.76\end{array}\right)$

• • iii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: binary logarithm of raw expression value of 219197_s_at, g₂: binary logarithm of raw expression value of 209291_at, and

${\mu }_1:=\left(\begin{array}{c}11.69\\ 9.34\end{array}\right),{\mu }_2:=\left(\begin{array}{c}9.76\\ 7.75\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}1.69& -0.55\\ -0.55& 2.12\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}1.60& -0.29\\ -0.29& 1.02\end{array}\right)$

• • For the separation of (ESR1+ FHL+, ESR1+ FHL++) against (ESR1+ LM), the following partial classifier is used:
  • i) With g₁ being the mean of the binary logarithms of the absolute expression level of 205479_s_at and 211668_s_at and g₂ being the binary logarithm of the absolute expression level of 203797_at, evaluate

$p_1:=\frac{1}{\sqrt{{\left(2-\pi \right)}^2-{\det \Sigma }_1}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_1\right)}^t{\Sigma }_1^{-1}\left(g-{\mu }_1\right)\right)$ $p_2:=\frac{1}{\sqrt{{\left(2-\pi \right)}^2-{\det \Sigma }_2}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_2\right)}^t{\Sigma }_2^{-1}\left(g-{\mu }_2\right)\right)$ $\mathrm{with}$ $g:=\left(\begin{array}{c}{g}_1\\ g_2\end{array}\right),{\mu }_1:=\left(\begin{array}{c}9.19\\ 8.61\end{array}\right),{\mu }_2:=\left(\begin{array}{c}10.01\\ 8.08\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.38& 0.11\\ 0.11& 0.28\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.62& 0.25\\ 0.25& 0.22\end{array}\right)$

• • If p₁>p₂, we assign the unknown sample to the first group of clusters, ESR1+ FHL+, ESR1+ FHL++, and if not, to the second cluster, ESR1+ LM.
  • ii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: binary logarithm of raw expression value of 212935_at, g₂: binary logarithm of raw expression value of 212494_at, and

${\mu }_1:=\left(\begin{array}{c}8.49\\ 9.15\end{array}\right),{\mu }_2:=\left(\begin{array}{c}9.30\\ 8.59\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.92& 0.11\\ 0.11& 0.29\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}1.04& 0.31\\ 0.31& 0.097\end{array}\right)$

• • iii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: binary logarithm of raw expression value of 221530_s_at, g₂: binary logarithm of raw expression value of 202177_at, and

${\mu }_1:=\left(\begin{array}{c}10.79\\ 9.23\end{array}\right),{\mu }_2:=\left(\begin{array}{c}10.13\\ 8.55\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.25& 0.026\\ 0.026& 0.23\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.081& -0.11\\ -0.11& 0.19\end{array}\right)$

• • For the separation of (ESR1+ FHL++) against (ESR1+ FHL+), the following partial classifier is used:
  • i) With g₁ being the binary logarithm of the absolute expression level of 209714_s_at and g₂ being the binary logarithm of the absolute expression level of 204259_at, evaluate

$p_1:=\frac{1}{\sqrt{{\left(2-\pi \right)}^2-{\det \Sigma }_1}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_1\right)}^t{\Sigma }_1^{-1}\left(g-{\mu }_1\right)\right)$ $p_2:=\frac{1}{\sqrt{{\left(2-\pi \right)}^2-{\det \Sigma }_2}}·\exp \left(-\frac{1}{2}·{\left(g-{\mu }_2\right)}^t{\Sigma }_2^{-1}\left(g-{\mu }_2\right)\right)$ $\mathrm{with}$ $g:=\left(\begin{array}{c}{g}_1\\ g_2\end{array}\right),{\mu }_1:=\left(\begin{array}{c}7.48\\ 10.03\end{array}\right),{\mu }_2:=\left(\begin{array}{c}8.12\\ 9.20\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.17& -0.074\\ -0.074& 0.21\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.31& 0.33\\ 0.33& 1.16\end{array}\right)$

• • If p₁>p₂, we assign the unknown sample to the first cluster, ESR1+ FHL++, and if not, to the second cluster, ESR1+ FHL+.
  • ii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: binary logarithm of raw expression value of 209200_at, g₂: binary logarithm of raw expression value of 204041_at, and

${\mu }_1:=\left(\begin{array}{c}9.07\\ 11.61\end{array}\right),{\mu }_2:=\left(\begin{array}{c}8.52\\ 10.20\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.24& 0.18\\ 0.18& 0.34\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.19& -0.011\\ -0.101& 2.29\end{array}\right)$

• • iii) Another choice for genes, μ₁, μ₂, Σ₁, and Σ₂ is g₁: mean of binary logarithms of raw expression values of 202954_at, 208079_s_at, and 204092_s_at, g₂: binary logarithm of raw expression value of 218644_at, and

${\mu }_1:=\left(\begin{array}{c}7.52\\ 8.15\end{array}\right),{\mu }_2:=\left(\begin{array}{c}8.24\\ 8.34\end{array}\right),{\Sigma }_1:=\left(\begin{array}{cc}0.16& -0.049\\ -0.049& 0.073\end{array}\right),{\Sigma }_2:=\left(\begin{array}{cc}0.25& -0.099\\ -0.099& 0.31\end{array}\right)$

2. Classification of an unknown sample is done by measuring the gene expression levels of some or all of the genes used in the partial classifiers (including an estrogen receptor related gene), determining the estrogen receptor state and then using one or more partial classifiers to subsequently assign the given unknown probe to one or more class or groups of classes using the partial classifiers obtained on a training set in step 1.

It is to be understood that alternative marker genes can be used for classification according to the present invention, in particular if said alternative marker genes show a similar expression pattern as show those used in the examples above. Alternative marker genes useful in methods and systems of the invention are listed in Tables 1-8 below.

TABLE 1
Genes useful for separation of ESR1-A, ESR1-B <-> ESR1-C, ESR1-D
Affymetrix	GenBank
Probe Set ID	Accession
HG U133A	No	Gene Symbol	Unigene ID
55616_at	AI703342	CAB2	Hs.91668
51158_at	AI801973	—	Hs.27373
32094_at	AB017915	CHST3	Hs.158304
222258_s_at	AF015043.1	SH3BP4	Hs.17667
222039_at	AA292789	LOC146909	Hs.433234
221922_at	AW195581	LGN	Hs.278338
221880_s_at	AI279819	—	Hs.27373
221811_at	BF033007	CAB2	Hs.91668
221521_s_at	BC003186.1	LOC51659	Hs.433180
221505_at	AW612574	LANPL	Hs.71331
221436_s_at	NM_031299	GRCC8	Hs.30114
221185_s_at	NM_025111	DKFZp434B227	Hs.334483
221024_s_at	NM_030777	SLC2A10	Hs.305971
220651_s_at	NM_018518	MCM10	Hs.198363
220625_s_at	AF115403.1	ELF5	Hs.11713
220559_at	NM_001426	EN1	Hs.271977
220425_x_at	NM_017578	ROPN1	Hs.194093
220192_x_at	NM_012391	PDEF	Hs.79414
219959_at	NM_017947	HMCS	Hs.157986
219918_s_at	NM_018123	ASPM	Hs.121028
219768_at	NM_024626	FLJ22418	Hs.36563
219735_s_at	NM_014553	LBP-9	Hs.114747
219582_at	NM_024576	FLJ21079	Hs.16512
219572_at	NM_017954	FLJ20761	Hs.107872
219498_s_at	NM_018014	BCL11A	Hs.130881
219497_s_at	NM_022893	BCL11A	Hs.130881
219157_at	NM_007246	KLHL2	Hs.122967
219148_at	NM_018492	TOPK	Hs.104741
218918_at	NM_020379	MAN1C1	Hs.8910
218870_at	NM_018460	ARHGAP15	Hs.177812
218807_at	NM_006113	VAV3	Hs.267659
218806_s_at	AF118887.1	VAV3	Hs.267659
218782_s_at	NM_014109	PRO2000	Hs.222088
218726_at	NM_018410	DKFZp762E1312	Hs.104859
218665_at	NM_012193	FZD4	Hs.19545
218542_at	NM_018131	C10orf3	Hs.14559
218502_s_at	NM_014112	TRPS1	Hs.26102
218353_at		RGS5	Hs.274368
218331_s_at	NM_017782	FLJ20360	Hs.26434
218298_s_at	NM_024952	FLJ20950	Hs.285673
218211_s_at	NM_024101	MLPH	Hs.297405
218009_s_at	NM_003981	PRC1	Hs.344037
217989_at	NM_016245	RetSDR2	Hs.12150
217901_at	BF031829	—	Hs.348710
216836_s_at	X03363.1	ERBB2	Hs.323910
216092_s_at	AL365347.1	SLC7A8	Hs.22891
215945_s_at	BC005016.1	TRIM2	Hs.12372
215726_s_at	M22976.1	CYB5	Hs.83834
215034_s_at	AI189753	TM4SF1	Hs.409060
214667_s_at	AK026607.1	PIG11	Hs.433813
214404_x_at	AI307915	PDEF	Hs.79414
213441_x_at	AI745526	PDEF	Hs.79414
213260_at	AU145890	—	Hs.284186
213226_at	AI346350	PMSCL1	Hs.91728
213122_at	AI096375	KIAA1750	Hs.173094
213060_s_at	U58515.1	CHI3L2	Hs.154138
212771_at	AU150943	LOC221061	Hs.66762
212730_at	AK026420.1	DMN	Hs.10587
212708_at	AV721987	—	Hs.184779
212594_at	N92498	—	Hs.326248
212510_at	AA135522	KIAA0089	Hs.82432
212458_at	AW138902	LOC200734	Hs.173108
212256_at	BE906572	GALNT10	Hs.107260
211709_s_at	BC005810.1	SCGF	Hs.425339
211657_at	M18728.1	CEACAM6	Hs.73848
210933_s_at	BC004908.1	MGC4655	Hs.381638
210761_s_at	AB008790.1	GRB7	Hs.86859
210605_s_at	BC003610.1	MFGE8	Hs.3745
210559_s_at	D88357.1	CDC2	Hs.334562
209897_s_at	AF055585.1	SLIT2	Hs.29802
209842_at	AI367319	SOX10	Hs.44317
209747_at	J03241.1	TGFB3	Hs.2025
209504_s_at	AF081583.1	PLEKHB1	Hs.380812
209396_s_at	M80927.1	CHI3L1	Hs.75184
209395_at	M80927.1	CHI3L1	Hs.75184
209387_s_at	M90657.1	TM4SF1	Hs.351316
209366_x_at	M22865.1	CYB5	Hs.83834
209173_at	AF088867.1	AGR2	Hs.91011
209071_s_at	AF159570.1	RGS5	Hs.24950
209070_s_at	AI183997	RGS5	Hs.24950
208998_at	U94592.1	UCP2	Hs.80658
208190_s_at	NM_015925	LISCH7	Hs.95697
208103_s_at	NM_030920	LANPL	Hs.71331
208072_s_at	NM_003648	DGKD	Hs.115907
208009_s_at	NM_014448	ARHGEF16	Hs.87435
207843_x_at	NM_001914	CYB5	Hs.83834
207828_s_at	NM_005196	CENPF	Hs.77204
207357_s_at	NM_017540	GALNT10	Hs.107260
206560_s_at	NM_006533	MIA	Hs.279651
205453_at	NM_002145	HOXB2	Hs.2733
205405_at	NM_003966	SEMA5A	Hs.27621
205240_at	NM_013296	LGN	Hs.278338
205044_at	NM_014211	GABRP	Hs.70725
204855_at	NM_002639	SERPINB5	Hs.55279
204825_at	NM_014791	MELK	Hs.184339
204822_at	NM_003318	TTK	Hs.169840
204751_x_at	NM_004949	DSC2	Hs.239727
204641_at	NM_002497	NEK2	Hs.153704
204613_at	NM_002661	PLCG2	Hs.75648
204288_s_at	NM_021069	ARGBP2	Hs.379795
204285_s_at	AI857639	PMAIP1	Hs.96
204259_at	NM_002423	MMP7	Hs.2256
204153_s_at	NM_002405	MFNG	Hs.31939
204146_at	BE966146	PIR51	Hs.24596
204030_s_at	NM_014575	SCHIP1	Hs.61490
204015_s_at	BC002671.1	DUSP4	Hs.2359
203764_at	NM_014750	DLG7	Hs.77695
203706_s_at	NM_003507	FZD7	Hs.173859
203705_s_at	AI333651	FZD7	Hs.173859
203693_s_at	NM_001949	E2F3	Hs.1189
203592_s_at	NM_005860	FSTL3	Hs.433827
203570_at	NM_005576	LOXL1	Hs.65436
203362_s_at	NM_002358	MAD2L1	Hs.79078
203358_s_at	NM_004456	EZH2	Hs.77256
203343_at	NM_003359	UGDH	Hs.28309
203214_x_at	NM_001786	CDC2	Hs.334562
203213_at	AL524035	CDC2	Hs.334562
202996_at	NM_021173	POLD4	Hs.82520
202991_at	NM_006804	STARD3	Hs.77628
202948_at	NM_000877	IL1R1	Hs.82112
202870_s_at	NM_001255	CDC20	Hs.82906
202752_x_at	NM_012244	SLC7A8	Hs.22891
202747_s_at	NM_004867	ITM2A	Hs.17109
202746_at	AL021786	ITM2A	Hs.17109
202589_at	NM_001071	TYMS	Hs.29475
202580_x_at	NM_021953	FOXM1	Hs.239
202412_s_at	AW499935	USP1	Hs.35086
202345_s_at	NM_001444	FABP5	Hs.153179
202342_s_at	NM_015271	TRIM2	Hs.12372
202236_s_at	NM_003051	SLC16A1	Hs.75231
202037_s_at	NM_003012	SFRP1	Hs.7306
202036_s_at	AF017987.1	SFRP1	Hs.7306
202035_s_at	AI332407	SFRP1	Hs.7306
201819_at	NM_005505	SCARB1	Hs.180616
201564_s_at	NM_003088	FSCN1	Hs.118400
201292_at	NM_001067.1	TOP2A	Hs.156346
201291_s_at	NM_001067.1	TOP2A	Hs.156346
201117_s_at	NM_001873	CPE	Hs.75360
201116_s_at	AI922855	CPE	Hs.75360
200824_at	NM_000852	GSTP1	Hs.226795
200783_s_at	NM_005563	STMN1	Hs.406269

TABLE 2
Genes useful for separation of ESR1-A <-> ESR1-B
Affymetrix	GenBank
Probe Set ID HG	Accession
U133A	No	Gene Symbol	Unigene ID
38149_at	D29642	KIAA0053	Hs.1528
34210_at	N90866	CDW52	Hs.276770
219812_at	NM_024070	MGC2463	Hs.323634
219716_at	NM_030641	APOL6	Hs.257352
219630_at	NM_005764	DD96	Hs.271473
219243_at	NM_018326	HIMAP4	Hs.30822
219157_at	NM_007246	KLHL2	Hs.122967
217236_x_at	S74639.1	IGHM	Hs.153261
215603_x_at	AI344075	GGT2	Hs.289098
215189_at	X99142.1	KRTHB6	Hs.278658
214916_x_at	BG340548	IGHM	Hs.153261
214777_at	BG482805	IGKC	Hs.406565
214765_s_at	AK024677.1	ASAHL	Hs.264330
214620_x_at	BF038548	PAM	Hs.83920
214617_at	AI445650	PRF1	Hs.411106
214433_s_at	NM_003944.1	SELENBP1	Hs.334841
214339_s_at	AA744529	MAP4K1	Hs.95424
214239_x_at	AI560455	LOC284106	Hs.184669
213958_at	AW134823	CD6	Hs.81226
213603_s_at	BE138888	RAC2	Hs.367740
213551_x_at	AI744229	LOC284106	Hs.184669
213539_at	NM_000732.1	CD3D	Hs.95327
213193_x_at	AL559122	TRB@	Hs.303157
213036_x_at	Y15724	ATP2A3	Hs.5541
213004_at	AF007150.1	ANGPTL2	Hs.8025
213001_at	AF007150.1	ANGPTL2	Hs.8025
212914_at	AV648364	CBX7	Hs.356416
212588_at	AI809341	PTPRC	Hs.170121
212587_s_at	AI809341	PTPRC	Hs.170121
212538_at	AL576253	zizimini 1	Hs.8021
212415_at	D50918.1	6-Sep	Hs.90998
212314_at	AB018289.1	KIAA0746	Hs.49500
212311_at	AB018289.1	KIAA0746	Hs.49500
212233_at	AL523076	—	Hs.82503
211998_at	NM_005324.1	H3F3B	Hs.180877
211902_x_at	L34703.1	TRA@	Hs.74647
211796_s_at	AF043179.1	TRB@	Hs.303157
211795_s_at	AF198052.1	FYB	Hs.58435
211742_s_at	BC005926.1	EVI2B	Hs.5509
211639_x_at	L23518.1	IGHM	Hs.153261
211417_x_at	L20493.1	—	Hs.352120
211339_s_at	D13720.1	ITK	Hs.211576
211277_x_at	BC004369.1	APP	Hs.177486
211138_s_at	BC005297.1	KMO	Hs.107318
210972_x_at	M15565.1	TRA@	Hs.74647
210915_x_at	M15564.1	TRB@	Hs.303157
210629_x_at	AF000425.1	LST1	Hs.380427
210140_at	AF031824.1	CST7	Hs.143212
210031_at	J04132.1	CD3Z	Hs.97087
210029_at	M34455.1	INDO	Hs.840
209919_x_at	L20490.1	GGTL4	Hs.352119
209879_at	AI741056	SELPLG	Hs.79283
209846_s_at	BC002832.1	BTN3A2	Hs.87497
209827_s_at	NM_004513.1	IL16	Hs.82127
209671_x_at	M12423.1	TRA@	Hs.74647
209670_at	M12959.1	TRA@	Hs.74647
209606_at	L06633.1	PSCDBP	Hs.270
209499_x_at	BF448647	TNFSF13	Hs.54673
209374_s_at	BC001872.1	IGHM	Hs.153261
209355_s_at	AB000889.1	PPAP2B	Hs.432840
209351_at	BC002690.1	KRT14	Hs.355214
209205_s_at	BC003600.1	LMO4	Hs.3844
209083_at	U34690.1	CORO1A	Hs.109606
208284_x_at	NM_013421	GGT1	Hs.401847
208078_s_at	NM_030751	TCF8	Hs.232068
207238_s_at	NM_002838	PTPRC	Hs.170121
207131_x_at	NM_013430	GGT1	Hs.401847
206978_at	NM_000647	CCR2	Hs.395
206666_at	NM_002104	GZMK	Hs.3066
206227_at	NM_003613	CILP	Hs.151407
206150_at	NM_001242	TNFRSF7	Hs.355307
206133_at	NM_017523	HSXIAPAF1	Hs.139262
206118_at	NM_003151	STAT4	Hs.80642
206082_at	NM_006674	P5-1	Hs.1845
205977_s_at	NM_005232	EPHA1	Hs.89839
205965_at	NM_006399	BATF	Hs.41691
205890_s_at	NM_006398	UBD	Hs.44532
205842_s_at	AF001362.1	JAK2	Hs.115541
205831_at	NM_001767	CD2	Hs.89476
205821_at	NM_007360	D12S2489E	Hs.74085
205798_at	NM_002185	IL7R	Hs.362807
205692_s_at	NM_001775	CD38	Hs.66052
205569_at	NM_014398	LAMP3	Hs.10887
205456_at	NM_000733	CD3E	Hs.3003
205306_x_at	AI074145	KMO	Hs.107318
205120_s_at	U29586.1	SGCB	Hs.77501
205060_at	NM_003631	PARG	Hs.91390
204951_at	NM_004310	ARHH	Hs.109918
204949_at	NM_002162	ICAM3	Hs.99995
204912_at	NM_001558	IL10RA	Hs.327
204891_s_at	NM_005356	LCK	Hs.1765
204855_at	NM_002639	SERPINB5	Hs.55279
204834_at	NM_006682	FGL2	Hs.351808
204774_at	NM_014210	EVI2A	Hs.70499
204677_at	NM_001795	CDH5	Hs.76206
204661_at	NM_001803	CDW52	Hs.276770
204655_at	NM_002985	CCL5	Hs.241392
204638_at	NM_001611	ACP5	Hs.1211
204613_at	NM_002661	PLCG2	Hs.75648
204502_at	NM_015474	SAMHD1	Hs.23889
204416_x_at	NM_001645	APOC1	Hs.268571
204279_at	NM_002800	PSMB9	Hs.381081
204205_at	NM_021822	APOBEC3G	Hs.250619
204192_at	NM_001774	CD37	Hs.153053
204141_at	NM_001069	TUBB	Hs.336780
204118_at	NM_001778	CD48	Hs.901
204116_at	NM_000206	IL2RG	Hs.84
203960_s_at	NM_016126	LOC51668	Hs.46967
203951_at	NM_001299	CNN1	Hs.21223
203923_s_at	NM_000397	CYBB	Hs.88974
203853_s_at	NM_012296	GAB2	Hs.30687
203793_x_at	NM_007144	ZNF144	Hs.184669
203760_s_at	U44403.1	SLA	Hs.75367
203233_at	NM_000418	IL4R	Hs.75545
203052_at	NM_000063	C2	Hs.2253
202957_at	NM_005335	HCLS1	Hs.14601
202902_s_at	NM_004079	CTSS	Hs.181301
202664_at	AI005043	—	Hs.24143
202575_at	NM_001878	CRABP2	Hs.183650
202528_at	NM_000403	GALE	Hs.76057
202409_at	X07868	—	Hs.251664
202307_s_at	NM_000593	TAP1	Hs.180062
202273_at	NM_002609	PDGFRB	Hs.76144
202240_at	NM_005030	PLK	Hs.433619
202147_s_at	NM_001550	IFRD1	Hs.7879
202146_at	AA747426	IFRD1	Hs.7879
201858_s_at	J03223.1	PRG1	Hs.1908
201694_s_at	NM_001964	EGR1	Hs.326035
201693_s_at	AV733950	EGR1	Hs.326035
201497_x_at	NM_022844	MYH11	Hs.78344
201450_s_at	NM_022037	TIA1	Hs.239489
201313_at	NM_001975	ENO2	Hs.146580
200824_at	NM_000852	GSTP1	Hs.226795
200632_s_at	NM_006096	NDRG1	Hs.75789
1405_i_at	M21121	CCL5	Hs.241392

TABLE 3
Genes useful for separation of ESR1-C <-> ESR1-D
Affymetrix
Probe Set ID	GenBank
HG U133A	Accession No	Gene Symbol	Unigene ID
58780_s_at	R42449	FLJ10357	Hs.22451
55616_at	AI703342	CAB2	Hs.91668
38149_at	D29642	KIAA0053	Hs.1528
37117_at	Z83838	ARHGAP8	Hs.102336
34210_at	N90866	CDW52	Hs.276770
221811_at	BF033007	CAB2	Hs.91668
221601_s_at	AI084226	TOSO	Hs.58831
220625_s_at	AF115403.1	ELF5	Hs.11713
220425_x_at	NM_017578	ROPN1	Hs.194093
220326_s_at	NM_018071	FLJ10357	Hs.22451
220192_x_at	NM_012391	PDEF	Hs.79414
219812_at	NM_024070	MGC2463	Hs.323634
219777_at	NM_024711	hIAN2	Hs.105468
219471_at	NM_025113	C13orf18	Hs.288708
219411_at	NM_024712	ELMO3	Hs.105861
219395_at	NM_024939	FLJ21918	Hs.282093
219388_at	NM_024915	FLJ13782	Hs.257924
219304_s_at	NM_025208	SCDGF-B	Hs.112885
219143_s_at	NM_017793	FLJ20374	Hs.8562
219127_at	NM_024320	MGC11242	Hs.36529
219010_at	NM_018265	FLJ10901	Hs.73239
218959_at	NM_017409	HOXC10	Hs.44276
218913_s_at	NM_016573	GMIP	Hs.49427
218856_at	NM_016629	TNFRSF21	Hs.159651
218816_at	NM_018214	LANO	Hs.35091
218807_at	NM_006113	VAV3	Hs.267659
218806_s_at	AF118887.1	VAV3	Hs.267659
218805_at	NM_018384	IAN4L1	Hs.26194
218678_at	NM_024609	FLJ21841	Hs.29076
218507_at	NM_013332	HIG2	Hs.61762
218380_at	NM_021730	PP1044	Hs.7212
218211_s_at	NM_024101	MLPH	Hs.297405
218186_at	NM_020387	RAB25	Hs.150826
218180_s_at	NM_022772	EPS8R2	Hs.55016
218145_at	NM_021158	C20orf97	Hs.26802
217904_s_at	NM_012104	BACE	Hs.49349
217767_at	NM_000064	C3	Hs.284394
217236_x_at	S74639.1	IGHM	Hs.153261
216836_s_at	X03363.1	ERBB2	Hs.323910
216381_x_at	AL035413	AKR7A3	Hs.284236
216033_s_at	S74774.1	FYN	Hs.169370
215785_s_at	AL161999.1	CYFIP2	Hs.258503
215726_s_at	M22976.1	CYB5	Hs.83834
215471_s_at	AJ242502.1	MAP7	Hs.146388
214617_at	AI445650	PRF1	Hs.411106
214581_x_at	BE568134	TNFRSF21	Hs.159651
214505_s_at	AF220153.1	FHL1	Hs.239069
214439_x_at	AF043899.1	BIN1	Hs.193163
214404_x_at	AI307915	PDEF	Hs.79414
214175_x_at	BE043700	RIL	Hs.424312
214038_at	AI984980	CCL8	Hs.271387
213620_s_at	AA126728	ICAM2	Hs.433303
213603_s_at	BE138888	RAC2	Hs.367740
213539_at	NM_000732.1	CD3D	Hs.95327
213508_at	AA142942	—	Hs.356665
213457_at	BF739959	—	Hs.379414
213441_x_at	AI745526	PDEF	Hs.79414
213375_s_at	N80918	CG018	Hs.22174
213338_at	BF062629	RIS1	Hs.35861
213193_x_at	AL559122	TRB@	Hs.303157
213160_at	D86964.1	DOCK2	Hs.17211
213005_s_at	D79994.1	KANK	Hs.77546
212827_at	X17115.1	IGHM	Hs.153261
212728_at	AB033058.1	DLG3	Hs.11101
212589_at	BG168858	RRAS2	Hs.206097
212588_at	AI809341	PTPRC	Hs.170121
212587_s_at	AI809341	PTPRC	Hs.170121
212458_at	AW138902	LOC200734	Hs.173108
212382_at	AK021980.1	—	Hs.289068
212187_x_at	NM_000954.1	PTGDS	Hs.8272
211796_s_at	AF043179.1	TRB@	Hs.303157
211795_s_at	AF198052.1	FYB	Hs.58435
211748_x_at	BC005939.1	PTGDS	Hs.8272
211742_s_at	BC005926.1	EVI2B	Hs.5509
211663_x_at	M61900.1	PTGDS	Hs.8272
211564_s_at	BC003096.1	RIL	Hs.424312
211527_x_at	M27281.1	VEGF	Hs.73793
211339_s_at	D13720.1	ITK	Hs.211576
211071_s_at	BC006471.1	AF1Q	Hs.75823
211056_s_at	BC006373.1	SRD5A1	Hs.552
210959_s_at	AF113128.1	SRD5A1	Hs.552
210915_x_at	M15564.1	TRB@	Hs.303157
210896_s_at	AF306765.1	ASPH	Hs.283664
210839_s_at	D45421.1	ENPP2	Hs.174185
210761_s_at	AB008790.1	GRB7	Hs.86859
210547_x_at	L21181.1	ICA1	Hs.167927
210513_s_at	AF091352.1	VEGF	Hs.73793
210399_x_at	U27336.1	FUT6	Hs.32956
210356_x_at	BC002807.1	MS4A1	Hs.89751
210347_s_at	AF080216.1	BCL11A	Hs.130881
210298_x_at	AF098518.1	FHL1	Hs.239069
209842_at	AI367319	SOX10	Hs.44317
209687_at	U19495.1	CXCL12	Hs.385710
209670_at	M12959.1	TRA@	Hs.74647
209633_at	L07590.1	PPP2R3A	Hs.28219
209606_at	L06633.1	PSCDBP	Hs.270
209584_x_at	AF165520.1	APOBEC3C	Hs.8583
209583_s_at	AF063591.1	MOX2	Hs.79015
209522_s_at	BC000723.1	CRAT	Hs.12068
209496_at	BC000069.1	RARRES2	Hs.37682
209392_at	L35594.1	ENPP2	Hs.174185
209366_x_at	M22865.1	CYB5	Hs.83834
209343_at	BC002449.1	FLJ13612	Hs.24391
209337_at	AF063020.1	PSIP2	Hs.82110
209293_x_at	U16153.1	ID4	Hs.34853
209291_at	NM_001546.1	ID4	Hs.34853
209213_at	BC002511.1	CBR1	Hs.88778
209200_at	N22468	MEF2C	Hs.78995
209199_s_at	N22468	MEF2C	Hs.78995
209135_at	AF289489.1	ASPH	Hs.283664
209083_at	U34690.1	CORO1A	Hs.109606
209016_s_at	BC002700.1	KRT7	Hs.23881
209008_x_at	U76549.1	KRT8	Hs.242463
208983_s_at	M37780.1	PECAM1	Hs.78146
208881_x_at	BC005247.1	IDI1	Hs.76038
208370_s_at	NM_004414	DSCR1	Hs.184222
208083_s_at	NM_000888	ITGB6	Hs.57664
207843_x_at	NM_001914	CYB5	Hs.83834
207842_s_at	NM_007359	MLN51	Hs.83422
207808_s_at	NM_000313	PROS1	Hs.64016
207540_s_at	NM_003177	SYK	Hs.74101
207339_s_at	NM_002341	LTB	Hs.890
207238_s_at	NM_002838	PTPRC	Hs.170121
206666_at	NM_002104	GZMK	Hs.3066
206560_s_at	NM_006533	MIA	Hs.279651
206481_s_at	NM_001290	LDB2	Hs.4980
206469_x_at	NM_012067	AKR7A3	Hs.284236
206364_at	NM_014875	KIF14	Hs.3104
206303_s_at	AF191653.1	NUDT4	Hs.355399
206150_at	NM_001242	TNFRSF7	Hs.355307
205980_s_at	NM_015366	ARHGAP8	Hs.102336
205968_at	NM_002252	KCNS3	Hs.47584
205961_s_at	NM_004682	PSIP2	Hs.82110
205926_at	NM_004843	WSX1	Hs.132781
205831_at	NM_001767	CD2	Hs.89476
205821_at	NM_007360	D12S2489E	Hs.74085
205798_at	NM_002185	IL7R	Hs.362807
205455_at	NM_002447	MST1R	Hs.2942
205405_at	NM_003966	SEMA5A	Hs.27621
205267_at	NM_006235	POU2AF1	Hs.2407
205079_s_at	NM_003829	MPDZ	Hs.169378
205049_s_at	NM_001783	CD79A	Hs.79630
205044_at	NM_014211	GABRP	Hs.70725
205024_s_at	NM_002875	RAD51	Hs.343807
204951_at	NM_004310	ARHH	Hs.109918
204949_at	NM_002162	ICAM3	Hs.99995
204942_s_at	NM_000695	ALDH3B2	Hs.87539
204912_at	NM_001558	IL10RA	Hs.327
204784_s_at	NM_022443	MLF1	Hs.85195
204731_at	NM_003243	TGFBR3	Hs.342874
204683_at	NM_000873	ICAM2	Hs.433303
204679_at	NM_002245	KCNK1	Hs.79351
204678_s_at	U90065.1	KCNK1	Hs.79351
204675_at	NM_001047	SRD5A1	Hs.552
204661_at	NM_001803	CDW52	Hs.276770
204615_x_at	NM_004508	IDI1	Hs.76038
204613_at	NM_002661	PLCG2	Hs.75648
204563_at	NM_000655	SELL	Hs.82848
204562_at	NM_002460	IRF4	Hs.82132
204446_s_at	NM_000698	ALOX5	Hs.89499
204442_x_at	NM_003573	LTBP4	Hs.85087
204396_s_at	NM_005308	GPRK5	Hs.211569
204345_at	NM_001856	COL16A1	Hs.26208
204220_at	NM_004877	GMFG	Hs.5210
204198_s_at	AA541630	RUNX3	Hs.170019
204197_s_at	NM_004350	RUNX3	Hs.170019
204192_at	NM_001774	CD37	Hs.153053
204153_s_at	NM_002405	MFNG	Hs.31939
204118_at	NM_001778	CD48	Hs.901
204116_at	NM_000206	IL2RG	Hs.84
204099_at	NM_003078	SMARCD3	Hs.71622
204083_s_at	NM_003289	TPM2	Hs.300772
204061_at	NM_005044	PRKX	Hs.147996
203936_s_at	NM_004994	MMP9	Hs.151738
203921_at	NM_004267	CHST2	Hs.8786
203911_at	NM_002885	RAP1GA1	Hs.433797
203685_at	NM_000633	BCL2	Hs.79241
203666_at	NM_000609	CXCL12	Hs.237356
203549_s_at	NM_000237	LPL	Hs.180878
203548_s_at	BF672975	LPL	Hs.180878
203281_s_at	NM_003335	UBE1L	Hs.16695
203216_s_at	NM_004999	MYO6	Hs.22564
202991_at	NM_006804	STARD3	Hs.77628
202957_at	NM_005335	HCLS1	Hs.14601
202931_x_at	NM_004305	BIN1	Hs.193163
202902_s_at	NM_004079	CTSS	Hs.181301
202890_at	T62571	MAP7	Hs.146388
202889_x_at	T62571	MAP7	Hs.146388
202862_at	NM_000137	FAH	Hs.73875
202790_at	NM_001307	CLDN7	Hs.278562
202555_s_at	NM_005965	MYLK	Hs.211582
202275_at	NM_000402	G6PD	Hs.80206
202147_s_at	NM_001550	IFRD1	Hs.7879
202146_at	AA747426	IFRD1	Hs.7879
202037_s_at	NM_003012	SFRP1	Hs.7306
202036_s_at	AF017987.1	SFRP1	Hs.7306
202035_s_at	AI332407	SFRP1	Hs.7306
201952_at	NM_001627.1	ALCAM	Hs.10247
201951_at	NM_001627.1	ALCAM	Hs.10247
201858_s_at	J03223.1	PRG1	Hs.1908
201849_at	NM_004052	BNIP3	Hs.79428
201688_s_at	BE974098	TPD52	Hs.2384
201650_at	NM_002276	KRT19	Hs.182265
201644_at	NM_003313	TSTA3	Hs.404119
201596_x_at	NM_000224	KRT18	Hs.406013
201540_at	NM_001449	FHL1	Hs.239069
201497_x_at	NM_022844	MYH11	Hs.78344
201211_s_at	AF061337.1	DDX3	Hs.380774
201058_s_at	NM_006097	MYL9	Hs.9615
201030_x_at	NM_002300	LDHB	Hs.234489
200962_at	AI348010	—	Hs.250367

TABLE 4
Genes useful for separation of ESR1++,
ESRl+ ER. ESR1+ EM <-> ESR1+ FHL++.
ESR1+ FHL+. ESR1+ LM
Affymetrix	GenBank
Probe Set ID HG	Accession
U133A	No	Gene Symbol	Unigene ID
38158_at	D79987	ESPL1	Hs.153479
221900_at	AI806793	COL8A2	Hs.353001
221731_x_at	J02814.1	CSPG2	Hs.81800
221730_at	NM_000393.1	COL5A2	Hs.82985
221729_at	NM_000393.1	COL5A2	Hs.82985
221671_x_at	M63438.1	IGKC	Hs.406565
221651_x_at	BC005332.1	IGKC	Hs.406565
221541_at	AL136861.1	DKF2P434B044	Hs.262958
221530_s_at	AB044088.1	BHLHB3	Hs.33829
221447_s_at	NM_031302	LOC83468	Hs.159993
219806_s_at	NM_020179	FN5	Hs.259737
219561_at	NM_016429	COPZ2	Hs.37482
219134_at	NM_022159	ETL	Hs.57958
219091_s_at	NM_024756	ENDOGLYX1	Hs.127216
218039_at	NM_016359	ANKT	Hs.279905
218009_s_at	NM_003981	PRC1	Hs.344037
217890_s_at	NM_018222	PARVA	Hs.44077
217525_at	AW305097	—	Hs.418738
217480_x_at	M20812	—
217428_s_at	X98568	—
217378_x_at	X51887	—
217281_x_at	AJ239383.1	IGHG3	Hs.300697
217157_x_at	AF103530.1	IGKC	Hs.381418
217148_x_at	AJ249377.1	IGLJ3	Hs.102950
217022_s_at	S55735.1	MGC27165	Hs.153261
216984_x_at	D84143.1	IGLJ3	Hs.102950
216576_x_at	AF103529.1	—	Hs.381417
216401_x_at	AJ408433	—
216207_x_at	AW408194	IGKV1D-13	Hs.390427
215646_s_at	R94644	—	Hs.81800
215446_s_at	L16895	LOX	Hs.348385
215388_s_at	X56210.1	HFL2	Hs.296941
215379_x_at	AV698647	IGLJ3	Hs.405944
215176_x_at	AW404894	IGKC	Hs.406565
215121_x_at	AA680302	IGLJ3	Hs.102950
215051_x_at	BF213829	AIF1	Hs.76364
214973_x_at	AJ275469	IGHG3	Hs.300697
214916_x_at	BG340548	IGHM	Hs.153261
214836_x_at	BG536224	IGKC	Hs.406565
214768_x_at	BG540628	IGKC	Hs.406565
214677_x_at	X57812.1	IGLJ3	Hs.102950
214669_x_at	BG485135	IGKC	Hs.406565
213800_at	X04697.1	HF1	Hs.250651
213790_at	W46291	—	Hs.352537
213502_x_at	X03529	LOC91316	Hs.350074
213194_at	BF059159	ROBO1	Hs.301198
213139_at	AI572079	SNAI2	Hs.93005
213095_x_at	AF299327.1	AIF1	Hs.76364
213071_at	AI146848	DPT	Hs.80552
213068_at	AI146848	DPT	Hs.80552
213004_at	AF007150.1	ANGPTL2	Hs.8025
212865_s_at	BF449063	COL14A1	Hs.403836
212764_at	U19969.1	TCF8	Hs.232068
212713_at	R72286	MFAP4	Hs.296049
212671_s_at	BG397856	HLA-DQA1	Hs.198253
212609_s_at	U79271.1	SDCCAG8	Hs.300642
212592_at	AV733266	IGJ	Hs.76325
212489_at	AI983428	COL5A1	Hs.146428
212488_at	AI983428	COL5A1	Hs.146428
212419_at	AL049949.1	FLJ90798	Hs.28264
212298_at	BE620457	NRP1	Hs.69285
212188_at	AF052169.1	LOC115207	Hs.109438
211896_s_at	AF138302.1	DCN	Hs.433989
211813_x_at	AF138303.1	DCN	Hs.433989
211798_x_at	AB001733.1	IGLJ3	Hs.102950
211645_x_at	M85256.1	IGKC	Hs.406565
211644_x_at	L14458.1	IGKC	Hs.406565
211643_x_at	L14457.1	IGKC	Hs.406565
211637_x_at	L23516.1	IGHM	Hs.153261
211571_s_at	D32039.1	CSPG2	Hs.81800
211368_s_at	U13700.1	CASP1	Hs.2490
210982_s_at	M60333.1	HLA-DRA	Hs.76807
210904_s_at	U81380.2	IL13RA1	Hs.285115
210839_s_at	D45421.1	ENPP2	Hs.174185
210072_at	U88321.1	CCL19	Hs.50002
209901_x_at	U19713.1	AIF1	Hs.76364
209687_at	U19495.1	CXCL12	Hs.385710
209542_x_at	M29644.1	IGF1	Hs.85112
209541_at	NM_000618.1	IGF1	Hs.85112
209540_at	NM_000618.1	IGF1	Hs.85112
209496_at	BC000069.1	RARRES2	Hs.37682
209436_at	AB018305.1	SPON1	Hs.5378
209392_at	L35594.1	ENPP2	Hs.174185
209374_s_at	BC001872.1	IGHM	Hs.153261
209335_at	AI281593	DCN	Hs.433989
209138_x_at	M87790.1	IGLJ3	Hs.102950
209047_at	AL518391	AQP1	Hs.76152
208937_s_at	D13889.1	ID1	Hs.75424
208850_s_at	AL558479	THY1	Hs.125359
208747_s_at	M18767.1	C1S	Hs.169756
208131_s_at	NM_000961	PTGIS	Hs.302085
208079_s_at	NM_003158	STK6	Hs.250822
207542_s_at	NM_000385	AQP1	Hs.76152
207480_s_at	NM_020149	MEIS2	Hs.104105
207266_x_at	NM_016837	RBMS1	Hs.241567
207238_s_at	NM_002838	PTPRC	Hs.170121
206584_at	NM_015364	LY96	Hs.69328
206102_at	NM_021067	KIAA0186	Hs.36232
206101_at	NM_001393	ECM2	Hs.35094
205941_s_at	AI376003	COL10A1	Hs.179729
205898_at	U20350.1	CX3CR1	Hs.78913
205392_s_at	NM_004166	CCL14	Hs.20144
205226_at	NM_006207	PDGFRL	Hs.170040
204964_s_at	NM_005086	SSPN	Hs.183428
204963_at	AL136756.1	SSPN	Hs.183428
204955_at	NM_006307	SRPX	Hs.15154
204927_at	NM_003475	C11orf13	Hs.72925
204897_at	NM_000958.1	PTGER4	Hs.199248
204619_s_at	BF590263	CSPG2	Hs.81800
204451_at	NM_003505	FZD1	Hs.94234
204359_at	NM_013231	FLRT2	Hs.48998
204298_s_at	NM_002317	LOX	Hs.432618
204222_s_at	NM_006851	GLIPR1	Hs.64639
204115_at	NM_004126	GNG11	Hs.83381
204092_s_at	NM_003600	STK6	Hs.250822
204052_s_at	NM_003014	SFRP4	Hs.105700
204051_s_at	AW089415	SFRP4	Hs.105700
204036_at	AW269335	EDG2	Hs.75794
203989_x_at	NM_001992	F2R	Hs.128087
203854_at	NM_000204	IF	Hs.36602
203748_x_at	NM_016839	RBMS1	Hs.241567
203666_at	NM_000609	CXCL12	Hs.237356
203325_s_at	AI130969	COL5A1	Hs.146428
203324_s_at	NM_001233	CAV2	Hs.139851
203323_at	BF197655	—	Hs.397414
203088_at	NM_006329	FBLN5	Hs.11494
203083_at	NM_003247	THBS2	Hs.108623
203065_s_at	NM_001753	CAV1	Hs.74034
202995_s_at	NM_006486	FBLN1	Hs.79732
202994_s_at	Z95331	FBLN1	Hs.79732
202954_at	NM_007019	UBE2C	Hs.93002
202766_s_at	NM_000138	FBN1	Hs.750
202723_s_at	AW117498	FOXO1A	Hs.170133
202705_at	NM_004701	CCNB2	Hs.194698
202503_s_at	NM_014736	KIAA0101	Hs.81892
202465_at	NM_002593	PCOLCE	Hs.202097
202381_at	NM_003816	ADAM9	Hs.2442
202311_s_at	NM_000088.1	COL1A1	Hs.434012
202283_at	NM_002615	SERPINF1	Hs.173594
202238_s_at	NM_006169	NNMT	Hs.364345
202095_s_at	NM_001168	BIRC5	Hs.1578
202075_s_at	NM_006227	PLTP	Hs.283007
201787_at	NM_001996	FBLN1	Hs.79732
201431_s_at	NM_001387	DPYSL3	Hs.74566
201430_s_at	W72516	DPYSL3	Hs.74566
201325_s_at	NM_001423	EMP1	Hs.79368

TABLE 5
Genes useful for separation of ESR1++ <-> ESR1+ ER, ESR1+ EM
Affymetrix	GenBank
Probe Set ID HG	Accession
U133A	No	Gene Symbol	Unigene ID
40016_g_at	AB002301	KIAA0303	Hs.432631
221824_s_at	AA770170	MGC26766	Hs.288156
218051_s_at	NM_022908	FLJ12442	Hs.84753
218002_s_at	NM_004887	CXCL14	Hs.24395
217875_s_at	NM_020182	TMEPAI	Hs.83883
213539_at	NM_000732.1	CD3D	Hs.95327
213288_at	AI761250	—	Hs.90797
213193_x_at	AL559122	TRB@	Hs.303157
212588_at	AI809341	PTPRC	Hs.170121
211996_s_at	BG256504	—	Hs.110613
210958_s_at	BC003646.1	KIAA0303	Hs.432631
210916_s_at	AF098641.1	—	Hs.306278
210915_x_at	M15564.1	TRB@	Hs.303157
210096_at	J02871.1	CYP4B1	Hs.687
210072_at	U88321.1	CCL19	Hs.50002
209374_s_at	BC001872.1	IGHM	Hs.153261
205831_at	NM_001767	CD2	Hs.89476
204897_at	NM_000958.1	PTGER4	Hs.199248
204655_at	NM_002985	CCL5	Hs.241392
204118_at	NM_001778	CD48	Hs.901
203895_at	AL535113	—	Hs.348724
203868_s_at	NM_001078	VCAM1	Hs.109225
203439_s_at	BC000658.1	STC2	Hs.155223
203438_at	AI435828	STC2	Hs.155223
202644_s_at	NM_006290	TNFAIP3	Hs.211600
201422_at	NM_006332	IFI30	Hs.14623
201369_s_at	NM_006887	ZFP36L2	Hs.78909

TABLE 6
Genes useful for separation of ESR1+ ER <-> ESR1+ EM
Affymetrix	GenBank
Probe Set ID HG	Accession		Unigene
U133A	No	Gene Symbol	ID
38158_at	D79987	ESPL1	Hs.153479
219197_s_at	AI424243	SCUBE2	Hs.105790
218613_at	NM_018422	DKFZp761K1423	Hs.236438
218469_at	NM_013372	CKTSF1B1	Hs.40098
218468_s_at	AF154054.1	CKTSF1B1	Hs.40098
217022_s_at	S55735.1	MGC27165	Hs.153261
216320_x_at	U37055	—	Hs.349110
215177_s_at	AV733308	ITGA6	Hs.227730
212741_at	AA923354	MAOA	Hs.183109
210559_s_at	D88357.1	CDC2	Hs.334562
209460_at	AF237813.1	NPD009	Hs.283675
209459_s_at	AF237813.1	NPD009	Hs.283675
209291_at	NM_001546.1	ID4	Hs.34853
207414_s_at	NM_002570	PACE4	Hs.170414
206102_at	NM_021067	KIAA0186	Hs.36232
203439_s_at	BC000658.1	STC2	Hs.155223
203438_at	AI435828	STC2	Hs.155223
203355_s_at	NM_015310	EFA6R	Hs.6763
203214_x_at	NM_001786	CDC2	Hs.334562
203213_at	AL524035	CDC2	Hs.334562
201656_at	NM_000210	ITGA6	Hs.227730
201627_s_at	NM_005542	INSIG1	Hs.56205
201037_at	NM_002627	PFKP	Hs.99910

TABLE 7
Genes useful for separation of ESR1+ FHL++,
ESR1+ FHL+ <-> ESR1+ LM
Affymetrix	GenBank
Probe Set ID HG	Accession
U133A	No	Gene Symbol	Unigene ID
222379_at	AI002715	—	Hs.172047
222250_s_at	AK001363.1	DKFZP434B168	Hs.48604
222043_at	AI982754	CLU	Hs.75106
222037_at	AI859865	—	Hs.319215
221872_at	AI669229	RARRES1	Hs.82547
221796_at	AA707199	NTRK2	Hs.47860
221653_x_at	BC004395.1	APOL2	Hs.241412
221645_s_at	M27877.1	ZNF83	Hs.305953
221530_s_at	AB044088.1	BHLHB3	Hs.33829
221521_s_at	BC003186.1	LOC51659	Hs.433180
221188_s_at	NM_014430	CIDEB	Hs.299867
220240_s_at	NM_017905	C13orf11	Hs.27337
219935_at	NM_007038	ADAMTS5	Hs.58324
219918_s_at	NM_018123	ASPM	Hs.121028
219777_at	NM_024711	hIAN2	Hs.105468
219304_s_at	NM_025208	SCDGF-B	Hs.112885
219077_s_at	NM_016373	WWOX	Hs.519
218976_at	NM_021800	JDP1	Hs.260720
218901_at	NM_020353	PLSCR4	Hs.182538
218819_at	NM_012141	DDX26	Hs.58570
218322_s_at	NM_016234	FACL5	Hs.11638
218236_s_at	NM_005813	PRKCN	Hs.143460
218039_at	NM_016359	ANKT	Hs.279905
218009_s_at	NM_003981	PRC1	Hs.344037
217784_at	BE384482	YKT6	Hs.296244
217763_s_at	NM_006868	RAB31	Hs.223025
217762_s_at	BE789881	RAB31	Hs.223025
217179_x_at	X79782.1	IGL@	Hs.405944
217148_x_at	AJ249377.1	IGLJ3	Hs.102950
216984_x_at	D84143.1	IGLJ3	Hs.102950
216384_x_at	AF257099	—
216320_x_at	U37055	—	Hs.349110
215603_x_at	AI344075	GGT2	Hs.289098
215504_x_at	AF131777.1	—	Hs.183475
214594_x_at	BG252666	ATP8B1	Hs.406187
214097_at	AW024383	RPS21	Hs.356317
214016_s_at	AL558875	SFPQ	Hs.180610
213693_s_at	AI610869	MUC1	Hs.89603
213577_at	AA639705	SQLE	Hs.71465
213554_s_at	BG257762	H41	Hs.283690
213158_at	AL049423.1	—	Hs.16193
213156_at	AL049423.1	—	Hs.16193
212981_s_at	BF791738	—	Hs.107479
212935_at	AB002360.1	MCF2L	Hs.25515
212915_at	AL569804	SEMACAP3	Hs.177635
212914_at	AV648364	CBX7	Hs.356416
212865_s_at	BF449063	COL14A1	Hs.403836
212774_at	AJ223321	ZNF238	Hs.69997
212494_at	AB028998.1	TENC1	Hs.6147
212444_at	AA156240	—	Hs.288660
212417_at	BF058944	SCAMP1	Hs.31218
212259_s_at	BF344265	HPIP	Hs.8068
212236_x_at	Z19574	KRT17	Hs.2785
212141_at	X74794.1	MCM4	Hs.154443
211698_at	AF349444.1	CRI1	Hs.75847
211695_x_at	AF348143.1	MUC1	Hs.89603
211668_s_at	K03226.1	PLAU	Hs.77274
211597_s_at	AB059408.1	HOP	Hs.13775
211430_s_at	M87789.1	IGHG3	Hs.300697
211417_x_at	L20493.1	—	Hs.352120
210605_s_at	BC003610.1	MFGE8	Hs.3745
210559_s_at	D88357.1	CDC2	Hs.334562
210235_s_at	U22815.1	PPFIA1	Hs.183648
209948_at	U61536.1	KCNMB1	Hs.93841
209919_x_at	L20490.1	GGTL4	Hs.352119
209906_at	U62027.1	C3AR1	Hs.155935
209897_s_at	AF055585.1	SLIT2	Hs.29802
209791_at	AL049569	PADI2	Hs.33455
209708_at	AY007239.1	DKFZP564G202	Hs.6909
209542_x_at	M29644.1	IGF1	Hs.85112
209541_at	NM_000618.1	IGF1	Hs.85112
209540_at	NM_000618.1	IGF1	Hs.85112
209505_at	AI951185	NR2F1	Hs.374991
209351_at	BC002690.1	KRT14	Hs.355214
209291_at	NM_001546.1	ID4	Hs.34853
209040_s_at	U17496.1	PSMB8	Hs.180062
209016_s_at	BC002700.1	KRT7	Hs.23881
208932_at	BC001416.1	PPP4C	Hs.2903
208767_s_at	AW149681	LAPTM4B	Hs.296398
208284_x_at	NM_013421	GGT1	Hs.401847
208029_s_at	NM_018407	LAPTM4B	Hs.296398
207961_x_at	NM_022870	MYH11	Hs.78344
207847_s_at	NM_002456	MUC1	Hs.89603
207480_s_at	NM_020149	MEIS2	Hs.104105
207131_x_at	NM_013430	GGT1	Hs.401847
206385_s_at	NM_020987	ANK3	Hs.75893
206049_at	NM_003005	SELP	Hs.73800
205882_x_at	AI818488	ADD3	Hs.324470
205875_s_at	NM_016381	TREX1	Hs.278408
205786_s_at	NM_000632	ITGAM	Hs.172631
205668_at	NM_002349	LY75	Hs.153563
205614_x_at	NM_020998	MST1	Hs.349110
205518_s_at	NM_003570	CMAH	Hs.24697
205479_s_at	NM_002658	PLAU	Hs.77274
205450_at	NM_002637	PHKA1	Hs.2393
205253_at	NM_002585	PBX1	Hs.155691
205159_at	AV756141	CSF2RB	Hs.285401
205157_s_at	NM_000422	KRT17	Hs.2785
205051_s_at	NM_000222	KIT	Hs.81665
204971_at	NM_005213	CSTA	Hs.2621
204894_s_at	NM_003734	AOC3	Hs.198241
204787_at	NM_007268	Z39IG	Hs.8904
204686_at	NM_005544	IRS1	Hs.96063
204641_at	NM_002497	NEK2	Hs.153704
204542_at	NM_006456	STHM	Hs.288215
204455_at	NM_001723	BPAG1	Hs.198689
204446_s_at	NM_000698	ALOX5	Hs.89499
204416_x_at	NM_001645	APOC1	Hs.268571
204359_at	NM_013231	FLRT2	Hs.48998
204348_s_at	NM_013410	AK3	Hs.274691
204115_at	NM_004126	GNG11	Hs.83381
204026_s_at	NM_007057	ZWINT	Hs.42650
204006_s_at	NM_000570	FCGR3B	Hs.372679
203954_x_at	NM_001306	CLDN3	Hs.25640
203953_s_at	BE791251	CLDN3	Hs.25640
203892_at	NM_006103	WFDC2	Hs.2719
203851_at	NM_002178	IGFBP6	Hs.274313
203797_at	AF039555.1	VSNL1	Hs.2288
203749_s_at	AI806984	RARA	Hs.361071
203726_s_at	NM_000227	LAMA3	Hs.83450
203698_s_at	NM_001463	FRZB	Hs.153684
203697_at	U91903.1	FRZB	Hs.153684
203590_at	NM_006141	DNCLI2	Hs.194625
203324_s_at	NM_001233	CAV2	Hs.139851
203214_x_at	NM_001786	CDC2	Hs.334562
203213_at	AL524035	CDC2	Hs.334562
203108_at	NM_003979	RAI3	Hs.194691
203065_s_at	NM_001753	CAV1	Hs.74034
203059_s_at	NM_004670	PAPSS2	Hs.274230
203038_at	NM_002844	PTPRK	Hs.79005
202870_s_at	NM_001255	CDC20	Hs.82906
202765_s_at	AI264196	FBN1	Hs.750
202760_s_at	NM_007203	AKAP2	Hs.42322
202705_at	NM_004701	CCNB2	Hs.194698
202555_s_at	NM_005965	MYLK	Hs.211582
202504_at	NM_012101	TRIM29	Hs.82237
202503_s_at	NM_014736	KIAA0101	Hs.81892
202242_at	NM_004615	TM4SF2	Hs.82749
202177_at	NM_000820	MGC5560	Hs.207251
201820_at	NM_000424	KRT5	Hs.433845
201787_at	NM_001996	FBLN1	Hs.79732
201753_s_at	NM_019903	ADD3	Hs.324470
201752_s_at	AI763123	ADD3	Hs.324470
201497_x_at	NM_022844	MYH11	Hs.78344
201461_s_at	NM_004759	MAPKAPK2	Hs.75074
201428_at	NM_001305	CLDN4	Hs.5372
201224_s_at	AU147713	SRRM1	Hs.18192
201212_at	D55696.1	LGMN	Hs.18069
201195_s_at	AB018009.1	SLC7A5	Hs.184601
201034_at	BE545756	ADD3	Hs.324470
200841_s_at	AI475965	EPRS	Hs.55921
200770_s_at	J03202.1	LAMC1	Hs.214982

TABLE 8
Genes useful for separation of ESR1+ FHL++ <-> ESR+ FHL+
Affymetrix	GenBank
Probe Set ID HG	Accession
U133A	No	Gene Symbol	Unigene ID
218644_at	NM_016445	PLEK2	Hs.39957
218451_at	NM_022842	CDCP1	Hs.146170
213364_s_at	AI052536	—	Hs.31834
212914_at	AV648364	CBX7	Hs.356416
210052_s_at	AF098158.1	C20orf1	Hs.9329
209714_s_at	AF213033.1	CDKN3	Hs.84113
209505_at	AI951185	NR2F1	Hs.374991
209200_at	N22468	MEF2C	Hs.78995
208079_s_at	NM_003158	STK6	Hs.250822
206754_s_at	NM_000767	CYP2B6	Hs.1360
204679_at	NM_002245	KCNK1	Hs.79351
204678_s_at	U90065.1	KCNK1	Hs.79351
204259_at	NM_002423	MMP7	Hs.2256
204092_s_at	NM_003600	STK6	Hs.250822
204041_at	NM_000898	MAOB	Hs.82163
202954_at	NM_007019	UBE2C	Hs.93002
201292_at	NM_001067.1	TOP2A	Hs.156346
201291_s_at	NM_001067.1	TOP2A	Hs.156346

LITERATURE

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Claims

1. Method of building a classificator for the classification of breast cancer samples into clinically relevant sub-classes, said method comprising (a) collecting data on the expression level of a plurality of genes in a plurality of breast tumor samples,(b) performing an unsupervised principle component analysis on data derived from said data collected under (a),(c) visualizing the outcome of said principle component analysis under (b),(d) visualizing categorical clinical information for individual samples in said visualization of step (c),(e) identifying clinically relevant sub-classes as regions in said visualization of step (d),(f) identifying marker genes and threshold values for expression levels of said marker genes, suitable for classification of said breast cancer samples into said clinically relevant breast cancer classes.

2. Method of claim 1, wherein said classification of said breast cancer samples is in a hierarchical classification tree.

3. Method of claim 2, wherein said hierarchical classification tree is built exclusively from binary classification steps.

4. Method of claim 1, wherein said data derived from said data collected under (a) is obtained by normalization of said collected data.

5. Method of claim 1, wherein the method further comprises filtering for genes that are technically well measurable and/or variably expressed in said plurality of breast tumor samples.

6. Method of claim 1, wherein said visualization is a visualization of a three-dimensional space, spanned by the first three principle components of said principle component analysis.

7. Method of claim 1, wherein said visualization of said categorical clinical information is by using a color code, a symbol code and/or a size code.

8. A system for building a classificator for the classification breast cancer samples into clinically relevant sub-classes, said system being adapted to perform the method of claim 1.

9. A system of claim 8, said system comprising (a) means for performing an unsupervised principle component analysis on data derived from gene expression data,(b) means for visualizing the outcome of said principle component analysis under (a) in a multidimensional space,(c) means for visualizing categorical clinical information of individual samples in said visualization of (b).

10. Method for the classification of a breast cancer from a sample of said tumor, said method comprising (a) assigning the sample to a first aggregate breast cancer class (2) if the sample is ESR(+), or to a second aggregate breast cancer class (3) if the sample is ESR(−),(b) if said sample is in the first aggregate breast cancer class (2), then (i) assigning the sample to a 3rd (4) or a 4th (5) aggregate breast cancer class, based on marker gene expression;(ii) if said sample is in the 3rd aggregate breast cancer class (4), then assigning the sample to a first (8) or a second (9) elementary breast cancer class, based on marker gene expression;(iii) if said sample is in the 4th aggregate breast cancer class (5), then assigning the sample to a third (10) or a fourth (11) elementary breast cancer class, based on marker gene expression;(c) if said sample is in the second aggregate breast cancer class (3), then (i) assigning the sample to a fifth (6) or a 6th (7) aggregate breast cancer class, based on marker gene expression,(ii) if said sample is in the fifth aggregate breast cancer class (6), then assigning the sample to a fifth elementary breast cancer class (12) or a 7th aggregate breast cancer class (13), based on marker gene expression,(iii) if said sample is in said 7th aggregate breast cancer class (13), then assigning the sample to a 6th (16) or 7th (17) elementary breast cancer class(iv) if said sample is in said 6th aggregate breast cancer class, then assigning said sample to an 8th aggregate breast cancer class (14) or to a 10th elementary breast cancer class (15),(v) if said sample is in said 8th aggregate breast cancer class (14), then assigning said sample to an 8th (18) or 9th (19) elementary breast cancer class.

11. Method of claim 10, wherein (a) said assigning said sample to a 3rd (4) or 4th (5) aggregate breast cancer class is based on a bivariate classifier using the expression level of two genes selected from Table 1,(b) said assigning said sample to a first (8) or second (9) elementary breast cancer class is based on a bivariate classifier using the expression level of two genes selected from Table 2,(c) said assigning said sample to a 3rd (10) or 4th (11) elementary breast cancer class is based on a bivariate classifier using the expression level of two genes selected from Table 3,(d) said assigning said sample to a 5th (6) or 6th (7) aggregate breast cancer class is based on a bivariate classifier using the expression level of two genes selected from Table 4,(e) said assigning said sample to a 5th elementary breast cancer class (12) or a 7th aggregate breast cancer class (13) is based on a bivariate classifier using the expression level of two genes selected from Table 5,(f) said assigning said sample to a 6th (16) or 7th (17) elementary breast cancer class is based on a bivariate classifier using the expression level of two genes selected from Table 6,(g) said assigning said sample to an 8th aggregate breast cancer class (14) or a 10th elementary breast cancer class (15) is based on a bivariate classifier using the expression level of two genes selected from Table 7,(h) said assigning said sample to an 8th (18) or 9th (19) elementary breast cancer class is based on a bivariate classifier using the expression level of two genes selected from Table 8.

12. Method of claim 10, wherein (a) said assigning said sample to a 3rd (4) or 4th (5) aggregate breast cancer class is based on a bivariate classifier using the expression level of two genes selected from the group consisting of 218211_s_at, 213441_x_at, 214404_x_at, 220192_x_at and 208190_s_at, or selected from the group consisting of 219572_at, 204641_at, 207828_s_at and 219918_s_at, or selected from the group consisting of 202580_x_at, 221436_s_at, 202035_s_at, 202036_s_at and 202037_s_at;(b) said assigning said sample to a first (8) or second (9) elementary breast cancer class is based on a bivariate classifier using the expression level of 206978_at and 203960_s_at or the absolute expression level of 204502_at and 214433_s_at, or the absolute expression level of 209374_s_at or 206133_at;(c) said assigning said sample to a 3rd (10) or 4th (11) elementary breast cancer class is based on a bivariate classifier using the expression level of two genes selected from the group consisting of 209392_at, 210839_s_at, 209135_at and 210896_s_at, or selected from the group consisting of 219777_at and 213508_at, or selected from the group consisting of 218806_s_at, 218807_at and 208370_s_at;(d) said assigning said sample to a 5th (6) or 6th (7) aggregate breast cancer class is based on a bivariate classifier using the absolute expression level of 208747_s_at and 38158_at, or 216401_x_at and 204222_s_at, or 214768_x_at and 202238_s_at;(e) said assigning said sample to a 5th elementary breast cancer class (12) or a 7th aggregate breast cancer class (13) is based on a bivariate classifier using the expression level of 213288_at and 204897_at, or the expression level of two genes selected from the group consisting of 203868_s_at, 203438_at and 203439_s_at, or the expression level of 209374_s_at and 203895_at;(f) said assigning said sample to a 6th (16) or 7th (17) elementary breast cancer class is based on a bivariate classifier using the absolute expression level of two genes selected from the group consisting of 218468_s_at, 218469_at, 203438_at and 203439_s_at, or selected from the group consisting of 201656_at, 215177_s_at and 201627_s_at, or selected from 219197_s_at and 209291_at;(g) said assigning said sample to an 8th aggregate breast cancer class (14) or a 10th elementary breast cancer class (15) is based on a bivariate classifier using the absolute expression level of two genes selected from the group consisting of 205479_s_at, 211668_s_at, 203797_at, or selected from the group consisting of 212935_at and 212494_at, or selected from the group consisting of 221530 s_at and 202177_at;(h) said assigning said sample to an 8th (18) or 9th (19) elementary breast cancer class is based on a bivariate classifier using the absolute expression level of two genes selected from the group consisting of 209714_s_at and 204259_at, or selected from 209200_at and 204041_at, or selected from the group consisting of 202954_at, 208079_s_at, 204092_s_at and 218644_at.