EP2059615A2 - Procédé de pronostic et diagnostic pour la thérapie d'une maladie - Google Patents

Procédé de pronostic et diagnostic pour la thérapie d'une maladie

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Publication number
EP2059615A2
EP2059615A2 EP07836975A EP07836975A EP2059615A2 EP 2059615 A2 EP2059615 A2 EP 2059615A2 EP 07836975 A EP07836975 A EP 07836975A EP 07836975 A EP07836975 A EP 07836975A EP 2059615 A2 EP2059615 A2 EP 2059615A2
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Prior art keywords
cancer
therapy
genes
expression
gene
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German (de)
English (en)
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Gennadi V. Glinksy
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Ordway Research Institute Inc
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Ordway Research Institute Inc
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Priority claimed from PCT/US2007/008088 external-priority patent/WO2007114896A2/fr
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to diagnostic and prognostic methods and kits for predicting therapy outcome based on the presence or absence in a subject of certain markers.
  • therapy outcome predictors and kits relating thereto can be used for any type of disease state or phenotype, including, but not limited to, cancers, metabolic disorders, immunologic disorders, gastrointestinal disorders, cardiovascular disorder, CNS disorders, circulatory system disorders, blood- related diseases, bone disorders, viral and bacterial disorders, chronic disorders such as arthritis, asthma, diabetes, heart disease, osteoporosis, and aging disorders including Alzheimer's.
  • a wide variety of treatment protocols for cancer and other disease states or phenotypes such as metabolic disorders, immunologic disorders, gastro-intestinal disorders, cardiovascular disorder, CNS disorders, circulatory system disorders, blood-related diseases, bone disorders, viral and bacterial disorders, chronic disorders such as arthritis, asthma, diabetes, heart disease, osteoporosis, and aging disorders including Alzheimer's have been developed in recent years.
  • very aggressive therapy is reserved for late stage diseases due to unwanted side effects produced by such therapy.
  • even such aggressive therapy commonly fails at such a late stage.
  • the ability to identify diseases responsive only to the most aggressive therapies at an earlier stage could greatly improve the prognosis for patients having such diseases.
  • U_5: 1503-1521 (2005) teaches that altered expression of the BMIl oncogene is functionally linked with self-renewal state of normal and leukemic stem cells as well as a poor prognosis profile of an 1 1 -gene death- from-cancer signature predicting therapy failure in patients with multiple types of cancer.
  • the present invention is directed to novel methods and kits for diagnosing the presence of disease states or phenotypes within a patient, such as cancer, metabolic disorders, immunologic disorders, gastro-intestinal disorders, cardiovascular disorder, CNS disorders, circulatory system disorders, blood-related diseases, bone disorders, viral and bacterial disorders, chronic disorders such as arthritis, asthma, diabetes, heart disease, osteoporosis, and aging disorders including Alzheimer's, and for determining whether a subject who has any of such disease states or phenotypes is susceptible to different types of treatment regimens.
  • the cancers to be tested include, but are not limited to, prostate, breast, lung, gastric, ovarian, bladder, lymphoma, mesothelioma, medullablastoma, glioma, and AML.
  • One embodiment of the present invention is directed to a method for diagnosing cancer or other diseases or phenotypes such as metabolic disorders, immunologic disorders, gastrointestinal disorders, cardiovascular disorder, CNS disorders, circulatory system disorders, blood- related diseases, bone disorders, viral and bacterial disorders, chronic disorders such as arthritis, asthma, diabetes, heart disease, osteoporosis, and aging disorders including Alzheimer's, or predicting disease-therapy outcome by detecting the expression levels of multiple markers in the same cell at the same time, and scoring their expression as being above a certain threshold, wherein the markers are from a particular pathway related to cancer, other pathways, or transregulatory SNPs, with the score being indicative of a disease state diagnosis or a prognosis for disease- therapy failure.
  • diseases or phenotypes such as metabolic disorders, immunologic disorders, gastrointestinal disorders, cardiovascular disorder, CNS disorders, circulatory system disorders, blood- related diseases, bone disorders, viral and bacterial disorders, chronic disorders such as arthritis, asthma, diabetes, heart disease, osteoporosis, and aging disorders including Alzheimer's
  • This method can be used to diagnose cancer or predict cancer-therapy outcomes for a variety of cancers.
  • the simultaneous co-expression of at least two markers in the same cell from a subject is a diagnostic for disease states including cancer and a predictor for the subject to be resistant to standard therapy for cancer or other disease states.
  • the markers can come from any pathway involved in the regulation of cancer, including specifically the PcG pathway and the "sternness" pathway.
  • the markers can be mRNA, DNA, or protein.
  • the markers can also be transregulatory SNPs as described herein.
  • markers are found within pathways related to cancer, other pathways, or in transregulatory SNPs, can be used as an assay to diagnose cancer disorders or other disease states and to predict whether a patient already diagnosed with cancer or other disease states will be therapy-responsive or therapy-resistant.
  • An element of the assay is that two or more markers are detected simultaneously within the same cell. Marker detection can be made through a variety of detection means, including bar-coding through immunofluorescence. The markers detected can be a variety of products, including mRNA, DNA, and protein. For mRNA based markers, PCR can be used as a detection means.
  • protein products or gene copy number can be identified through detection means known in the art.
  • the markers detected can be from a variety of pathways related to cancer. Suitable pathways for markers within the scope of the present invention include any pathways related to oncogenesis and metastasis, and more specifically include the Polycomb group (PcG) chromatin silencing pathway and the "sternness" pathway. Additional suitable markers include transregulatory SNPs.
  • the invention is directed to a method for diagnosing cancer or predicting cancer-therapy outcome in a subject, said method comprising the steps of: a) obtaining a sample from the subject, b) selecting a marker from a pathway related to cancer, c) screening for a simultaneous aberrant expression level of two or more markers in the same cell from the sample, and d) scoring their expression level as being aberrant when the expression level detected is above or below a certain detection threshold coefficient, wherein the detection threshold coefficient is determined by comparing the expression levels of the samples obtained from the subjects to values in a reference database of sample phenotypes obtained from subjects with either a known diagnosis or known clinical outcome after therapy, wherein the presence of an aberrant expression level of two or more markers in individual cells and presence of cells aberrantly expressing two or more such markers is indicative of a cancer diagnosis or a prognosis for cancer-therapy failure in the subject.
  • the subset of markers to be used within the methods of the present invention include any markers associated with cancer pathways.
  • the markers can be selected from the genes identified in Figures 27-38.
  • the markers can comprise anywhere ranging from two markers listed within each table up to the whole set of genes listed within each of these tables.
  • the markers can comprise any percentage of genes selected from each of these tables, including 90%, 80%, 70%, 60%, or 50% of the genes identified in Figures 27-38-
  • the markers are transregulatory SNPs, which are shown in Figure 48.
  • an aberrant co-expression level of the markers can be indicative of the presence of cancer in the subject, or predictive of cancer-therapy failure in the subject.
  • the markers can be selected from any suitable cancer pathway, including in preferred embodiments markers from the Polycomb or "sternness" pathway.
  • markers can be genes selected from the group consisting of ADA, AMACR+p63, ANK3, BCL2L1, BIRC5, BMI-I, BUBl, CCNBl, CCNDl, CESl, CHAFlA, CRIPl, CRYAB, ESMl, EZH2, FGFR2, FOS, Gbx2, HCFCl, IER3, ITPRl, JUNB, KLF6, KI67, KNTC2, MGC5466, Phcl, RNF2, Suzl2, TCF2, TRAPlOO, USP22, Wnt5A and ZFP36.
  • the markers are selected from the group consisting of BMIl, Ezh2, H2A, H3, transcription factors, and methylation patterns.
  • the aberrant co-expression level detected is of BMIl and Ezh2, and in another preferred embodiment the aberrant co-expression level detected is of H2A and H3.
  • the markers being detected are in the form of either mRNA, DNA, or protein.
  • the sample phenotypes are selected from the group consisting of cancer, non-cancer, recurrence, non-recurrence, relapse, non-relapse, invasiveness, non-invasiveness, metastatic, non-metastatic, localized, tumor size, tumor grade, Gleason score, survival prognosis, lymph node status, tumor stage, degree of differentiation, age, hormone receptor status, PSA level, histologic type, and disease free survival.
  • the aberrant expression level of two or more markers can be detected by any detection means known in the art, including, but not limited to, subjecting the cells to an analysis selected from the group consisting of multicolor quantitative immunofluorescence co-localization analysis, fluorescence in situ hybridization, and quantitative RT-PCR analysis.
  • the present invention is directed to a method of determining a detection threshold coefficient for classifying a sample phenotype from a subject, the method comprising the steps of: a) obtaining a sample from the subject, b) selecting two or more markers from a pathway related to cancer, c) screening for a simultaneous aberrant expression level of the two or more markers in the same cell from the sample; d) scoring the marker expression in the cells by comparing the expression levels of the samples obtained from the subjects to values in a reference database of samples obtained from subjects with either a known diagnosis or known clinical outcome after therapy, and e) determining the detection threshold coefficient for the sample classification accuracy at different detection thresholds using reference database of samples from subjects with known phenotypes.
  • Detection threshold coefficients which are indicative of a cancer diagnosis or a prognosis for cancer-therapy failure have an absolute value within the range of .gtoreq.0.5. to .gtoreq.0.999.
  • Preferred levels of detection threshold coefficients which are indicative of a cancer diagnosis or a prognosis for cancer-therapy failure have an absolute value of .gtoreq.0.5, .gtoreq.0.6, .gtoreq.0.7, .gtoreq.0.8, .gtoreq.0.9, .gtoreq.0.95, .gtoreq.0.99, .gtoreq.0.995. 5 and .gtoreq.0.999.
  • the method further comprises determining the best performing magnitude of said detection threshold and using said magnitude to assess the reliability of said established detection threshold in classifying a sample phenotype. In another embodiment, the method further comprises using the best performing magnitude of said detection threshold to score an unclassified sample and assign a sample phenotype to said sample.
  • the present invention is directed to a method for simultaneously detecting an aberrant co-expression level of two or more markers a single cell, said method comprising the steps of: a) obtaining a sample of tissue, b) selecting a marker defined by a pathway, c) screening for a simultaneous aberrant expression level of the two or more markers, and d) scoring their expression level as being aberrant when the expression level detected is above or below a certain detection threshold coefficient, wherein the detection threshold coefficient is determined by comparing the expression levels of the samples obtained from the subjects to values in a reference database of sample phenotypes obtained from subjects with either a known diagnosis or known clinical outcome after therapy.
  • the method of diagnosing involves performing a Kaplan-Meier survival analysis, wherein the performance of each of the SNP-based signatures of the subject are assessed.
  • the invention involves a method of generating a subset of CTOP genes for use in predicting a phenotype in a subject comprising the steps of analyzing SNP patterns of a gene, correlating the SNP patterns with CTOP genes, and identifying a subset of CTOP genes.
  • the SNPs to be analyzed are generally defined by features present of geographic population differentiation SNPs.
  • the geographic populations are selected from the group consisting of American, Asian, European, African, and Australian.
  • the application also discloses a method of determining the phenotypic relevance of a set of SNPs which are selected from trans-regulatory SNPs, comprising the steps of building a CTOP gene expression signature, wherein the CTOP genes are regulated by trans-regulatory SNPs; and determining the SNPs that regulate the CTOP genes within the CTOP gene expression signature, wherein the SNPs that regulate the CTOP genes are phenotypically relevant.
  • the subset of genes or SNPs are use in predicting a phenotype of a subject.
  • Another embodiment includes a composition comprising a set of probes that hybridize to at least two of the genes presented in any one of the gene sets presented in Figures 27-38, 48 and 56-57 or a combination of gene or SNP subsets, wherein said combination comprises at least two of the subsets presented in Figures 27-38, 48 and 56-57.
  • the combination of gene subsets of claim 20, wherein each subset of said combination comprises at least one gene or SNP of any of said subsets identified in Figures 27-38, 48 and 56-57.
  • the present invention is also directed to kits useful in detecting the simultaneous aberrant co-expression levels of two or more markers in a single cell.
  • Figure 1 shows HapMap analysis revealing population-specific profiles of genotype and allele frequencies of SNPs associated with cancer therapy outcome predictor (CTOP) genes comprising an 1 1-gene death-from-cancer signature.
  • COP cancer therapy outcome predictor
  • B, E Annotated haplotypes associated with the BMIl (B) and BUBl (E) genes in CEU, YRI, CHB, and JPT HapMap populations. Arrows indicate SNPs with population-specific profiles of genotype and allele frequencies.
  • C, D, F - H Bar graph plots demonstrating population-specific profiles of genotype and allele frequencies in different HapMap populations for individual SNPs associated with genes comprising an 1 1-gene death-from-cancer signature.
  • Figure 2 shows HapMap analysis revealing population-specific profiles of genotype and allele frequencies of SNPs associated with CTOP genes predicting the likelihood of disease relapse in prostate cancer patients after radical prostatectomy.
  • A Chromosomal locations of genes encoding transcripts comprising prostate cancer recurrence predictor signatures.
  • B - D Bar graph plots demonstrating population-specific profiles of genotype and allele frequencies in different HapMap populations for individual SNPs associated with genes comprising prostate cancer recurrence predictor signatures. For each SNP the frequencies shown within each set of bar graphs in the following order (from left to right): CEU, CHB, JPT, YRI. B, KLF6 (COPEB) gene; C, Wnt5, TCF2, CHAFlA, and KIAA0476 genes; D, PPFIA3, CDS2, FOS, and CHAFlA genes.
  • CEU CEU
  • CHB JPT
  • YRI. B KLF6 (COPEB) gene
  • C Wnt5, TCF2, CHAFlA, and KIAA0476 genes
  • D PPFIA3, CDS2, FOS, and CHAFlA genes.
  • Figure 3 shows HapMap analysis revealing population-specific profiles of genotype and allele frequencies of SNPs associated with cancer therapy outcome predictor (CTOP) genes comprising a 50-gene proteomics-based cancer therapy outcome signature.
  • COP cancer therapy outcome predictor
  • Figure 4 shows HapMap analysis identifying non-synonymous coding SNPs associated with CTOP genes and manifesting population-specific profiles of genotype and allele frequencies.
  • a - D Annotated haplotypes associated with the TRAF3IP2 (A), PXN (B), MKI67 (C), and RAGE (D) genes in CEU, YRI, CHB, and JPT HapMap populations. Arrows indicate non- synonymous coding SNPs with population-specific profiles of genotype and allele frequencies.
  • Figure 5 shows population-specific profiles of genotype and allele frequencies of SNPs associated with oncogenes and tumor suppressor genes.
  • Figure 6 shows that SNP-based gene expression signatures predict therapy outcome in prostate and breast cancer patients.
  • a - D Genes expression of which is regulated by SNP variations in normal individuals provide gene expression models predicting therapy outcome in breast (A, C) and prostate (B, D) cancer patients.
  • E - H Genes containing high-population differentiation non-synonymous SNPs (E, F) and genes representing loci in which natural selection most likely occurred (G, H) provide gene expression- based therapy outcome prediction models for breast (E, G) and prostate (F, H) cancer patients.
  • E, F Kaplan-Meier analysis of therapy outcome classification performance in breast cancer (E) and prostate cancer (F) patients of gene expression-based CTOP models generated from genetic loci containing high-population differentiation non-synonymous SNPs.
  • G H. Kaplan-Meier analysis of therapy outcome classification performance in breast cancer (G) and prostate cancer (H) patients of gene expression-based CTOP models generated from genetic loci in which natural selection most likely occurred.
  • I, J Kaplan-Meier analysis of therapy outcome classification performance in breast cancer (I) and prostate cancer (J) patients of gene expression-based CTOP models generated from genetic loci regulated by SNP variations in normal individuals.
  • K L. Kaplan-Meier analysis of therapy outcome classification performance in breast cancer (E) and prostate cancer (F) patients of gene expression-based CTOP models generated from genetic loci selected based on similarity of SNP profiles with population specific SNP profiles of known CTOP genes.
  • Figure 7 shows microarray analysis identifying clinically relevant cooperating oncogenic pathways in human prostate and breast cancers.
  • Plots D and H show Kaplan-Meier analysis based on patients' stratification taking into account evidence for activation of multiple pathways in individual tumors.
  • Gene expression signature-based patients' stratification for Kaplan-Meier survival analysis were performed as described in Glinsky et al., J. Clin. Invest. 115: 1503-1521 (2005) and Glinskyet al., J. Clin. Invest.
  • Figure 8 shows how comparative cross-species translational genomics integrates knowledge written in two languages (DNA sequence variations and mRNA expression levels) and three writing systems reflecting defined phenotype/gene expression pattern associations (SNP variations; transgenic mouse models of cancers; genomics of stem cell biology).
  • Figure 9 shows Q-RT-PCR analysis of mRNA abundance levels of a representative set of genes comprising the BM-1-pathway signature in BM-I siRNAitreayed PC-3-32 human prostate carcinoma cells.
  • Figure 10 shows siRNA-mediated changes of the transcript abundance levels of 11 genes comprising BM-1-pathway signature.
  • Figure 11 shows EZH2 siRNA-mediated changes of the transcript abundance levels of 11 genes comprising the BM-1-pathway signature.
  • Figure 12 shows siRNA-mediated changes of the transcript abundance levels of 11 genes comprising BM-1-pathway signature.
  • Figure 13 shows expression profiles of 11 gene MM-I -signature in distant metastatic lesions of the TRAMP transgenic mouse model of prostate cancer and PNS neurospheres.
  • Figure 14 shows increased DNA copy numbers of the BM-I and Ezh2 genes in human prostate carcinoma cells selected for high metastatic potential.
  • Figure 15 shows the quadruplicon of prostate cancer progression in the LNCap progression model.
  • Figure 16 shows the quadruplicon of prostate cancer progression in the PC-3 progression model.
  • Figure 17 shows the quadruplicon of prostate cancer progression in the PC-3 bone metastasis progression model.
  • Figure 18 shows expression levels in PC-3-32 and PC-3 cells.
  • Figure 19 shows cytoplasmic AMACR and nuclear p63 expression in parental PC-3 human prostate carcinoma cells and PC-3-32 human prostate carcinoma metastasis precursor cells.
  • Figure 20 shows that high expression levels of the BMIl and Ezh2 oncoproteins in human prostate carcinoma metastasis precursor cells are associated with marked accumulation of a dual-positive high BMIl/Ezh2-expressing cell population and increased DNA copy number of the BMIl and Ezh2 genes.
  • A-D A quantitative immunofluorescence co-localization analysis of the BMIl (mouse monoclonal antibody) and Ezh2 (rabbit polyclonal antibody) oncoproteins in PC-3-32 human prostate carcinoma metastasis precursor cells and parental PC-3 cells.
  • BMIl mouse monoclonal antibody
  • Ezh2 rabbit polyclonal antibody
  • the protein expression differences and the accumulation of dual-positive high BMIl/Ezh2-expressing cells were confirmed using a second distinct combination of antibodies: rabbit polyclonal antibodies for BMIl detection and mouse monoclonal antibodies for Ezh2 detection.
  • A immunof ⁇ uorescent analysis of PC-3-32 cells
  • B immunofluorescent analysis of PC-3 cells
  • C the histograms representing typical distributions of the BMIl (top panels) and Ezh2 (bottom panels) expression levels in PC-3 and PC-3-32 cells
  • D the plots illustrating the levels of dual positive high BMIl/Ezh2-expressing cells in metastatic PC-3-32 cells (22.4%; top panel) and parental PC-3 cells (1.5%; bottom panel). The results of one of two independent experiments are shown.
  • Figure 21 shows results of activation of the PcG chromatin silencing pathway in metastatic human prostate carcinoma cells.
  • a quantitative immunofluorescence co-localization analysis was utilized to measure the expression of the BMIl, Ezh2, H3metK27, and UbiH2A markers in human prostate carcinoma cells and calculate the numbers of dual-positive cells expressing various two-marker combinations.
  • Figure 22 shows that targeted reduction of the BMIl (3A) or Ezh2 (3B) expression increases sensitivity of human prostate carcinoma metastasis precursor cells to anoikis.
  • Anoikis- resistant PC-3-32 prostate carcinoma cells were treated with BMIl- or £zA2-targeting siRNAs and continuously monitored for expression levels of the various mRNAs, BMI and Ezh2 oncoproteins, as well as cell growth and viability under various culture conditions.
  • PC-3-32 cells with reduced expression of either BMIl or Ezh2 oncoproteins acquired sensitivity to anoikis as demonstrated by the loss of viability and increased apoptosis compared to the control LUC siRNA-treated cultures growing in detached conditions.
  • Figure 23 shows that treatment of human prostate carcinoma metastasis precursor cells with stable siRNAs targeting either BMIl or Ezh2 gene products depletes a sub-population of dual positive high BMIl/Ezh2-expressing cells.
  • Blood-borne PC-3-32 prostate carcinoma cells were treated with chemically modified resistant to degradation LUC-, BMIl-, or £zA2-targeting stable siRNAs and continuously monitored for expression levels of the BMI and Ezh2 oncoproteins.
  • Two consecutive applications of the stable siRNAs caused a sustained reduction of the BMIl and Ezh2 expression and depletion of the sub-population of dual positive high BMIl/Ezh2-expressing carcinoma cells. The results at the 11-day post-treatment time point are shown.
  • Figure 24 shows that human prostate carcinoma metastasis precursor cells depleted for a sub-population of dual positive high BMIl/Ezh2-expressing cells manifest a dramatic loss of malignant potential in vivo.
  • Adherent cultures of blood-borne PC-3-GFP-39 prostate carcinoma cells were treated with chemically modified degradation-resistant stable siRNAs targeting BMIl or Ezh2 mRNAs or control LUC siRNA.
  • 24 hrs after second treatment 1.5 x i O 6 cells were injected into prostates of nude mice. Note that all control animals developed highly aggressive rapidly growing metastatic prostate cancer and died within 50 days of experiment. Only 20% of mice in the BMIl- and Ezh2-targeting therapy groups developed less malignant more slowly growing tumors.
  • FIG 25 shows that tissue microarray analysis (TMA) of primary prostate tumors from patients diagnosed with prostate adenocarcinomas reveals increased levels of dual-positive BMI1/Ezh2 high-expressing cells.
  • BMIl and Ezh2 oncoprotein expression were measured in prostate TMA samples from cancer patients and healthy donors using a quantitative co- localization immunofluorescence method and the number of dual positive high BMI1/Ezh2- expressing nuclei was calculated for each sample.
  • primary prostate tumors from patients diagnosed with prostate adenocarcinomas manifest a diverse spectrum of accumulation of dual positive BMI1/Ezh2 high-expressing cells and patients with higher levels of BMIl or Ezh2 expression in prostate tumors manifest therapy-resistant malignant phenotype (Figure 26).
  • FIG. 26 shows that Increased BMIl and Ezh2 expression is associated with high likelihood of therapy failure and disease relapse in prostate cancer patients after radical prostatectomy.
  • Kaplan-Meier survival analysis demonstrates that cancer patients with more significant elevation of the BMIl and Ezh2 expression [having higher tumor (T) to adjacent normal tissue (N) ratio, T/N: Figure 26A; or having tumors with higher levels of BMIl (28B) or Ezh2 (28C) expression) are more likely to fail therapy and develop a disease recurrence after radical prostatectomy.
  • Figure 26E shows the Kaplan-Meier survival analysis of 79 prostate cancer patients stratified into five sub-groups using eight-covariate cancer therapy outcome (CTO) algorithm.
  • CTO algorithm integrates individual prognostic powers of BMIl and Ezh2 expression values and six clinico-pathological covariates (preoperative PSA, Gleason score, surgical margins, extra-capsular invasion, seminal vesicle invasion, and age).
  • Figure 27 shows breast cancer CTOP signatures in Affymetrix format, with predictive outcomes.
  • Figure 28 shows breast cancer CTOP signatures in Agilent Rosetta Chip format, with predictive outcomes.
  • Figure 29 shows prostate cancer CTOP signatures in Affymetrix format, with predictive outcomes.
  • Figure 30 shows PI3K pathway CTOP signatures.
  • Figure 31 shows SNP based CTOP signatures NG2007.
  • Figure 32 shows the parent methylation Signatures.
  • Figure 33 shows the histones H3 and H2A CTOP signatures.
  • Figure 34 shows the CTOP gene expression signatures for prostate cancer.
  • Figure 35 shows the CTOP gene expression signatures for breast cancer.
  • Figure 36 shows the CTOP gene expression signature and survival data for lung cancer.
  • Figure 37 shows the CTOP gene expression signature for ovarian cancer.
  • Figure 38 shows the CTOP gene expression signatures for breast cancer.
  • Figure 39 shows examples of the evaluation of the CMAPOOO and CMAPl 1 drug combinations in prostate cancer and the CMAP 19 drug combination in breast cancer.
  • Figure 40 shows CTOP scores for lung cancer.
  • Figure 41 shows Kaplan-Meier survival analysis of seventy-nine prostate cancer patients stratified into sub-groups with distinct expression profiles of the individual Polycomb pathway ESC signatures (top six panels) or six ESC signatures algorithm (bottom panel) in primary prostate tumors.
  • top six panels or six ESC signatures algorithm (bottom panel) in primary prostate tumors.
  • bottom 50% scores are the values of the corresponding signature CTOP scores and divided into poor prognosis (top 50% scores) and good prognosis sub-groups.
  • patients were sorted in descending order based on the values of the cumulative CTOP scores and divided into poor prognosis (top 50% scores) and good prognosis (bottom 50% scores) sub-groups.
  • the cumulative CTOP scores represent the sum of the six individual CTOP scores calculated for each patient.
  • Figure 42 shows Kaplan-Meier survival analysis of two-hundred eighty-six early-stage LN negative breast cancer patients stratified into sub-groups with distinct expression profiles of the individual Polycomb pathway ESC signatures (top six panels) or six ESC signatures algorithm (single middle panel) in primary breast tumors. Bottom four panels show patients' classification performance of the six ESC signatures algorithm in four different breast cancer therapy outcome data sets. Patients * stratification was performed using either individual CTOP scores (top six panels) or cumulative CTOP scores (bottom five panels) as described in the legend to the Figure 41.
  • FIG 43 shows bivalent chromatin domain-containing transcription factors (BCD-TF) manifest "sternness" expression profiles in therapy-resistant prostate and breast tumors.
  • Chromatin context identified by the presence of histones harboring specific modifications of the histone tails defines mutually exclusive transcriptionally active or silent states of corresponding genetic loci in genomes of most cells.
  • ESC multiple chromosomal regions were identified simultaneously harboring both "silent" (H3K27met3) and “active” (H3K4) histone marks and ⁇ 100 transcription factor (TF) encoding genes are residing within these bivalent chromatin domain-containing chromosomal regions.
  • TF encoding genes in ESC including bivalent chromatin domain-containing TF genes (BCD-TF), maintenance of a "sternness” state, and transition to differentiated phenotypes is regulated by the balance of the "sternness” TFs (Nanog, Sox2, Oct4) and Polycomb group (PcG) proteins bound to the promoters of target genes.
  • BCD-TF bivalent chromatin domain-containing TF genes
  • PcG Polycomb group
  • Eight-gene BCD-TF signature (derived from thirteen-gene BCD-TF signatures) manifesting highly concordant expression profiles (r — 0.716; p ⁇ 0.001 ) in ESC and therapy-resistant breast and prostate tumors.
  • Kaplan-Meier analysis demonstrates that prostate and breast cancer patients with tumors harboring ESC-like expression profiles of the eight-gene BCD-TF signature are more likely to fail therapy (bottom two panels).
  • Gene expression profiles of clinical samples were independently generated for therapy-resistant breast and prostate tumors using multivariate Cox regression analysis of microarrays of tumor samples from 286 breast cancer and 79 prostate cancer patients with known log-term clinical outcome after therapy.
  • Figure 44 shows Kaplan-Meier survival analysis of two-hundred eighty-six early-stage LN negative breast cancer patients (top four panels) and seventy-nine prostate cancer patients (bottom four panels) stratified into sub-groups with distinct expression profiles of the individual CTOP signatures [bivalent chromatin domain transcription factors (BCD-TF) and ESC pattern 3 signatures], eight ESC signatures algorithm, and nine "sternness" signatures algorithm in primary breast or prostate tumors. Patients' stratification was performed using either individual CTOP scores (for individual signatures) or cumulative CTOP scores (for CTOP algorithms) as described in the legend to the Figure 41.
  • Figure 45 shows Kaplan-Meier survival analysis of seventy-nine prostate cancer patients (top four panels) and ninety-seven early-stage LN negative breast cancer patients (middle four panels) stratified into sub-groups with distinct expression profiles of the individual CTOP signatures [histones H3 and H2A signatures; Polycomb (PcG) pathway methylation signature] and two signatures PcG methylation/histones H3/H2A algorithm (bottom two panels) in primary prostate and breast tumors. Patients' stratification was performed using either individual CTOP scores (for individual signatures) or cumulative CTOP scores (for CTOP algorithm) as described in the legend to the Figure 41.
  • Figure 46 shows Kaplan-Meier survival analysis of two-hundred eighty-six early-stage LN negative breast cancer patients (top left panel), seventy-nine prostate cancer patients (top right panel), ninety-one early-stage lung cancer patients (bottom left panel), and one-hundred thirty-three ovarian cancer patients (bottom right panel) stratified into sub-groups with distinct expression profiles of the nine "sternness" signatures algorithm in primary breast, prostate, lung, and ovarian tumors. Patients' stratification was performed using cumulative CTOP scores of the nine "sternness"' signatures as described in the legend to the Figure 41. Patients were sorted in descending order based on the values of the cumulative CTOP scores and divided into five subgroups at 20% increment of the cumulative CTOP score values.
  • Figure 47 shows validation of the Polycomb pathway activation in metastatic and therapy-resistant human prostate cancer.
  • Blood-borne PC-3-32 human prostate carcinoma cells contain increased levels of CD44+/CD24- cancer stem cell-like population of dual-positive BMI1/Ezh2 high-expressing cells (middle panel) with increased levels of H3met3K27 and H2AubiKl 19 histones (bottom two FACS figures).
  • CD44+CD24- cancer stem cell-like populations were isolated using sterile FACS sorting from parental PC-3 and blood-borne PC-3-32 metastasis precursor cells and subjected to multicolor quantitative immunofluorescence co-localization analysis (18) for BMIl and Ezh2 Polycomb proteins (middle panel) or Polycomb pathway substrates H3met3K27 and H2AubiKl 19 histones (bottom two FACS figures).
  • Multi-color FISH analysis reveals marked enrichment of blood-borne human prostate carcinoma metastasis precursor cells for cell population with co-amplification of both BMIl and Ezh2 genes.
  • nuclei of diploid hTERT-immortalized human fibroblasts containing two copies of the BMIl and Ezh2 genes are shown.
  • Bottom two panels present quantitative FISH analysis of the DNA copy numbers of BMIl and Ezh2 genes in parental PC-3 and blood-borne PC-3-32 human prostate carcinoma ceils.
  • Prostate cancer TMA Kaplan-Meier survival analysis of seventy-one prostate cancer patients with distinct levels of dual-positive BMI1/Ezh2 high expressing cells in primary prostate tumors.
  • Prostate cancer TMA were subjected to multi-color quantitative immunofluorescence co-localization analysis of expression of the BMIl and Ezh2 proteins.
  • Prostate cancer patients having > 1% of dual-positive BMI1/Ezh2 high expressing cells manifested statistically significant increased likelihood of therapy failure after radical prostatectomy.
  • Figure 48 shows a list of gene expression regulatory SNPs associated with CTOP signatures for prostate and breast cancer.
  • Figure 49 is a graph showing the classification performance of the 49-transcript SNP- associated CTOP signature on a data set comprising 286 early-stage LN negative breast cancer patients.
  • Figure 50 is a graph showing the classification performance of the 36-transcript SNP- associated CTOP signature on a data set comprising 79 prostate cancer patients after a radical prostatectomy.
  • Figure 51 is a graph of the expression profiles of the 9-gene Alzheimer's signature in different groups of patients.
  • Figure 52 is a graph of the expression profiles of the 1 l-gene Alzheimer's signature in different groups of patients.
  • Figure 53 is a graph of the expression profiles of the 23-gene Alzheimer's signature in different groups of patients.
  • Figure 54 is a graph of the 38-gene longevity signature.
  • Figure 55 is a graph of the 57-gene longevity signature.
  • Figure 56 shows Alzheimer's CTOP signatures in Affymetrix format, with predictive outcomes.
  • Figure 57 shows the CTOP gene expression signatures for Alzheimer's disease.
  • the present invention is directed to novel methods and kits for diagnosing the presence of a disease state or phenotype, including, but not limited to, cancers, metabolic disorders, immunologic disorders, gastro-intestinal disorders, cardiovascular disorder, CNS disorders, circulatory system disorders, blood-related diseases, bone disorders, viral and bacterial disorders, chronic disorders such as arthritis, asthma, diabetes, heart disease, osteoporosis, and aging disorders including Alzheimer's within a patient, and for determining whether a subject who has such disease state is susceptible to different types of treatment regimens.
  • a disease state or phenotype including, but not limited to, cancers, metabolic disorders, immunologic disorders, gastro-intestinal disorders, cardiovascular disorder, CNS disorders, circulatory system disorders, blood-related diseases, bone disorders, viral and bacterial disorders, chronic disorders such as arthritis, asthma, diabetes, heart disease, osteoporosis, and aging disorders including Alzheimer's within a patient, and for determining whether a subject who has such disease state is susceptible to different types of treatment regimens.
  • the cancers to be tested include, but are not limited to, prostate, breast, lung, gastric, ovarian, bladder, lymphoma, mesothelioma, medullablastoma, glioma, mantle cell lymphoma, and AML.
  • kits and methods of the present invention can be used to predict various different types of clinical outcomes.
  • the invention can be used to predict recurrence of disease state after therapy, non-recurrence of a disease state after therapy, therapy failure, short interval to disease recurrence (e.g., less than two years, or less than one year, or less than six months), short interval to metastasis in cancer (e.g., less than two years, or less than one year, or less than six months), invasiveness, non-invasiveness, likelihood of metastasis in cancer, likelihood of distant metastasis in cancer, poor survival after therapy, death after therapy, disease free survival and so forth.
  • markers refers to genes, RNA, DNA, mRNA, or SNPs,.
  • a “set or markers” refers to a group of markers.
  • a "set of genes” refers to a group of genes.
  • a “set of genes” or a “set of markers” according to the invention can be identified by any method now known or later developed to assess gene, RNA, or DNA expression, including but not limited to measurements relating to the biological processes of nucleic acid amplification, transcription, RNA splicing, and translation.
  • a "set of genes” or a "set of markers” refers to a group of genes or markers that are differentially expressed in a first sample as compared to a second sample.
  • a "set of genes” or a “set or markers” refers to at least one gene or marker, for example, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more genes or markers.
  • a "set" refers to at least one.
  • differentially expressed refers to the existence of a difference in the expression level of a nucleic acid or protein as compared between two sample classes, for example a first sample and a second sample as defined herein. Differences in the expression levels of "differentially expressed” genes preferably are statistically significant. Preferably, there is a 2-fold or more (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more) increase or decrease in the expression levels of differentially expressed nucleic acid or protein.
  • there is at least a 5% (for example 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100%) increase or decrease in the expression levels of differentially expressed nucleic acid or protein.
  • expression refers to any one of RNA, cDNA, DNA, or protein expression.
  • “Expression values” refer to the amount or level of expression of a nucleic acid or protein according to the invention. Expression values are measured by any method known in the art and described herein. As used herein, “increased” refers to 2-fold or more (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more) greater than. “Increased” also refers to at least 5% or more (for example 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100%) greater than.
  • “decreased” refers to 2-fold or more (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more) less than. “Decreased” also refers to at least 5% or more (for example 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100%) less than.
  • a "subset of genes” refers to at least one gene of a "set of genes” as defined herein.
  • a subset of genes is predictive of a particular phenotype, for example, disease outcome, diagnosis of a particular disease of interest, prognosis of a particular disease of interest, recurrence, non-recurrence, invasiveness, non-invasiveness, metastatic, non- metastatic, localized, organ confined, tumor grade, Gleason score, survival prognosis, lymph node status, tumor stage, degree of differentiation, age, hormone receptor status, PSA level, histologic type, disease free survival, disease progression, remission, biochemical recurrence, metastatic recurrence, local recurrence, response to therapy, disease relapse, non-relapse, therapy failure and cure.
  • predictive means that a set of genes or a subset of genes according to the invention, is indicative of a particular phenotype of interest (for example disease outcome, diagnosis of a particular disease of interest, prognosis of a particular disease of interest, recurrence, non-recurrence, invasiveness, non-invasiveness, metastatic, non- metastatic, localized, organ confined, tumor grade, Gleason score, survival prognosis, lymph node status, tumor stage, degree of differentiation, age, hormone receptor status, PSA level, histologic type, disease free survival, disease progression, remission, biochemical recurrence, metastatic recurrence, local recurrence, response to therapy, disease relapse, non-relapse, therapy failure and cure).
  • a particular phenotype of interest for example disease outcome, diagnosis of a particular disease of interest, prognosis of a particular disease of interest, recurrence, non-recurrence, invasiveness, non-invasiveness, metastatic,
  • a subset of genes, according to the invention that is "predictive" of a particular phenotype correlates with a particular phenotype at least 10% or more, for example 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100%.
  • a "phenotype" refers to any detectable characteristic of an organism.
  • a "phenotype" refers to disease outcome, diagnosis of a particular disease of interest, prognosis of a particular disease of interest, recurrence, non-recurrence, invasiveness, non-invasiveness, metastatic, non-metastatic, localized, organ confined, tumor grade, Gleason score, survival prognosis, lymph node status, tumor stage, degree of differentiation, age, hormone receptor status, PSA level, histologic type, disease free survival, disease progression, remission, biochemical recurrence, metastatic recurrence, local recurrence, response to therapy, disease relapse, non-relapse, therapy failure and cure.
  • diagnosis refers to a process of determining if an individual is afflicted with a disease or ailment.
  • Prognosis refers to a prediction of the probable occurrence and/or progression of a disease or ailment, as well as the likelihood of recovery from a disease or ailment, or the likelihood of ameliorating symptoms of a disease or ailment or the likelihood of reversing the effects of a disease or ailment. "Prognosis” is determined by monitoring the response of a patient to therapy.
  • first sample refers to a sample from a normal subject or individual, or a normal cell line.
  • an “individual” “or “subject” includes a mammal, for example, human, mouse, rat, dog, cow, pig, sheep etc...
  • a “subject” includes both a patient and a normal individual.
  • patient refers to a mammal who is diagnosed with a disease or ailment.
  • normal refers to an individual who has not shown any disease or ailment symptoms or has not been diagnosed by a medical doctor.
  • a “second sample” refers to a sample from a patient or an unclassified individual, or an animal model for a disease of interest.
  • a “second sample” also refers to a sample from a cell line that is a model for a disease of interest, for example a tumor cell line.
  • Tumor is to be construed broadly to refer to any and all types of solid and diffuse malignant neoplasias including but not limited to sarcomas, carcinomas, leukemias, lymphomas, etc., and includes by way of example, but not limitation, tumors found within prostate, breast, colon, lung, and ovarian tissues.
  • a “tumor cell line” refers to a transformed cell line derived from a tumor sample. Usually, a “tumor cell line” is capable of generating a tumor upon explant into an appropriate host.
  • a “tumor cell line” line usually retains, in vitro, properties in common with the tumor from which it is derived, including, e.g., loss of differentiation or loss of contact inhibition, and will undergo essentially unlimited cell divisions in vitro.
  • control cell line refers to a non-transformed, usually primary culture of a normally differentiated cell type.
  • tissue of origin it is preferable to use a "control cell line” and a “tumor cell line” that are related with respect to the tissue of origin, to improve the likelihood that observed gene expression differences or differences in RNA or protein levels, are related to gene expression changes underlying the transformation from control cell to tumor.
  • An “unclassified sample” refers to a sample for which classification is obtained by applying the methods of the present invention.
  • An “unclassified sample” may be one that has been classified previously using the methods of the present invention, or through the use of other molecular biological or pathohistological analyses. Alternatively, an “unclassified sample” may be one on which no classification has been carried out prior to the use of the sample for classification by the methods of the present invention.
  • the fold expression change or differential expression data are logarithmically transformed.
  • logarithmically transformed means, for example, 1Og 10 transformed.
  • multivariate analysis refers to any method of determining the incremental, statistical power of the members of a set of genes to predict a phenotype of interest.
  • Methods of "multivariate analysis” useful according to the invention include but are not limited to multivariate Cox analysis.
  • multivariate Cox analysis refers to Cox proportional hazard survival regression analysis as performed by using the program presented at the world wide web at http://members.aol.com/johnp71/prophaz.html, and as described in Glinsky et al., 2005, J. Clin. Investig. 115:1503.
  • “survival analysis” refers to a method of verifying that a set of genes or a subset of genes according to the invention is “predictive”, as defined herein, of a particular phenotype of interest. “Survival analysis” takes the survival times of a group of subjects (usually with some kind of medical condition) and generates a survival curve, which shows how many of the members remain alive over time. Survival time is usually defined as the length of the interval between diagnosis and death, although other "start” events (such as surgery instead of diagnosis), and other "end” events (such as recurrence instead of death) are sometimes used.
  • Survival is often influenced by one or more factors, called “predictors” or “covariates”, which may be categorical (such as the kind of treatment a patient received) or continuous (such as the patient's age, weight, or the dosage of a drug).
  • predictors or “covariates”
  • continuous such as the patient's age, weight, or the dosage of a drug.
  • a “baseline” survival curve is the survival curve of a hypothetical "completely average” subject ⁇ someone for whom each predictor variable is equal to the average value of that variable for the entire set of subjects in the study.
  • This baseline survival curve does not have to have any particular formula representation; it can have any shape whatever, as long as it starts at 1.0 at time 0 and descends steadily with increasing survival time.
  • the baseline survival curve is then systematically "flexed” up or down by each of the predictor variables, while still keeping its general shape.
  • the proportional hazards method (for example Cox Multivariate analysis) computes a "coefficient", or "relative weight coefficient" for each predictor variable that indicates the direction and degree of flexing that the predictor has on the survival curve. Zero means that a variable has no effect on the curve - - it is not a predictor at all; a positive variable indicates that larger values of the variable are associated with greater mortality. Knowing these coefficients, a "customized" survival curve for any particular combination of predictor values is constructed. More importantly, the method provides a measure of the sampling error associated with each predictor's coefficient. This allows for assessment of which variables' coefficients are significantly different from zero; that is: which variables are significantly related to survival.
  • Multivariate Cox analysis is used to generate a "relative weight coefficient".
  • a "relative weight coefficient” is a value that reflects the predictive value of each gene comprising a gene set of the invention.
  • Multivariate Cox analysis computes a "relative weight coefficient" for each predictor variable; for example, each gene of a gene set, that indicates the direction and degree of flexing that the predictor has on a survival curve. Zero means that a variable has no effect on the curve and is not a predictor at all. A positive variable indicates that larger values of the variable are associated with greater mortality. Knowing these "relative weight coefficients" a survival curve can be constructed for any combination of predictor values.
  • a “correlation coefficient” means a number between -1 and 1 which measures the degree to which two variables are linearly related. If there is perfect linear relationship with positive slope between the two variables, there is a correlation coefficient of 1; if there is positive correlation, whenever one variable has a high (low) value, so does the other. If there is a perfect linear relationship with negative slope between the two variables, there is a correlation coefficient of -1; if there is negative correlation, whenever one variable has a high (low) value, the other has a low (high) value. A correlation coefficient of 0 means that there is no linear relationship between the variables.
  • correlation coefficients include the correlation coefficient, pX;y; that ranges between -1 and+1, such as is generated by Microsoft Excel's CORREL function, the Pearson product moment correlation coefficient, r, that also ranges between- 1 and+1, that reflects the extent of a linear relationship between two data sets, such as is generated by Microsoft Excel's PEARSON function, or the square of the Pearson product moment correlation coefficient, r ⁇ 2>, through data points in known y's and known x's, such as is generated by Microsoft Excel's RSQ function.
  • the r ⁇ 2> value can be interpreted as the proportion of the variance in y attributable to the variance in x.
  • a correlation coefficient, px,y is greater than or equal to 0.8, or is greater than or equal to 0.9, or is greater than or equal to 0.95, or is greater than or equal to 0.995.
  • transformations e.g. natural log transformations
  • correlation coefficients either mathematically, or empirically using samples of known classification.
  • the magnitude of the correlation coefficient can be used as a threshold for classification.
  • the appropriate threshold can be determined through the use of test data that seek to classify samples of known classification using the methods of the present invention. The threshold is adjusted so that a desired level of accuracy (e.g., greater than about 70% or greater than about 80%, or greater than about 90% or greater than about 95% or greater than about 99% accuracy is obtained). This accuracy refers to the likelihood that an assigned classification is correct.
  • the tradeoff for the higher confidence is an increase in the fraction of samples that are unable to be classified according to the method. That is, the increase in confidence comes at the cost of a loss in sensitivity.
  • the expression value, or logarithmically transformed expression value for each member of a set of genes is multiplied by a "relative weight coefficient", as defined herein and as determined by multivariate Cox analysis, to provide an "individual survival score" for each member of a set of genes.
  • a "survival score” refers to the sum of the individual survival scores for each member of a set of genes of the invention.
  • Kaplan-Meier survival analysis includes but is not limited to Kaplan-Meier Survival Analysis.
  • Kaplan-Meier survival analysis is carried out using GraphPad Prism version 4.00 software (GraphPad Software) or as described in Glinsky et al., 2005, supra.
  • Statistical significance of the difference between the survival curves for different groups of patients is assessed using Chi square and Logrank tests.
  • a p-value according to the invention is less than or equal to 0.25, preferably less than or equal to 0.1 and more preferably, less than or equal to 0.075, for example, 0.075, 0.070, 0.065, 0.060, 0.055, 0.050 etc... and most preferably less than or equal to 0.05, for example, 0.05, 0.045, 0.040, 0.035, 0.020, 0.010 etc...
  • p-value refers to a p-value generated for a set of genes by multivariate Cox analysis.
  • a "p-value” as used herein also refers to a p-value for each member of a set of genes.
  • a “p-value” also refers to a p-value derived from Kaplan-Meier analysis, as defined herein.
  • a "p-value" of the invention is useful for determining if a set of genes or a subset of genes of the invention is predictive of a phenotype.
  • a “combination of gene sets” refers to at least two gene sets according to the invention.
  • a “combination of gene subsets” refers to at least two gene subsets according to the invention.
  • the term “probe” refers to a labeled oligonucleotide which forms a duplex structure with a gene in a gene set or gene subset of the invention, due to complementarity of at least one sequence in the probe with a sequence in the gene.
  • Probes useful for the formation of a cleavage structure according to the invention are between about 17-40 nucleotides in length, preferably about 17-30 nucleotides in length and more preferably about 17-25 nucleotides in length.
  • a "primer” or an “oligonucleotide primer” refers to a single stranded DNA or RNA molecule that is hybridizable to a gene in a gene set or gene subset of the invention and primes enzymatic synthesis of a second nucleic acid strand.
  • Oligonucleotide primers useful according to the invention are between about 10 to 100 nucleotides in length, preferably about 17-50 nucleotides in length and more preferably about 17-45 nucleotides in length.
  • One embodiment of the present invention is directed to a method for diagnosing any type of disease state or phenotype, including, but not limited to, cancers, metabolic disorders, immunologic disorders, gastro-intestinal disorders, cardiovascular disorder, CNS disorders, circulatory system disorders, blood-related diseases, bone disorders, viral and bacterial disorders, chronic disorders such as arthritis, asthma, diabetes, heart disease, osteoporosis, and aging disorders including Alzheimer's or predicting disease-therapy outcome by detecting the expression levels of multiple markers in the same cell at the same time, and scoring their expression as being above a certain threshold, wherein the markers are from a particular pathway related to cancer, other pathways, or transregulatory SNPs, with the score being indicative or a disease state diagnosis or a prognosis for disease-therapy failure.
  • cancers metabolic disorders, immunologic disorders, gastro-intestinal disorders, cardiovascular disorder, CNS disorders, circulatory system disorders, blood-related diseases, bone disorders, viral and bacterial disorders, chronic disorders such as arthritis, asthma, diabetes, heart disease, osteoporosis, and aging
  • This method can be used to diagnose cancer or predict cancer-therapy outcomes for a variety of cancers.
  • the simultaneous co-expression of at least two markers in the same cell from a subject is a diagnostic for cancer or other disease states and a predictor for the subject to be resistant to standard therapy for cancer or other diseases.
  • the markers can come from any pathway involved in the regulation of cancer, including specifically the PcG pathway and the "sternness" pathway.
  • the markers can be mRNA (messenger RNA), DNA, microRNA, protein, or transregulatory SNPs.
  • markers to be used within the methods of the present invention include any markers associated with cancer pathways.
  • the markers can be selected from the genes identified in Figures 27-38.
  • the markers can comprise anywhere ranging from two markers listed within each table up to the whole set of genes listed within each of these tables.
  • the markers can comprise any percentage of genes selected from each of these tables, including 90%, 80%, 70%, 60%, or 50% of the genes identified in each of Figures 27-38.
  • Marker detection can be made through a variety of detection means, including bar-coding through immunofluorescence.
  • the markers detected can be a variety of products, including mRNA, DNA, microRNA, and protein.
  • PCR can be used as detection means.
  • protein products, gene expression, or gene copy number can be identified through detection means known in the art.
  • Detection means in case of a nucleic acid probe, include measuring the level of mRNA or cDNA to which a probe has been engineered to bind, where the probe binds the intended species and provides a distinguishable signal.
  • the probes are affixed to a solid support, such as a microarray.
  • the probes are primers for nucleic acid amplification of a set of genes.
  • Q-RT-PCR amplification can be used.
  • Detecting expression for measurement or determining protein expression levels can also be accomplished by using a specific binding reagent, such as an antibody.
  • expression levels of the markers can be analyzed by any method now known or later developed to assess gene expression, including but not limited to measurements relating to the biological processes of nucleic acid amplification, transcription, RNA splicing, and translation.
  • transcript concentration e.g., as by Northern blotting, expression array measurements, quantitative RT-PCR, or comparative genomic hybridization
  • protein concentration e.g., as by quantitative 2D gel electrophoresis, mass spectrometry, Western blotting, ELISA, or other method for determining protein concentration
  • affinity reagents could be used with the present invention, such as one or more antibodies (monoclonal or polyclonal) and the invention can include using techniques, such as ELISA, for the analysis.
  • specific antibodies specific to the markers to be detected can be used in a kit and in methods of the present invention.
  • the kit would include reagents and instructions for use, where the reagents could be protein-specific differentially-labeled fluorescent antibodies; protein-specific antibodies from different species (mouse, rabbit, goat, chicken, etc.) and differentially labeled species-specific antibodies; DNA and RNA-based probes with different fluorescent dyes; bar-coded nucleic acid- and protein-specific probes (each probes having a unique combination of colors).
  • the reagents could be protein-specific differentially-labeled fluorescent antibodies; protein-specific antibodies from different species (mouse, rabbit, goat, chicken, etc.) and differentially labeled species-specific antibodies; DNA and RNA-based probes with different fluorescent dyes; bar-coded nucleic acid- and protein-specific probes (each probes having a unique combination of colors).
  • Expression values for any member of a gene set, marker set, or subset according to the invention can be obtained by any method now known or later developed to assess gene or marker expression, including but not limited to measurements relating to the biological processes of nucleic acid amplification, transcription, RNA splicing, and translation.
  • Direct and indirect measures of gene or marker copy number e.g., as by fluorescence in situ hybridization or other type of quantitative hybridization measurement, or by quantitative PCR
  • transcript concentration e.g., by Northern blotting, expression array measurements or quantitative RT-PCR
  • protein concentration e.g., by quantitative 2-D gel electrophoresis, mass spectrometry, Western blotting, ELISA, or other method for determining protein concentration
  • the markers detected can be from a variety of pathways, including those related to cancer. Suitable pathways for markers within the scope of the present invention include any pathways related to oncogenesis and metastasis, and more specifically include the Polycomb group (PcG) chromatin silencing pathway and the "sternness" pathway.
  • PcG Polycomb group
  • Representative cancer pathways within the context of the present invention include but are not limited to, the Polycomb pathway, the Polycomb pathway target genes, "sternness" pathways, DNA methylation pathways, BMIl, Ezh2, Suzl2, Suzl2/PolII, EED, PcG-TF, BCD- TF, TEZ, Nanog/Sox2/Oct4, Myc, He2/neu, CCNDl, E2F3, PBK, beta-catenin, ras, src, PTEN, p53, Rb, pl6/ARF, p21, Wnt, and Hh pathways.
  • the Polycomb group (PcG) gene BMIl is required for the proliferation and self-renewal of normal and leukemic stem cells.
  • Over-expression o ⁇ Bmil oncogene causes neoplastic transformation of lymphocytes and plays an essential role in the pathogenesis of myeloid leukemia.
  • Another PcG protein, Ezh2 has been implicated in metastatic prostate and breast cancers, suggesting that PcG pathway activation is relevant for epithelial malignancies.
  • activation of the BMIl oncogene-associated PcG pathway plays an essential role in metastatic prostate cancer, thus mechanistically linking the pathogenesis of leukemia, self- renewal of stem cells, and prostate cancer metastasis.
  • the methods of the present invention provide for the diagnosis, prognosis, and treatment strategy for a patient with a disorder of the above mentioned types.
  • Treatment includes determining whether a patient has an expression pattern of markers associated with the disorder and administering to the patient a therapeutic adapted to the treatment of the disorder.
  • the method can include the identification of increased BMI 1 and Ezh2 expression and the formulation of a treatment plan specific to this phenotype.
  • the detection of appropriate or inappropriate activation of "sternness” genetic pathways can be used to diagnose cancer or other disorders and to predict the likelihood of therapy success or failure.
  • Inappropriate activation of "sternness” genes in cancer cells may be associated with aggressive clinical behavior and increased likelihood of therapy failure.
  • a sub-set of human prostate tumors represents a genetically distinct highly malignant sub-type of prostate carcinoma with high propensity toward metastatic dissemination even at the early stage of disease. Such a high propensity toward metastatic dissemination of this type of prostate tumors is associated with the early engagement of normal stem cells into malignant process. Elucidation of such inappropriate activation of "sternness” gene expression can help tailor cancer therapy to a patient's individual needs.
  • the invention is directed to prognostic assays for therapy for cancer and other disease states that can be used to diagnose cancer and other disease states and to predict the resistance of various disease states to standard therapeutic regimens.
  • the invention is directed to methods and compositions for predicting the outcome of disease therapy for individual patients. In one embodiment, the method is used to predict whether a particular patient will be therapy-responsive or therapy-resistant.
  • the invention can be used with a variety of cancers, including but not limited to, breast, prostate, ovarian, lung, glioma, and lymphoma.
  • the invention is directed to personalized medicine for patients with cancer or other disease states or phenotypes, such as metabolic disorders, immunologic disorders, gastrointestinal disorders, cardiovascular disorder, CNS disorders, circulatory system disorders, blood- related diseases, bone disorders, viral and bacterial disorders, chronic disorders such as arthritis, asthma, diabetes, heart disease, osteoporosis, and aging disorders including Alzheimer's, and encompasses the selection of treatment options with the highest likelihood of successful outcome for individual patients.
  • the present invention is directed to the use of an assay to predict the outcome after therapy in patients with early stage disease and provide additional information at the time of diagnosis with respect to likelihood of therapy failure.
  • the detection of the state of transcription factors can be used to diagnose the presence of cancer or other disease states or phenotypes and to predict the likelihood of therapy success or failure.
  • the determination of a common pattern of the transcription factor expression can be used as a profile to help determine clinical outcome.
  • the invention is also directed to a particular sub-set of BCD-TF genes defined here as the eight gene BCD-TF signature that manifests "sternness" expression profiles in therapy-resistant prostate and breast tumors ( Figure 43).
  • the detection of the methylation state of target genes can be used to diagnose cancer or other disease states or phenotypes and to predict the likelihood of therapy success or failure. More particularly, PcG target genes with promoters frequently hypermethylated in cancer manifest distinct expression profiles associated with therapy-resistant and therapy-sensitive prostate and breast cancers ( Figure 44), implying that differences in gene expression between tumors with distinct outcome after therapy may be driven, in part, by the distinct promoter hypermethylation patterns of the PcG target genes. These differences can be exploited to generate highly informative gene expression signatures of the PcG target genes hypermethylated in cancer for stratification of prostate and breast cancer patients into sub-groups with statistically distinct likelihood of therapy failure (Figure 44).
  • the invention involves both a method to classify patients into sub-groups predicted to be either therapy-responsive or therapy-resistant, and a method for determining alternate therapies for patients who are classified as resistant to standard therapies.
  • the method of the present invention is based on an accurate classification of patients into subgroups with poor and good prognosis reflecting a different probability of disease recurrence and survival after standard therapy.
  • the invention relates to a method for diagnosing cancer or predicting cancer-therapy outcome in a subject, said method comprising the steps of: a) obtaining a sample from the subject, b) selecting a marker from a pathway related to cancer, c) screening for a simultaneous aberrant expression level of two or more markers in the same cell from the sample, and d) scoring their expression level as being aberrant when the expression level detected is above or below a certain detection threshold coefficient, wherein the detection threshold coefficient is determined by comparing the expression levels of the samples obtained from the subjects to values in a reference database of samples obtained from subjects with either a known diagnosis or known clinical outcome after therapy, wherein the presence of an aberrant expression level of two or more markers in individual cells and presence of cells aberrantly expressing two or more such markers is indicative of a cancer diagnosis or a prognosis for cancer-therapy failure in the subject.
  • An aberrant expression level is a level of expression that can either be higher or lower than the expression level as compared to reference samples.
  • the reference samples can have a variety of phenotypes, including both diseased phenotypes and non-diseased phenotypes.
  • the sample phenotypes within the scope of the present invention include, but are not limited to, cancer, non-cancer, recurrence, non-recurrence, relapse, non-relapse, invasiveness, non- invasiveness, metastatic, non-metastatic, localized, tumor size, tumor grade, Gleason score, survival prognosis, lymph node status, tumor stage, degree of differentiation, age, hormone receptor status, PSA level, histologic type, and disease free survival.
  • a detection threshold coefficient within the context of the present invention is a value above which or below which a patient or sample can be classified as either being indicative of a cancer diagnosis or a prognosis for cancer-therapy failure.
  • the detection threshold coefficients are defined by a plurality of measurements of samples in the reference database; sorting the samples in descending order of the values of measurements; assignment of the probability of samples having a phenotype in sub-groups of samples defined at different increments of the values of measurements (e.g., samples comprising top 10%; 20%; 30%; 40%; 50%; 60%; 70%; 80%; 90% of the values); selecting the statistically best-performing detection threshold coefficient defined as the value of measurements segregating samples with the values below and above the threshold into subgroups with statistically distinct probability of having a phenotype (cancer vs non-cancer; therapy failure vs cure; etc.), ideally, segregating patients into subgroups with 100% probability of therapy failure and with 100% probability of a cure or as close to this probability values as practically possible.
  • This value of markers measurements is defined as the best performing magnitude of the detection threshold.
  • the samples of unknown phenotype are then placed into corresponding subgroups based on the values of markers measurements and assigned the corresponding probability of having a phenotype.
  • one skilled in the art can utilize different statistical programs and approaches such as the univariate and multivariate Cox regression analysis and Kaplan-Meier survival analysis.
  • Detection threshold coefficients which are indicative of a disease diagnosis or a prognosis for therapy failure have an absolute value within the range of .gtoreq.0.5. to .gtoreq.0.999.
  • Preferred levels of detection threshold coefficients which are indicative of a disease diagnosis or a prognosis for therapy failure have an absolute value of .gtoreq.0.5, .gtoreq.0.6, .gtoreq.0.7, .gtoreq.0.8, .gtoreq.0.9, .gtoreq.0.95, .gtoreq.0.99, .gtoreq.0.995., and .gtoreq.0.999.
  • the present invention is also directed to a method of determining detection threshold coefficients for classifying a sample phenotype from a subject.
  • This method comprises the steps of selecting two or more markers from a pathway related to cancer, other pathway, or transregulatory SNPs, screening for a simultaneous aberrant expression level of the two or more markers in the same cell from the sample and scoring the marker expression in the cells by comparing the expression levels of the samples obtained from the subjects to values in a reference database of samples obtained from subjects with either a known diagnosis or known clinical outcome after therapy, and determining the sample classification accuracy at different detection thresholds using reference database of samples from subjects with known phenotypes.
  • the method of determining detection threshold coefficients for classifying a sample phenotype from a subject further comprises the additional step of determining the best performing magnitude of said detection threshold and using said magnitude to assess the reliability of said established detection threshold in classifying a sample phenotype.
  • Selection of the statistically best-performing detection threshold coefficient is defined as the value of measurements of the segregating samples with the values below and above the threshold, which are then split into subgroups with a statistically distinct probability of. having a phenotype (cancer vs non-cancer; therapy failure vs cure, etc.). More preferably, patients or samples can be segregated into subgroups with 100% probability of therapy failure and with 100% probability of a cure, or as close to this probability values as practically possible.
  • This value of markers measurements is defined as the best performing magnitude of the detection threshold. Additionally, the best performing magnitude of the detection threshold coefficient can be used to score an unclassified sample and assign a sample phenotype to said sample.
  • multivariate analysis is multivariate Cox analysis as described in Glinsky et al., 2005 J. Clin. Invest. 1 15 : 1503.
  • multivariate Cox analysis refers to Cox proportional hazard survival regression analysis as performed by using the program presented at the world wide web at http://members.aol.com/johnp71/prophaz.html, and as described in Glinsky et al., 2005, J. Clin, rnvestig. 1 15:1503.
  • Weighted survival score analysis reflects the incremental statistical power of individual covariates as predictors of therapy outcome based on a multicomponent prognostic model. For example, microarray-based or Q-RT-PCR-derived gene expression values are normalized and log-transformed on a base 10 scale. The log-transformed normalized expression values for each data set are analyzed in a multivariate Cox proportional hazard regression model, with overall survival or event-free survival as the dependent variable.
  • the log-transformed normalized gene expression value measured for each gene are multiplied by a coefficient derived from the multivariate Cox proportional hazard regression analysis, for example a relative weight coefficient, as defined herein.
  • Final survival predictor score comprises a sum of scores for individual genes and reflects the relative contribution of each of the genes in the multivariate analysis.
  • the negative weighting values indicate that higher expression correlates with longer survival and favorable prognosis, whereas the positive score values indicate that higher expression correlates with poor outcome and shorter survival.
  • the weighted survival predictor model is based on a cumulative score of the weighted expression values of all of the genes of a set of genes.
  • the invention provides for an individual survival score for each member of a set of genes, calculated by multiplying the expression value or the logarithmically transformed expression value for each member of a set of genes by a relative weight coefficient or a correlation coefficient, as determined by multivariate Cox analysis.
  • the invention also provides for a survival score, wherein a survival score is the sum of the individual survival scores for each member of a set of genes.
  • Survival analysis refers to a method of verifying that a set of genes or a subset of genes according to the invention is "predictive", as defined herein, of a particular phenotype of interest. Survival analysis includes but is not limited to Kaplan-Meier survival analysis. In one embodiment, the Kaplan-Meier survival analysis is carried out using the Prism 4.0 software. Statistical significance of the difference between the survival curves for different groups of patients was assessed using Chi square and Logrank tests.
  • the Kaplan-Meier survival analysis is carried out using GraphPad Prism version 4.00 software' (GraphPad Software).
  • the endpoint for survival analysis in prostate cancer is the biochemical recurrence defined by the serum prostate-specific antigen (PSA) increase after therapy.
  • Disease-free interval is defined as the time period between the date of radical prostatectomy (RP) and the date of PSA relapse (for the recurrence group) or the date of last follow-up (for the non-recurrence group).
  • RP radical prostatectomy
  • RP radical prostatectomy
  • RP date of PSA relapse
  • last follow-up for the non-recurrence group.
  • Statistical significance of the difference between the survival curves for different groups of patients is assessed using X ⁇ 2> and log-rank tests.
  • the major mathematical complication with survival analysis is that you usually do not have the luxury of waiting until the very last subject has died of old age; you normally have to analyze the data while some subjects are still alive. Also, some subjects may have moved away, and may be lost to follow-up. In both cases, the subjects were known to have survived for some amount of time (up until the time the one performing the analysis last saw them). However, the one performing the analysis may not know how much longer a subject might ultimately have survived.
  • Several methods have been developed for using this "at least this long" information to preparing unbiased survival curve estimates, the most common being the Life Table method and the method of Kaplan and Meier Analysis, as defined herein.
  • the present invention is also directed to a kit to detect the presence of two or more markers from a pathway related to cancer, from another pathway, or from transregulatory SNPs as specified herein.
  • the kit can contain as detection means protein-specific differentially-labeled fluorescent antibodies; protein-specific antibodies from different species (mouse, rabbit, goat, chicken, etc.) and differentially labeled species-specific antibodies; DNA and RNA-based probes with different fluorescent dyes; bar-coded nucleic acid- and protein-specific probes (each probes having a unique combination of colors), and any other detection means known in the art.
  • the kit can include a marker sample collection means and a means for determining whether the sample expresses in the same cell at the same time two or more markers from a pathway related to cancer.
  • the kit contains a standard and/or an algorithmic device for assessing the results and additional reagents and components including for example DNA amplification reagents, DNA polymerase, nucleic acid amplification reagents, restrictive enzymes, buffers, a nucleic acid sampling device, DNA purification device, deoxynucleotides, oligonucleotides (e.g. probes and primers) etc.
  • a standard and/or an algorithmic device for assessing the results and additional reagents and components including for example DNA amplification reagents, DNA polymerase, nucleic acid amplification reagents, restrictive enzymes, buffers, a nucleic acid sampling device, DNA purification device, deoxynucleotides, oligonucleotides (e.g. probes and primers) etc.
  • DFI disease-free interval
  • FBS fetal bovine serum
  • NPEC normal prostate epithelial cells
  • PC prostate cancer
  • PSA prostate specific antigen
  • Q-RT-PCR quantitative reverse-transcription polymerase chain reaction
  • RP radical prostatectomy
  • SKCC Sidney Kimmel Cancer Center
  • AMACR alpha-methylacyl-coenzyme A racemase
  • Ezh2 enhancer of zeste homolog 2
  • FACS fluorescence activated cell sorting.
  • a CTOP algorithm can be built which combines the prognostic power of multiple gene expression-based CTOP models.
  • Application of a CTOP algorithm to large databases of early-stage breast and prostate tumors identifies cancer patients with 100% probability of a cure with existing cancer therapies as well as patients with nearly 100% likelihood of treatment failure, thus providing a clinically feasible framework essential for the introduction of rational evidence-based individualized therapy selection and prescription protocols.
  • CTOP genes manifest a common feature of SNP patterns reflected in population-specific profiles of SNP genotype and allele frequencies.
  • a majority of population-specific SNPs associated with CTOP genes represented by YRI population-differentiation SNPs, perhaps, reflecting a general trend of higher level of low- frequency alleles in the YRI population compared to CEU, CHB, and JPT populations due to bottlenecks in history of non-YRI populations.
  • five non-synonymous coding SNPs were identified that represented good candidates for follow-up functional studies.
  • population-specific SNP patterns are readily discernable for genes with well-established causal role in cancer as oncogenes or tumor suppressor genes, implying that the genes are targets for geographically localized form of natural selection (Figure 5).
  • Figure 5 the data suggests the presence of population differentiation-associated cancer-related patterns of SNPs spanning across multiple chromosomal loci and, perhaps, forming a genome-scale cancer haplotype pattern.
  • PC prostate carcinoma
  • M metastasis signature
  • R recurrence signature
  • DFC death-from-cancer signature.
  • Differential expression of genes listed in the table was confirmed by the Q-RT-PCR method using LCM dissected samples of malignant and adjacent normal tissues from prostate tumor samples.
  • SNP-based gene expression signatures predict therapy outcome in prostate and breast cancer patients.
  • Our analysis demonstrates that CTOP genes are distinguished by a common population specific SNP pattern and potential utility as molecular predictors of cancer treatment outcome based on distinct profiles of mRNA expression.
  • All gene expression models designed to predict cancer therapy outcome were developed using phenotype-based signature discovery protocols, e.g., genetic loci comprising the predictive models were selected based on association of their expression profiles with clinically relevant phenotype of interest.
  • phenotype-based signature discovery protocols e.g., genetic loci comprising the predictive models were selected based on association of their expression profiles with clinically relevant phenotype of interest.
  • heritable genetic variations driven by geographically localized form of natural selection determining population differentiations may have a significant impact on cancer treatment outcome by influencing the individual's gene expression profile.
  • genes, expression levels of which are known to be regulated by SNP variations may provide good candidates for building gene expression-based CTOP models.
  • Microarray analysis identifies clinically relevant cooperating oncogenic pathways associated with cancer therapy outcome.
  • BiId et al., Nature 439: 353-357 (2006) provides compelling evidence of the power of microarray gene expression analysis in identifying multiple clinically relevant oncogenic pathways activated in human cancers. It provides mechanistic explanation to mounting experimental data demonstrating that there are multiple gene expression signatures predicting cancer therapy outcome in a given set of patients diagnosed with a particular type of cancer: presence of multiple CTOP models is most likely reflect deregulation of multiple oncogenic pathways, perhaps, cooperating in development of an oncogenic state.
  • intronic SNPs A majority of SNPs identified in this study is represented by intronic SNPs, suggesting that intronic SNPs may influence gene expression by yet unknown mechanism. Theoretically, intronic SNPs may influence gene expression by affecting a variety of processes such as chromatin silencing and remodeling, alternative splicing, transcription of microRNA genes, processivity of RNA polymerase, etc. Most likely mechanism of action would entail effect on stability and affinity of interactions between DNA molecule and corresponding multi-subunit complexes. Comparative genomics analysis has shown that about 5% of the human sequence is highly conserved across species, yet less than half of this sequence spans known functional elements such as exons.
  • conserved non-genic sequences lack diversity because of selective constraint due to purifying selection; alternatively, such regions may be located in cold-spots for mutations. Most recent evidence shows that conserved non-genic sequences are not mutational cold-spots, and thus represent high interest for functional study. It would be of interest to determine whether population differentiation intronic SNPs overlap with such highly evolutionary conserved non-genic sequences.
  • Our analysis provides a possible clue with regard to mechanisms of genesis and evolution of disease-causing loci and translation of SNP variations in disease phenotypes. Geographically localized form of natural selection drives evolution of population differentiation SNP profiles which is translated in phenotypic diversity by determining individual gene expression variations.
  • Preferred markers within the context of the present invention include the double positive BMI1/Ezh2 from the PcG pathway.
  • the Polycomb group (PcG) gene BMIl is required for the proliferation and self-renewal of normal and leukemic stem cells.
  • PcG Polycomb group
  • Bmil oncogene causes neoplastic transformation of lymphocytes and plays essential role in pathogenesis of myeloid leukemia.
  • Another PcG protein, Ezh2 was implicated in metastatic prostate and breast cancers, suggesting that PcG pathway activation is relevant for epithelial malignancies. Whether an oncogenic role of the BMIl and PcG pathway activation may be extended beyond the leukemia and may affect progression of solid tumors has previously remained unknown.
  • activation of the BMIl oncogene-associated PcG pathway plays an essential role in metastatic prostate cancer, thus mechanistically linking the pathogenesis of leukemia, self-renewal of stem cells, and prostate cancer metastasis.
  • TMA tissue microarray
  • Quantitative immunofluorescence co-localization analysis and expression profiling experiments documented increased BMIl and Ezh2 expression in clinical prostate carcinoma samples and demonstrated that high levels of BMIl and Ezh2 expression are associated with markedly increased likelihood of therapy failure and disease relapse after radical prostatectomy.
  • Gene-silencing analysis reveals that activation of the PcG pathway is mechanistically linked with highly malignant behavior of human prostate carcinoma cells and is essential for in vivo growth and metastasis of human prostate cancer. It is concluded that the results of experimental and clinical analyses indicate the important biological role of the PcG pathway activation in metastatic prostate cancer.
  • the PcG pathway activation is a common oncogenic event in pathogenesis of metastatic solid tumors and provides the basis for development of small molecule inhibitors of the PcG chromatin silencing pathway as a novel therapeutic modality for treatment of metastatic prostate cancer.
  • the PcG pathway activation hypothesis implies that individual cells with activated chromatin silencing pathway would exhibit a concomitant nuclear expression of both BMIl and Ezh2 proteins. Furthermore, cells with activated PcG pathway would manifest the increased expression levels of protein substrates targeted by the activation of corresponding enzymes to catalyze the H2A-K119 ubiquitination (BMIl -containing PRCl complex) and H3-K27 methylation (Ezh2-containing PRC2 complex). Observations that increased BMIl expression is associated with metastatic prostate cancer suggest that the PcG pathway might be activated in metastatic human prostate carcinoma cells. Consistent with this idea, previous independent studies documented an association of the increased Ezh2 expression with metastatic disease in prostate cancer patients. Therefore, immunofluorescence analysis was applied to measure the expression of protein markers of the PcG pathway activation in prostate cancer metastasis precursor cells isolated from blood of nude mice bearing orthotopic human prostate carcinoma xenografts.
  • Immunofluorescence analysis reveals that expression of all four individual protein markers of PcG pathway activation is elevated in blood-bome human prostate carcinoma metastasis precursor cells compared to the parental cells comprising a bulk of primary tumors ( Figures 20 & 21).
  • the quantitative immunofluorescence co-localization analysis allowing for a simultaneous detection and quantification of several markers in a single cell was carried out.
  • the quantitative immunofluorescence co-localization analysis demonstrates a marked enrichment of the population of blood-borne human prostate carcinoma metastasis precursor cells with the dual positive high BMIl/Ezh2-expressing cells (Figure 20A).
  • the level of gene amplification as determined by the measurement of DNA copy number for both BMIl and Ezh2 genes is higher in metastatic cancer cell variants compared to the non-metastatic or less malignant counterparts, suggesting that gene amplification may play a casual role in elevation of the BMIl and Ezh2 oncoprotein expression levels and high BMIl/Ezh2-expressing cells may acquire a competitive survival advantage during tumor progression.
  • PcG pathway activation renders circulating human prostate carcinoma metastasis precursor cells resistant to anoikis.
  • human prostate carcinoma metastasis precursor cells were isolated from the blood of nude mice bearing orthotopic human prostate carcinoma xenografts, transfected with BMIl, Ezh2, or control siRNAs, and continuously monitored for mRN A and protein expression levels of BMIl, Ezh2, and a set of additional genes and protein markers using inmmunofluorescence analysis, RT-PCR, and Q-RT-PCR methods.
  • Q-RT-PCR and RT-PCR analyses showed that siRNA-mediated BMIl -silencing caused ⁇ 90% inhibition of the endogenous BMIl mRNA expression.
  • results of the experiments demonstrate that a population of highly metastatic prostate carcinoma cells is markedly enriched for cancer cells expressing increased levels of multiple markers of the PcG pathway activation. These data suggest that carcinoma cells with activated PcG pathway may manifest a highly malignant behavior in vivo characteristic of cancer cell variants selected for increased metastatic potential.
  • blood-borne human prostate carcinoma metastasis precursor cells were treated with chemically modified stable siRNA targeting either BMIl or Ezh2 mRNAs to generate a cancer cell population with diminished levels of dual positive high BMI l/Ezh2-expressing carcinoma cells.
  • Stable siRNA- treated prostate carcinoma cells continue to grow in adherent culture in vitro for several weeks allowing for expansion of siRNA-treated cultures in quantities sufficient for in vivo analysis.
  • Microarray analysis demonstrates that cancer patients with high levels of BMIl and Ezh2 mRNA expression in prostate tumors have a significantly worst relapse-free survival after radical prostatectomy (RP) compared with the patients having low levels of BMIl and Ezh2 expression (Figure 26), suggesting that more profound alterations of the PcG protein chromatin silencing pathway in carcinoma cells are associated with therapy resistant clinically lethal prostate cancer phenotype.
  • Figure 26E shows the Kaplan-Meier survival analysis of 79 prostate cancer patients stratified into five sub-groups using eight-covariate cancer therapy outcome (CTO) algorithm (Table 2, below). Table 2. 8-covariate prostate cancer recurrence predictor model
  • BMIl expression level remains a statistically significant prognostic marker in the multivariate analysis (Table 3).
  • Application of the 8-covariate prostate cancer recurrence model combining the incremental statistical power of individual prognostic markers appears highly informative in stratification of prostate cancer patients into sub-groups with differing likelihood of therapy failure and disease relapse after radical prostatectomy ( Figure 26).
  • One of the distinctive features of this model is that it identifies a sub-group of prostate cancer patients comprising bottom 20% of recurrence predictor score and manifesting no clinical or biochemical evidence of disease relapse ( Figure 26).
  • 80% of patients in a sub-group comprising top 20% of recurrence predictor score failed therapy within five year period after radical prostatectomy.
  • the prognostic power of the 11-gene signature was validated in multiple independent therapy outcome sets of clinical samples obtained from more than 2,500 cancer patients diagnosed with 12 different types of cancer, including six epithelial (prostate; breast; lung; ovarian; gastric; and bladder cancers) and five non-epithelial (lymphoma; mesothelioma; medulloblastoma; glioma; and acute myeloid leukemia, AML) malignancies.
  • Presence of dual positive high BMIl/Ezh2-expressing cells appears essential for maintenance of tumorigenic and metastatic potential of human prostate carcinoma cells in vivo, since targeted depletion of dual positive high BMIl/Ezh2-expressing cells from a population of highly metastatic human prostate carcinoma cells treated with stable siRNAs generates a cancer cell population with dramatically diminished malignant potential in vivo.
  • the BMIl and Ezh2 proteins are members of the Polycomb group protein (PcG) chromatin silencing complexes conferring genome scale transcriptional repression via covalent modification of histones.
  • the BMIl PcG protein is a component hPRClL complex (human Polycomb repressive complex 1-like) which was recently identified as the E3 ubiquitin ligase complex that is specific for histone H2A and plays a key role in Polycomb silencing.
  • Ubiquitination/deubiquitination cycle of histones H2A and H2B is important in regulating chromatin dynamics and transcription mediated, in part, via 'cross-talk' between histone ubiquitination and methylation.
  • Rnf2 one of the up-regulated genes in the 11-gene death- from-cancer signature profile (Rnf2) plays a central role in the PRC 1 complex formation and function thus complementing the BMI-I function in the PRCl complex.
  • Rnf2 expression plays a crucial non-redundant role in development during a transient contact formation between PRCl and PRC2 complexes via Rnf2 as described for Drosophila.
  • the Ezh2 protein is a member of the Polycomb PRC2 and PRC3 complexes with a histone lysine methyltransferase (HKMT) activity that is associated with transcriptional repression due to chromatin silencing.
  • the HKMT-Ezh2 activity targets lysine residues on histones Hl and H3 (H3-K27 or H1-K26).
  • H3-K27 methylation conferred by an active HKMT- Ezh2-containing complex is one of the key molecular events essential for chromatin silencing in vivo.
  • Ezh2 associates with different EED isoforms thereby determining the specificity of histone methyltransferase activity toward histone H3-K27 or histone H 1 -K26.
  • PRCl, PRC2, and PRC3 complexes implying a coordinate regulation of expression of their essential components such as BMIl and Ezh2 oncoproteins. It follows that dual positive high BMIl/Ezh2-expressing carcinoma cells with elevated expression of the H2AubiKl 19 and H3metK27 histones should be regarded as cells with activated PcG protein chromatin silencing pathway.
  • the BMIl -containing PcG complex forms a unique discrete nuclear structure that was termed the PcG bodies, the size and number of which in nuclei significantly varied in different cell types.
  • the nuclei of dual positive high BMIl/Ezh2-expressing cells almost uniformly contain six prominent discrete PcG bodies, perhaps, reflecting the high level of the BMIl expression and indicating the active state of the PcG protein chromatin silencing pathway.
  • the new type of the PcG chromatin silencing complex is formed containing the Sirtl protein. This suggests that in high Ezh2-expressing carcinoma cells a distinct set of genetic loci could be repressed due to activation of the Ezh2/Sirtl -containing PcG chromatin silencing complex.
  • a cancer stem cell hypothesis proposes that the presence of rare stem cell-resembling tumor cells among the heterogeneous mix of cells comprising a tumor is essential for tumor progression and metastasis of epithelial malignancies.
  • One of the implications of a cancer stem cell hypothesis is that similar genetic regulatory pathways might define critical stem cell-like functions in both normal and tumor stem cells.
  • BMIl oncogene-driven pathway(s) as one of the key regulatory mechanisms of "sternness" functions in both normal and cancer stem cells.
  • the Polycomb group (PcG) gene BMIl influences the proliferative potential of normal and leukemic stem cells and is required for the self-renewal of hematopoietic and neural stem cells. Self-renewal ability is one of the essential defining properties of a pluripotent stem cell phenotype.
  • BMIl oncogene is expressed in all primary myeloid leukemia and leukemic cell lines analyzed so far and over-expression of BMIl causes neoplastic transformation of lymphocytes.
  • Recent clinical genomics data provide a powerful evidence supporting a cancer stem cell hypothesis and suggest that gene expression signatures associated with the "sternness" state of a cell (defined as phenotypes of self-renewal, asymmetrical division, and pluripotency) might be informative as molecular predictors of cancer therapy outcome.
  • a mouse/human comparative cross-species translational genomics approach was utilized to identify an 1 1-gene signature that distinguishes stem cells with normal self-renewal function from stem cells with drastically diminished self-renewal ability due to the loss of the BMIl oncogene as well as consistently displays a normal stem cell-like expression profile in distant metastatic lesions as revealed by the analysis of metastases and primary tumors in both a transgenic mouse model of prostate cancer and cancer patients.
  • Kaplan-Meier analysis confirmed that a stem cell-like expression profile of the 11-gene signature in primary tumors is a consistent powerful predictor of a short interval to disease recurrence, distant metastasis, and death after therapy in cancer patients diagnosed with twelve distinct types of cancer.
  • These data suggest the presence of a conserved BMIl oncogene-driven pathway, which is similarly activated in both normal stem cells and a clinically lethal therapy- resistant subset of human tumors diagnosed in a wide range of organs and uniformly exhibiting a marked propensity toward metastatic dissemination.
  • Cancer stem cells may indeed constitute metastasis precursor cells since most of the early disseminated carcinoma cells detected in the bone marrow of breast cancer patients manifest a breast cancer stem cell phenotype.
  • ESC embryonic stem cells
  • This analysis reveals multiple gene expression signatures of the ESC regulatory circuitry which appear highly informative in stratification of the early-stage breast, lung, and prostate cancer patients into subgroups with dramatically distinct likelihood of therapy failure.
  • Genetic signatures of regulatory circuitry of embryonic stem cells (ESC) identify therapy- resistant phenotypes in cancer patients diagnosed with multiple types of epithelial malignancies.
  • CTOP Cancer therapy outcome predictor
  • Detection of failures was calculated as the number of cases that actually failed therapy and were classified by the CTOP algorithm into poor prognosis groups (top 50% scores) with relation to the total number of therapy failure cases in the data set. Microarray data sets and associated clinical information were reported elsewhere (5). Und, undefined due to the 100% cure rate in the good prognosis group.
  • Gene expression signatures of the ESC regulatory circuitry predict therapy failure in multiple independent data sets of breast cancer patients.
  • Detection of failures was calculated as the number of cases that actually failed therapy and were classified by the CTOP algorithm into poor prognosis groups (top 50% scores) with relation to the total number of therapy failure cases in the data set. Microarray data sets and associated clinical information were reported elsewhere.
  • Detection of failures was calculated as the number of cases that actually failed therapy and were classified by the CTOP algorithm into poor prognosis groups (top 50% scores) with relation to the total number of therapy failure cases in the data set. Microarray data sets and associated clinical information were reported elsewhere.
  • Ten "sternness” signatures (six ESC signatures plus BCD-TF, BMIl -pathway, PcG methylation, and Histones H3/H2A signatures). Detection of failures (the number and percentage) was calculated as the number of cases that actually failed therapy and were classified by the CTOP algorithm into poor prognosis groups (top 50% scores) with relation to the total number of therapy failure cases in the data set. Microarray data sets and associated clinical information were reported elsewhere.
  • the present invention can also be used to analyze the level of transcription factors as either an indicator of the presence of cancer or other diseases or phenotypes or as a predictor of therapy outcome. Details of transcription factor analysis are below.
  • BCD-TF bivalent chromatin domain transcription factor genes
  • nucleosomal compositions of histones harboring specific modifications of the histone tails defines mutually exclusive transcriptionally active or silent states of the chromatin.
  • Transcriptional status of corresponding genetic loci in genomes of most cells is governed by the nucleosome-defmed chromatin patterns and strictly follows activation/repression rules.
  • H3K27met3 "silent"
  • H3K4 "active"
  • TF transcription factor
  • Many of the bivalent chromatin domain (BCD) - containing genes were previously identified as the Polycomb Group (PcG) protein-target genes in both human and mouse ESC and are repressed or transcribed at low levels in ESC.
  • "sternness" gene expression profiles of BCD-TF in mouse ESC were derived by comparing microarray analyses of pluripotent self-renewing ESC (control ESC cultures treated with HP siRNA) versus ESC treated with Esrrb siRNA (day 6).
  • Esrrb siRNA-treated ESC does not manifest "sternness” phenotype and form colonies of differentiated cells.
  • Kaplan-Meier analysis demonstrates that prostate and breast cancer patients with tumors harboring ESC-like expression profiles of the eight-gene BCD-TF signature are more likely to fail therapy (bottom two panels), suggesting that a sub-set of BCD-TF genes defined here as the eight gene BCD-TF signature manifests "sternness" expression profiles in therapy-resistant prostate and breast tumors (Figure 43).
  • therapy-resistant and therapy-sensitive tumors manifest distinct pattern of association with "stemness'Vdifferentiation pathways engaged in ESC during transition from pluripotent self-renewing state to differentiated phenotypes.
  • One of the major implications of this hypothesis is the prediction that therapy-resistant and therapy-sensitive tumors develop within genetically distinct "stemness'Vdifferentiation programs. This prediction was tested by interrogating the prognostic power of genes comprising the ESC pattern 3 "stemness'Vdifferentiation program recently identified by a combination of the RNA interference and gene expression analyses.
  • the present invention can also be used to analyze the DNA promoter methylation patterns of genes as either an indicator of the presence of cancer or other diseases or phenotypes or as a predictor of therapy outcome. Details of the analysis of DNA promoter methylation patterns of genes are below. Is therapy-resistant phenotype of human epithelial malignancies associated with distinct methylation patterns of the Polycomb target genes?
  • Multivariate Cox regression analysis demonstrates that PcG target genes with promoters frequently hypermethylated in cancer manifest distinct expression profiles associated with therapy-resistant and therapy-sensitive prostate and breast cancers ( Figure 45), implying that differences in gene expression between tumors with distinct outcome after therapy may be driven, in part, by the distinct promoter hypermethylation patterns of the PcG target genes. These differences can be exploited to generate highly informative gene expression signatures of the PcG target genes hypermethylated in cancer for stratification of prostate and breast cancer patients into sub-groups with statistically distinct likelihood of therapy failure (Figure 45). This analysis suggests that therapy-resistant and therapy-sensitive tumors are likely to manifest different profiles of the promoter hypermethylation of PcG target genes and these differences can be utilized for development of DNA-based diagnostic, prognostic, and individualized therapy selection tests.
  • Post-translational modifications of the histones H3 and H2A in particular, trimethylation of the lysine 27 residue (H3met3K27) by the Ezh2-containing PRC2 complex and ubiquitination of the histone H2A by the BMIl -containing PRCl complex, are consistently linked to the transcriptional silencing mediated by the PcG proteins and a cross-talk between Polycomb targeting and DNA promoter hypermethylation. It was therefore tested whether therapy-resistant and therapy-sensitive tumors would manifest distinct expression profiles of the histones H3 and H2 A variants. Multivariate Cox regression analysis demonstrates that activation and inhibition of expression of distinct variants of the H3 and H2A histones are associated with tumors manifesting different outcome after therapy.
  • the present invention can also be used to analyze the patterns of transregulatory SNPs as markers for either an indicator of the presence of cancer or other disease states or phenotypes or as a predictor of disease therapy outcome.
  • Transregulatory SNPs are intronic SNPs which regulate the gene expression of genes in a different loci than the SNPs themselves. These SNPs are not part of a gene, they are located in non-coding sections of DNA. For example, SNPs located on a non-coding section of chromosome 1 have been found to regulate the expression of genes on chromosome 5, 7, and 1 1. These transregulatory SNPs that control gene expression at a distance are also ones that contribute to a disease phenotype and can thus be used as predictors of therapy outcome.
  • Such SNP profiles can be used to design association studies (which reduces the sample size) and can also be linked with cancer or other disease state therapy predictors. Such studies resulted in the discovery of a class of intronic SNPs that control gene expression at a distance (transregulatory SNPs) and which also can be used as predictors of therapy outcome in any disease state. More particularly, a set of SNPs have been discovered which can be used as treatment outcome predictors for breast cancer and prostate cancer. Such SNPs are shown in Figure 48.
  • Kaplan-Meier survival analysis was performed as described in Example 14 to assess the patients' stratification performance of each of the SNP-based signatures. Patients were sorted in descending order based on the numerical values of the CTOP scores and survival curves were generated by designating the patients with top 50% scores and bottom 50% scores into poor prognosis and good prognosis groups, respectively. These analytical protocols were independently carried out for a 79-patient prostate cancer data set and a 286-patient breast cancer data set. The survival analysis using these transregulatory SNPs as predictors of treatment outcome in breast cancer and prostate cancer are shown in Figures 49 and 50, respectively.
  • Additional markers within the scope of the present invention include longevity-related genes as markers for either an indicator of the presence of aging or Alzheimer's or as a predictor of aging or Alzheimer's therapy outcome. These signatures include a 9-gene, 1 1-gene, and 23- gene Alzheimer's signatures as well as a 38-gene and 57-gene longevity signatures, which are shown in Figures 51-55 and in Figures 56 and 57. These gene expression signatures have been identified as those associated with the "centarian" phenotype of Homo sapiens.
  • Such gene expression signatures and markers thereof can be used to identify promising therapeutic modalities (including genetic, biological, and small molecule effectors), which can be used to induce in human cells expression changes resembling the expression patterns of the "centarian" phenotype.
  • the potential therapeutic utility of such identified effectors can then be used to extend the life span of mammalian species.
  • “Sternness” CTOP algorithm identifies therapy-resistant phenotypes and predicts the likelihood of treatment failure in prostate, breast, ovarian, and lung cancer patients.
  • the Affimetrix-based CTOP algorithms were developed using the Netherlaqnds-286 data set and tested using the MSKCC-95 and Duke- 169 data sets.
  • the Agilent-based CTOP algorithms were developed using the Nethcrlads-97 data set and tested using the Netherlands-295 data set.
  • the CTOP algorithms based on the cancer-specific death after therapy were developed using the Netherlands-295 data set (last row).
  • the end-points are the overall survival and cancer-specific death.
  • the end- points are the relapse-free survival.
  • the Netherlands-97 data set the end-points are metastasis-free survival.
  • CTOP algorithm identified using breast cancer data set was applied to the lung cancer data set and ovarian cancer data set.
  • the end-points are the overall survival and cancer-specific death.
  • the breast cancer data set the end-points are the disease-free survival.
  • the prostate cancer data set the end point is the relapse-free survival.
  • poor prognosis groups include patients with top 50% values of the cumulative CTOP scores in a given data set. Und, undefined due to the 100% cure rate in the good prognosis group. See text for details. Table 10. Classification performance of the CTOP algorithm comprising nine "sternness" signatures in predicting clinical outcome in prostate, breast, lung, and ovarian cancer atients
  • CTOP algorithm identified using breast cancer data set was applied to the lung cancer data set and ovarian cancer data set.
  • the end-points are the overall survival and cancer-specific death.
  • the end-points are the disease-free survival.
  • the end point is the relapse-free survival.
  • poor prognosis groups include patients with top 60% values of the cumulative CTOP scores in a given data set.
  • the poor prognosis group includes patients with top 80% cumulative CTOP score values. Und, undefined due to the 100% cure rate in the good prognosis group. See Figure 6 and text for details.
  • CTOP algorithm identified using breast cancer data set was applied to the lung cancer data set and ovarian cancer data set.
  • the end-points are the overall survival and cancer-specific death.
  • the end-points are the disease-free survival.
  • the end point is the relapse- free survival.
  • poor prognosis groups include patients with top 60% values of the cumulative CTOP scores in a given data set. Und, undefined due to the 100% cure rate in the good prognosis group. See text for details.
  • CTOP cancer therapy outcome predictor
  • a "sternness” CTOP algorithm demonstrates nearly 100% prognostic accuracy for a majority of patients in retrospective analysis of large cohorts of breast, prostate, lung, and ovarian cancer patients, suggesting that therapy-resistant and therapy-sensitive tumors develop within genetically distinct "stemness'Vdifferentiation programs driven by engagement of the PcG proteins chromatin silencing pathway.
  • the signatures of the PcG pathway appear highly informative in stratification of the early-stage breast, lung, and prostate cancer patients into subgroups with dramatically distinct likelihood of therapy failure.
  • the analysis further supports the existence of transcriptionally discernable type of human cancer detectable in a sub-group of early-stage cancer patients diagnosed with distinct epithelial malignancies appearing in multiple organs.
  • These early-stage carcinomas of seemingly various origins appear to exhibit a poor therapy outcome gene expression profile, which is uniformly associated with increased propensity to develop metastasis, high likelihood of treatment failure, and increased probability of death from cancer after therapy.
  • Cancer patients who fit this transcriptional profile might represent a genetically, biologically, and clinically distinct type of cancer exhibiting highly malignant clinical behavior and therapy resistance phenotype even at the early stage of tumor progression.
  • Enrichment of primary tumors with NSCs increases likelihood of horizontal genomic transfer (large-scale transfer of DNA and chromatin) between NSCs and tumor cells via cell fusion and/or uptake of apoptotic bodies.
  • Reprogrammed somatic hybrids of tumor cells and NSCs acquire transformed phenotype and epigenetic self-renewal program.
  • Postulated progeny of hybrid cells contains a sub-population of self-renewing cancer stem cells with epigenetic and transcriptional markers of NSCs and high propensity toward metastatic dissemination.
  • Recent experimental observations demonstrate direct involvement of the bone marrow-derived cells in development of breast and colon cancers in transgenic mouse cancer models suggesting that cancer stem cells can originate from the bone marrow-derived cells.
  • CMAP-based search for cancer therapeutics targeting "sternness” pathways in solid tumors reveals drug combinations causing transcriptional reversal of "sternness” signatures associated with therapy-resistant phenotypes of epithelial cancers.
  • CMAP analysis demonstrates that a combination of the PI3K pathway inhibitor, estrogen receptor (ER) antagonist, and mTOR inhibitor causes transcriptional reversal of "sternness” signatures in 35 of 37 (95%) patients diagnosed with therapy-resistant prostate cancer.
  • CMAP-based design of target-tailored individualized breast cancer therapies reveals drug combinations causing transcriptional reversal of "sternness' signatures in 91 of 107 (85%) of the early-stage breast cancer patients with therapy-resistant disease phenotypes.
  • a combination of PI3K pathway inhibitor, ER antagonist, and HDAC inhibitor causes transcriptional reversal of "sternness" pathways in 53 of 107 (49.5%) patients diagnosed with the early-stage therapy- resistant breast cancer.
  • CMAP-based analysis of target-tailored individualized therapies for lung cancer reveals drug combinations causing transcriptional reversal of "sternness' signatures in 39 of 45 (87%) of the early-stage lung cancer patients with therapy-resistant tumor phenotypes.
  • the connectivity map-based approach to discovery of small molecule drugs targeting clinical phenotype-associated gene expression signatures may be useful for multiple therapeutic applications beyond therapy-resistant human malignancies.
  • Two clinical outcome sets comprising 21 (outcome set 1) and 79 (outcome set 2) samples were utilized for analysis of the association of the therapy outcome with expression levels of the BMIl and Ezh2 genes and other clinico-pathological parameters.
  • Expression profiling data of primary tumor samples obtained from 1243 microarray analyses of eight independent therapy outcome cohorts of cancer patients diagnosed with four types of human cancer were analyzed in this study. Microarray analysis and associated clinical information for clinical samples analyzed in this work were previously published and are publicly available.
  • Prostate tumor tissues comprising clinical outcome data set were obtained from 79 prostate cancer patients undergoing therapeutic or diagnostic procedures performed as part of routine clinical management at the Memorial Sloan-Kettering Cancer Center (MSKCC). Clinical and pathological features of 79 prostate cancer cases comprising validation outcome set are presented elsewhere. Median follow-up after therapy in this cohort of patients was 70 months. Samples were snap-frozen in liquid nitrogen and stored at — 8O 0 C. Each sample was examined histologically using H&E-stained cryostat sections. Care was taken to remove nonneoplastic tissues from tumor samples. Cells of interest were manually dissected from the frozen block, trimming away other tissues. Overall, 146 human prostate tissue samples were analyzed in this study, including forty-six samples in a tissue microarray (TMA) format. TMA samples analyzed in this study were exempt according to the NIH guidelines.
  • TMA tissue microarray
  • the cancer therapy outcome database includes 3,176 therapy outcome samples from patients diagnosed with thirteen distinct types of cancers (Table 3): prostate cancer (220 patients); breast cancer (1 171 patients); lung adenocarcinoma (340 patients); ovarian cancer (216 patients); gastric cancer (89 patients); bladder cancer (31 patients); follicular lymphoma (191 patients); diffuse large B-cell lymphoma (DLBCL, 298 patients); mantle cell lymphoma (MCL, 92 patients); mesothelioma (17 patients); medulloblastoma (60 patients); glioma (50 patients); acute myeloid leukemia (AML, 401 patients).
  • Table 3 prostate cancer (220 patients); breast cancer (1 171 patients); lung adenocarcinoma (340 patients); ovarian cancer (216 patients); gastric cancer (89 patients); bladder cancer (31 patients); follicular lymphoma (191 patients); diffuse large B-cell lymphoma (DLBCL, 298 patients); mantle cell lymphom
  • LNCap- and PC-3-derived cell lines were developed by consecutive serial orthotopic implantation, either from metastases to the lymph node (for the LN series), or reimplanted from the prostate (Pro series). This procedure generated cell variants with differing tumorigenicity, frequency and latency of regional lymph node metastasis. Except where noted, cell lines were grown in RPMI 1640 supplemented with 10% FBS and gentamycin (Gibco BRL) to 70-80% confluence and subjected to serum starvation as described, or maintained in fresh complete media, supplemented with 10% FBS.
  • Cells were harvested by 5-min digestion with 0.25% trypsin/0.02% EDTA (Irvine Scientific, Santa Ana, CA, USA), washed and resuspended in serum free medium. Cells at concentration 1.7 x 10 s cells/well in 1 ml of serum free medium were plated in 24-well ultra low attachment polystyrene plates (Corning Inc., Corning, NY, USA) and incubated at 37°C and 5% CO 2 overnight. Viability of cell cultures subjected to anoikis assays were > 95% in Trypan blue dye exclusion test.
  • Apoptotic cells were identified and quantified using the Annexin V-FITC kit (BD Biosciences Pharmingen) per manufacturer instructions. The following controls were used to set up compensation and quadrants: 1) Unstained cells; 2) Cells stained with Annexin V-FITC (no PI); 3) Cells stained with PI (no Annexin V-FITC). Each measurements were carried out in quadruplicate and each experiments were repeated at least twice. Annexin V-FITC positive cells were scored as early apoptotic cells; both Annexin V-FITC and PI positive cells were scored as late apoptotic cells; unstained Annexin V-FITC and PI negative cells were scored as viable or surviving cells. In selected experiments apoptotic cell death was documented using the TUNEL assay.
  • Cells were washed in cold PBS phosphate-buffered saline and stained according to manufacturer's instructions using the Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, San Jose, CA, USA) or appropriate antibodies for cell surface markers. Flow analysis was performed by a FACS Calibur instrument (BD Biosciences, San Jose, CA, USA). Cell Quest Software was used for data acquisition and analysis. All measurements were performed under the same instrument setting, analyzing 10 3 — 10 4 cells per sample.
  • RNA and mRNA extraction For gene expression analysis, cells were harvested in lysis buffer 2 hrs after the last media change at 70-80% confluence and total RNA or mRNA was extracted using the RNeasy (Qiagen, Chatsworth, CA) or FastTract kits (Invitrogen, Carlsbad, CA). Cell lines were not split more than 5 times prior to RNA extraction, except where noted. Detailed protocols were described elsewhere.
  • Asymetrix arrays The protocol for mRNA quality control and gene expression analysis was that recommended by Affymetrix. In brief, approximately one microgram of mRNA was reverse transcribed with an oligo(dT) primer that has a T7 RNA polymerase promoter at the 5 1 end. Second strand synthesis was followed by cRNA production incorporating a biotinylated base. Hybridization to Affymetrix U95Av2 arrays representing 12,625 transcripts overnight for 16 h was followed by washing and labeling using a fluorescently labeled antibody. The arrays were read and data processed using Affymetrix equipment and software as reported previously.
  • the significance of the overlap between the lists of stem cell-associated and prostate cancer-associated genes was calculated by using the hypergeometric distribution test.
  • the Multiple Experiments Viewer (MEV) software version 3.0.3 of the Institute for Genomic Research (TIGR) was used for clustering algorithm data analysis and visualization.
  • Polycomb pathway "sternness" signatures The initial analysis was performed using two cancer therapy outcome data sets: 79-patients prostate cancer data set and 286-patients breast cancer data set. For each parent signature (Table 4), the multivariate Cox regression analysis was carried out. Consistent with the concept that therapy resistant and therapy sensitive tumors develop within distinct Polycomb-driven "stemness'Vdifferentiation programs, all signatures generate statistically significant models of cancer therapy outcome were found. The number of predictors in each signature, we removed from further analysis all probe sets with low independent predictive values were removed from further analysis to eliminate redundancy (typically, with the p > 0.1 in multivariate Cox regression analysis).
  • the weighted survival score analysis was implemented to reflect the incremental statistical power of the individual covariates as predictors of therapy outcome based on a multi- component prognostic model.
  • the microarray-based or Q-RT-PCR-derived gene expression values were normalized and log-transformed on a base 10 scale.
  • the log-transformed normalized expression values for each data set were analyzed in a multivariate Cox proportional hazards regression model, with overall survival or event-free survival as the dependent variable.
  • To calculate the survival/prognosis predictor score for each patient the log-transformed normalized gene expression value measured for each gene by a coefficient derived from the multivariate Cox proportional hazard regression analysis was multiplied.
  • Final survival predictor score comprises a sum of scores for individual genes and reflects the relative contribution of each of the eleven genes in the multivariate analysis.
  • the negative weighting values indicate that higher expression correlates with longer survival and favorable prognosis, whereas the positive score values indicate that higher expression correlates with poor outcome and shorter survival.
  • the weighted survival predictor model is based on a cumulative score of the weighted expression values of eleven genes.
  • CTOP score (-0.403xGbx2) + (1.2494xKI67) + (-0.3105xCyclin Bl) + (-0.1226xBUBl) + (0.0077xHEC) + (0.0369xKIAA1063) + (-1.7493xHCFCl) + (-1.1853xRNF2) + (1.5242xANK3) + (- 0.5628xFGFR2) + (-0.4333xCESl).
  • Images were collected on an inverted microscope (OlympusIX70) equipped with a DeltaVision imaging system using a x 40 objective. Images were processed by softWoRx v.2.5 software (Applied Precision Inc., Issaquah, WA) and images were quantified with using ImageJ 1.29x software.
  • TMAs human prostate cancer tissue microarrays
  • Analysis was carried- out on the prostate cancer TMAs from Chemicon (Temecula, CA; TMA # 3202-4; four cancer cases and two cases of normal tissue; and TMA # 1202-4; twenty five cases of cancer and five cases of normal tissue) and TMA of 10 cases of prostate cancer from the SKCC tumor bank (San Diego, CA).
  • TMAs contain two 2.0 mm cores of each case and haematoxylin-and-eosin (H&E) sections which were used for visual selection of the pathological tissues, histological diagnosis, and grading by the pathologists of TMA providers.
  • H&E haematoxylin-and-eosin
  • paraffin-embedded sections were baked at 56°C for 1 hour, allowed to cool for about 5 minutes, dewaxed in xylene, and rehydrated in a series of graded alcohols.
  • Antigen retrieval was achieved by boiling slides in 1OmM sodium citrate buffer, 0.05% Tween 20, pH 6.0 in a water bath for 30 minutes. The sections were washed with PBS, incubated in 100 mM glycine/PBS for 10 minutes, blocked in 0.5% BSA/0.05% gelatine cold water fish skin/PBS and incubated with primary antibody overnight.
  • the primary antibodies were rinsed off with PBS and slides were incubated with secondary antibodies at 1 :300 dilutions for 1 hour at room temperature.
  • Secondary antibodies (chicken antirabbit Alexa 594, goat antimouse Alexa 488, goat antimouse IgGl Alexa 350, and donkey antigoat Alexa 488 conjugates) were from Molecular Probes (Eugene, OR).
  • the slides were washed four times in PBS for five minutes each wash, rinsed in distilled water and the specimen were coversliped with Prolong Gold Antifade Reagent (Molecular Probes, Eugene, OR) containing DAPI.
  • Prolong Gold Antifade Reagent Molecular Probes, Eugene, OR
  • the comparison thresholds for each marker combination were defined at the 90-95% exclusion levels for dual positive cells in corresponding control samples (parental low metastatic cells).
  • the comparison thresholds for each marker combination were defined at the 99% or greater exclusion levels for dual positive cells in corresponding control samples (normal epithelial cells in TMA experiments). All individual immmunofluorescent assay experiments (defined as the experiments in which the corresponding comparisons were made) were carried out simultaneously using the same reagents and included all experimental samples and controls utilized for a quantitative analysis. Statistical significance of the measurements was ascertained and consistency of the findings was confirmed in multiple independent experiments, including several independent sources of the prostate cancer TMA samples.
  • Orthotopic xenografts of human prostate PC-3 cells and prostate cancer metastasis precursor sublines used in this study were developed by surgical orthotopic implantation as previously described in Glinsky et al (2003), supra. Briefly, 2 x 10 6 cultured PC-3 cells or sublines were injected subcutaneously into male athymic mice, and allowed to develop into firm palpable and visible tumors over the course of 2 - 4 weeks. Intact tissue was harvested from a single subcutaneous tumor and surgically implanted in the ventral lateral lobes of the prostate gland in a series often athymic mice per cell line subtype as described in Glinsky et al (2003), supra. During orthotopic cell inoculation experiments, a single-cell suspension of 1.5 x 10 6 cells was injected into mouse prostate gland in a series often athymic mice per therapy group.
  • PC3 human prostate adenocarcinoma cell line, derived subline PC3-32 and diploid human fibroblast BJl-hTERT cells were used for the assessment of gene amplification status.
  • the cyanine-3 or cyanine-5 labeled BAC clone RPl 1-28C14 was used for the EZH2 locus (7q35- q36)
  • the BAC clone RPl 1-232K21 was used for the BMIl locus (lOpl 1.23)
  • the BAC clone RP11-440N18 was used for the Myc locus (8q24.12-q24.13)
  • the BAC clone RPl 1-1112H21 was used for the LPL locus (8p22).
  • FISH analysis was done accordingly protocol as described previously.
  • Methanol/glacial acetic acid cell fixation Cell cultures were synchronized with 4 ug/ml aphidicolin (Sigma Chemical Co.) for 17 hour at 37 0 C. Synchronized cells were subjected to hypotonic treatment in 0.56% KCl for 20 minute at 37 0 C, followed by fixation in Carnoy's fixative (3:1 methanol : glacial acetic acid). Cell suspension was dropped onto glass slides, air dried. The slides are treated for 30 minutes with 0.005% pepsin in 0.0 IN HCl at room temperature and then are dehydrated through a series washes in 70%, 85%, and 100% ethanol.
  • Denaturation of DNA is performed by plunging the slide in a coplin jar containing 70% formamide/2x SSC (pH 7.0) for 30 min at 75°C. The slide immediately are plunged into ice-cold 2x SSC and then dehydrated as earlier.
  • Fluorescence in situ hybridization All BAC clones were obtained from the Rosewell Park Cancer Institute (RPCI, Buffalo, NY). The BAC DNA was labeled with Cy3- dCTP or Cy5-dCTP (Perkin Elmer Life Sciences, Inc.) using BioPrime DNA Labeling System (Invitrogen). The resultant probes are purified with QlAquick PCR Purification Kit (Qiagen). DNA recovery and the amount of incorporated Cy3 or Cy 5 are verified by Nanodrop spectrophotometry.
  • the probe Prior to hybridization the probe is precipitated with 20 ug competitor human Cot-1 DNA (per 18x18 mm coverslip) and washed in 70% ethanol. The dried pellet is thoroughly resuspended in 10 ul hybridization buffer (2x SSC, 20% dextran sulfate, 1 mg/ml BSA; NEB Inc.). The denaturated probe solution is deposited onto cells on slide. Hybridization was carried outovernight at 42°C in a dark humidified chamber.
  • siRNAs stable siRNAs
  • control luciferase siRNAs were purchased from Dharmacon Research, Inc. siRNAs were transfected into human prostate carcinoma cells according to the manufacturer's protocols. Cell cultures were continuously monitored for growth and viability and assayed for mRNA expression levels of BMIl, Ezh2, and selected set of genes using RT-PCR and Q-RT-PCR methods. Eight individual siRNA sequences comprising the SMART pools (four sequences for each gene, BMIl and Ezh2) were tested and a single most effective siRNA sequence was selected for synthesis in the chemically modified stable siRNA form for each gene.
  • the siRNA treatment protocol [two consecutive treatments of cells in adherent cultures with 100 nM (final concentration) of Dharmacon degradation-resistant siRNAs at day 1 and 4 after plating], as designed, caused only moderate reduction in the average BMI 1 and Ezh2 protein expression levels (20-50% maximal effect) and having no or only marginal effect on cell proliferation in the adherent cultures (at most ⁇ 25% reduction in cell proliferation).
  • the real time PCR methods measures the accumulation of PCR products by a fluorescence detector system and allows for quantification of the amount of amplified PCR products in the log phase of the reaction.
  • Total RNA was extracted using RNeasy mini-kit (Qiagen, Valencia, CA, USA) following the manufacturer's instructions. A measure of 1 ⁇ g (tumor samples), or 2 ⁇ g and 4 ⁇ g (independent preparations of reference cDNA and DNA samples from cell culture experiments) of total RNA was used then as a template for cDNA synthesis with Superscript II (Invitrogen, Carlsbad, CA, USA). cDNA synthesis step was omitted in the DNA copy number analysis (32).
  • Q-PCR primer sequences were selected for each cDNA and DNA with the aid of Primer ExpressTM software (Applied Biosystems, Foster City, CA, USA). PCR amplification was performed with the gene-specific primers. Q-PCR reactions and measurements were performed with the SYBR-Green and ROX as a passive reference, using the ABI 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Conditions for the PCR were as follows: one cycle of 10 min at 95°C; 40 cycles of 0.20 min at 94° C; 0.20 min at 6O 0 C and 0.30 min at 72°C. The results were normalized to the relative amount of expression of an endogenous control gene GAPDH.
  • mRNA messenger RNA
  • GPDH endogenous control gene
  • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH: 5'- CCCTCAACGACCACTTTGTCA-3' and 5'- TTCCTCTTGTGCTCTTGCTGG- 3') was used as the endogenous RNA and cDNA quantity normalization control.
  • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH: 5'- CCCTCAACGACCACTTTGTCA-3' and 5'- TTCCTCTTGTGCTCTTGCTGG- 3') was used as the endogenous RNA and cDNA quantity normalization control.
  • Glyceraldehyde-3-phosphate dehydrogenase (GAPDH: 5'- CCCTCAACGACCACTTTGTCA-3' and 5'- TTCCTCTTGTGCTCTTGCTGG- 3') was used as the endogenous RNA and cDNA quantity normalization control.
  • cDNA prepared from primary in vitro cultures of normal human prostate epithelial cells cDNA
  • the Kaplan-Meier survival analysis was carried out using the GraphPad Prism version 4.00 so ⁇ tware (GraphPad Software, San Diego, CA).
  • the end point for survival analysis in prostate cancer was the biochemical recurrence defined by the serum PSA increase after therapy.
  • Disease-free interval was defined as the time period between the date of radical prostatectomy (RP) and the date of PSA relapse (recurrence group) or date of last follow-up (non- recurrence group).
  • DFI Disease-free interval
  • RP radical prostatectomy
  • recurrence group date of PSA relapse
  • non- recurrence group date of last follow-up

Abstract

La présente invention concerne de nouveaux procédés et nécessaires pour diagnostiquer la présence de cancer chez u patient, et pour déterminer si un sujet cancéreux est capable de réagir à différents types de schémas thérapeutiques. Les cancers à tester sont notamment ceux de la prostate, des seins, des poumons, de l'estomac, des ovaires, de la vessie, les lymphomes, les mésothéliomes, les médulloblastomes, les gliomes et la leucémie myéloïde aigüe (AML). L'identification de patients résistant à la thérapie au début de leur schéma thérapeutique peut conduire à une modification de la thérapie de façon à obtenir une issue plus favorable. Un mode de réalisation de l'invention concerne un procédé de diagnostic de cancer ou de prédiction d'issue de thérapie anticancéreuse par détection des niveaux d'expression de plusieurs marqueurs dans la même cellule au même moment, et par notation de leur expression comme se situant au-dessus d'un certain seuil, les marqueurs provenant d'une voie particulière se rapportant au cancer, la note étant une indication ou un diagnostic de cancer, ou un pronostic d'échec de thérapie anticancéreuse. Ce procédé convient pour le diagnostic du cancer ou la prédiction des issues des traitements anticancéreux pour divers cancers. Les marqueurs peuvent provenir de n'importe quelle voie impliquée dans la régulation du cancer, et notamment spécifiquement de la voie PcG et de la voie de 'souchitude' au sens de 'caractéristique des cellules souches'. Les marqueurs peuvent être de l'ARNm, de l'ARNmicro, de l'ADN, ou de la protéine.
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