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WO2012166700A2 - Profilage moléculaire d'un microenvironnement de tumeur létale - Google Patents

Profilage moléculaire d'un microenvironnement de tumeur létale Download PDF

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WO2012166700A2
WO2012166700A2 PCT/US2012/039814 US2012039814W WO2012166700A2 WO 2012166700 A2 WO2012166700 A2 WO 2012166700A2 US 2012039814 W US2012039814 W US 2012039814W WO 2012166700 A2 WO2012166700 A2 WO 2012166700A2
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tumor
cancer
stromal
gene
cav
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WO2012166700A3 (fr
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Michael P. Lisanti
Federica Sotgia
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    • 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
    • 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
    • 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
    • C12Q2600/118—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
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the biological markers that are useful in the course of detection and/or treatment of cancer. BACKGROUND OF THE FNVENTION
  • Cancer is one of the most significant diseases confronting centuries, and even though progress has been made in cancer treatment, particularly in the medical therapy of cancer, many challenges remain.
  • the various anticancer agents for suppressing the growth of cancer cells that have been developed suppress the growth of not only cancer cells, but also normal cells, causing various side effects including nausea and vomiting, hair loss, myelosuppression, kidney damage, and nerve damage. Consequently, understanding the origins of these malignancies and their microenvironments will facilitate development of more precise diagnostic tools and more effective therapeutic strategies that minimize maximize therapeutic benefits and minimize toxicity for the patients undergoing treatment for cancer.
  • a bioactive substance such as PDGF (platelet-derived growth factor), TGF- ⁇ (transforming growth factor- ⁇ ), HGF (hepatocyte growth factor), or SDF- 1 (stromal cell-derived factor- 1) produced in the interstitium is involved in such growth of a tumor (Micke et al, Expert Opin Ther Targets. 2005, 9(6), 1217-1233).
  • PDGF platelet-derived growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • HGF hepatocyte growth factor
  • SDF- 1 stromal cell-derived factor- 1
  • stromal caveolin-1 can be a useful biomarker of a "lethal" tumor microenvironment.
  • a loss of stromal Cav-1 was associated with a five-year survival rate of less than 10%.
  • triple negative patients from the same cohort, but having high stromal Cav-1 levels had a twelve-year survival rate of greater than 75%.
  • DCIS ductal carcinoma in situ
  • Witkiewicz A.K. "Towards a new "stromal-based” classification system for human breast cancer prognosis and therapy” Cell Cycle 2009, 8, 1654-8; Witkiewicz A.K, "Stromal caveolin-1 levels predict early DCIS progression to invasive breast cancer” Cancer Biol. Ther.
  • the present disclosure provides a prognostic signature (also referred to herein as a biological marker or biomarker) for assessing cancer prognosis in a subject, comprising a set of one or more genes (or gene products) derived from a genome -wide transcriptional profiling of a cancer tumor stroma.
  • the prognostic signature is a Cav-1 -deficient tumor stromal signature and comprises a set of at least one or more gene transcripts selected from the gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combinations thereof.
  • the Cav-1 -deficient tumor stromal signature comprises a first set of one or more gene transcripts that are up-regulated in the tumor stromal material and/or a second set of one or more gene transcripts that is down- regulated in the tumor stromal material.
  • said tumor stromal signature comprises a set of one or more gene transcripts that is up-regulated in the tumor stromal material, wherein said set of at least one or more up-regulated gene transcripts is selected from the up- regulated gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combinations thereof.
  • said tumor stromal signature comprises a set of one or more gene transcripts that is down-regulated in the tumor stromal material, wherein said set of at least one or more down-regulated gene transcripts is selected from down-regulated gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combinations thereof.
  • said tumor stromal signature comprises a first set of one or more gene transcripts that is up-regulated in the tumor stromal material, wherein said first set of at least one or more up-regulated gene transcripts is selected from the up-regulated gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combinations thereof; and a second set of one or more gene transcripts that is down-regulated in the tumor stromal material, wherein said second set of at least one or more down-regulated gene transcripts is selected from down-regulated gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combinations thereof.
  • the Cav-1 -deficient tumor stromal signature is derived from a breast cancer tumor stroma.
  • the Cav-1 -deficient tumor stromal signature is derived from a breast cancer stroma, wherein the breast cancer is of a subtype selected from the group consisting of ER positive (or ER(+) ), ER negative (or ER(-), and HER2 positive (or HER2(+)).
  • the present disclosure provides a method of assessing cancer prognosis, wherein the method comprises: (a) determining a genome-wide transcriptional profile of a first stromal material, wherein the first stromal material is derived from a biological sample obtained from a subject afflicted with cancer or suspected of having cancer; (b) comparing the genome-wide transcriptional profile (or a set of gene transcripts subgenome -transcriptional profile derived therefrom) to a Cav-1 -deficient tumor stromal signature, wherein the Cav-1 - deficient tumor stromal signature comprises a set of at least one or more gene transcripts selected from the gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combintinations thereof, whereby an overlap of from about 25% to about 75% between the genome -wide transcriptional profile (or a subgenome-transcriptional profile derived therefrom) and the Cav-1 -deficient tumor stromal signature indicates a poor prognosis (e.g.
  • the method of assessing cancer prognosis comprises: (a) determining a genome-wide transcriptional profile of a biological sample, wherein the biological sample is obtained from a subject afflicted with cancer or suspected of having cancer; (b) comparing the genome-wide transcriptional profile (or a set one or more gene transcripts comprising gene transcripts up-regulated and/or gene transcripts down-regulated or both gene transcriipts up-regulated and gene transcripts down-regulated in the biological sample) to a Cav- 1 -deficient tumor stromal signature, wherein the Cav-1 -deficient tumor stromal signature comprises at least a set of one or more gene transcripts selected from the gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combintinations thereof, whereby an overlap of from about 25% to about 75% between the genome -wide transcriptional profile and the Cav-1 -deficient tumor stromal signature indicates a poor prognosis (e.g., risk of recurrence the
  • the present disclosure provides a method of monitoring progression of cancer, including breast cancer, in a subject, the method comprising: (a) obtaining a first sample from a subject at a first time point and a second sample from said subject at a second time point; (b) determining a genome-wide transcriptional profile of said first and second samples; (c) comparing the genome-wide transcriptional profile of said first sample to the genome-wide transcriptional profile of said second sample; and (d) thereafter, comparing any difference in transcriptional profile between the genome -wide transcriptional profile of said first sample and the genome-wide transcriptional profile of said second sample to a Cav-1 -deficient tumor stromal signature, wherein the Cav-1 -deficient tumor stromal signature comprises a set of at least one or more gene transcripts having gene names and/or gene symbols listed in
  • Supplemental Tables 1, 3, or combintinations thereof whereby an overlap of from about 25% to about 75%o is indicative of a poor clinical outcome (e.g., recurrence of the cancer in said subject, or decreased overall survival of the cancer by the subject, or is an indication that the cancer has progressed in said subject, or is an indication that the cancer has metastasized in said subject).
  • a poor clinical outcome e.g., recurrence of the cancer in said subject, or decreased overall survival of the cancer by the subject, or is an indication that the cancer has progressed in said subject, or is an indication that the cancer has metastasized in said subject.
  • the present disclosure provides a method of stratifying a subject or a group of subjects having a cancer tumor for a clinical trial.
  • the method comprises: (a) roviding a stromal material, wherein first stromal material is derived from a biological sample obtained from a subject having a cancer tumor; (b) determining a genome-wide transcriptional profile of the stromal material; (c) comparing the genome-wide transcriptional profile of said stromal material to a Cav-1 -deficient tumor stromal signature, wherein the Cav-1 - deficient tumor stromal signature comprises at least a set one or more gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combintinations thereof; and (c) stratifying the subject for a clinical trial based on the results of the comparison step (c).
  • an overlap of from about 25% to about 75% is indicative of stratification class of the subjects.
  • the method of stratifying a subject or a group of subjects having a cancer tumor for a clinical trial comprises: (a) roviding a biological sample, wherein biological sample is obtained from a subject having a cancer tumor; (b) determining a genome-wide transcriptional profile of the biological sample; (c) comparing the genome -wide transcriptional profile of said said biological sample to a Cav-1 -deficient tumor stromal signature, wherein the Cav-1 -deficient tumor stromal signature comprises at least one or more gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combintinations thereof; and (c) stratifying the subject for a clinical trial based on the results of the comparison step (c).
  • overlap of from about 25% to about 75% is indicative of the stratification class of the subjects.
  • Figure 2 illustrates heatmaps of gene transcripts associated with myofibroblast differentiation, autophagy, lysosomal degradation, and glycolysis;
  • Figure 3 illustrates heatmaps of gene transcripts associated with the response to hypoxia and mitochondria
  • Figure 4 illustrates heatmaps of gene transcripts associated with inflammation and redox/stress signaling
  • Figure 5 illustrates heatmaps of gene transcripts associated with dna damage and repair
  • Figure 6 illustrates heatmaps of gene transcripts associated with aging, apoptosis, and brca 1 -mutation positive breast cancer patients
  • Figure 7 illustrates heatmaps of gene transcripts associated with ER-negative breast cancers
  • Figure 8 illustrates heatmaps of gene transcripts associated with neural stem cells
  • Figure 9 illustrates Venn diagrams for the intersection of the cav-1 -deficient stromal gene signature with other breast cancer tumor stromal gene sets
  • Figure 10 illustrates the Cav-1 -deficient stromal gene signature is up-regulated in breast cancer and is associated with tumor recurrence
  • Figure 11 illustrates the Cav-1 -deficient stromal gene signature is associated with tumor recurrence and poor survival in ER(+) and luminal A breast cancer patients.
  • Figure 12 illustrates understanding the hierarchy of the cellular processes associated with a Cav-1 -deficient tumor microenvironment.
  • Supplemental Table 7 Intersection with Breast Tumors Derived from BRCA 1 - positive Patients
  • Supplemental Table 7 Intersection with Breast Tumors Derived from BRCA1 - positive Patients
  • Supplemental Table 8 Intersection with Breast Tumors Derived from ER-negative Patients
  • Supplemental Table 10 Intersection with Glycolysis, Hypoxia, and Oxidative Stress
  • Supplemental Table 11 Intersection with Genes Upregulated During DNA Damage and Repair
  • Supplemental Table 14 Intersection with Aging Associated Genes
  • Supplemental Table 15 contains the 3,459 unique genes that were up-regulated in the stroma of tumors showing a loss of stromal Cav-1 expression
  • Supplemental Table 16 contains the 1 ,297 unique genes that were up-regulated in the stroma of tumors showing a loss of stromal Cav-1 expression.
  • Transcriptional profiling includes analysis of the relative abundance of messenger RNA transcribed from different genes. Transcriptional profiling can be used to facilitate the understanding of patterns of gene expression that are associated with all biological processes, including development, differentiation, response to environmental stresses, and other cellular and organismal functions of interest. The ability to analyze patterns of gene expression can lead to discovery of new genes associated with biological processes. A detailed understanding of gene regulation at the transcriptional level is also a premier concern of the pharmaceutical industry, enabling identification of genetic targets for drug development and leading to the understanding of the well known heterogenity in the way different individuals respond to pharmaceutical interventions. Transcriptional profiling may be conducted by the techniques of "differential display" (see, for example, Liang, P. and Pardee, A. B.
  • the present disclosure provides new transcriptional gene signatures that can effectively be used for prognostic and therapeutic stratification of cancer tumors:
  • said transcriptional gene signatures comprise at least a set of one or more gene transcripts, wherein the set of one or more gene transcripts is selected from the group consisting of gene transcripts having gene names and/or gene symbols listed in Table 1, or any one of Supplementary Tables 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or combintinations thereof.
  • transcriptional gene signatures are used to unambiguously stratify patients based on their stromal Cav-1 status, into Cav-1 (+) and Cav-1 (-) sub-groups, thus, greatly facilitating prediction of their prognosis.
  • the transcriptional gene signatures of the present invention are used in therapeutic stratification of cancer patients. The present inventors have used transcriptional profiling of cancer patients to facilitate the
  • mechanistic insights into how the status of Cav-1 in the breast cancer tumor stroma modulates cancer tumor progression, recurrence of cancer tumor and/or metastasis of cancer is provided.
  • the present inventors isolated tumor stroma from breast cancer patients with a loss of stromal Cav-1 and patients with high stromal Cav-1, for direct comparison and demonstrated that the Cav-1 deficient tumor stroma from human breast cancer patients shows transcriptional evidence for the same biological processes that have been implicated in the "Autophagic Tumor Stroma Model of Cancer". These biological processes include oxidative stress/aging, hypoxia, a compensatory increase in mitochondria-associated genes, NFkB -activation, inflammation, DNA damage, aerobic glycolysis, autophagy/apoptosis, lysosomal degradation, and "sternness" (summarized in Figure 12).
  • the present inventors also observed strong association between Cav-1 deficient tumor stroma and ER(-) breast cancer.
  • gene profiles from Cav-1 deficient tumor stroma were elevated in all breast cancers, relative to normal healthy breast tissue.
  • ER(-) negative breast cancers that were identified either by ER IHC or via ESR1 transcriptional analysis (summarized in Figure 10).
  • GSEA gene set enrichment analysis
  • one aspect of the present invention relates to use of the stromal Cav-1 deficient gene signature disclosed herein to predict clinical outcome in cancer patients, including breast cancer patients.
  • the stromal Cav-1 deficient gene signature in accordance with the present invention is used to predict recurrence of cancer, or progression of cancer and/or decreased overall survival in cancer patients, including ER(+) and luminal A breast cancer patients.
  • the present inventors have shown that the Cav-1 - deficient stromal signature of the present invention is strongly associated with increased recurrence and decreased overall survival in ER(+) and luminal A breast cancer patients, despite the fact that these breast cancer-derived tumors were not subjected to laser capture
  • tumor stromal signatures of the present invention can be used either alone or in conjunction with other diagnostic tests, including immuno-histochemical (IHC) staining.
  • IHC immuno-histochemical
  • Tumors with a "fibrotic focus” or "central scar” are reportedly associated with a poor prognosis 36 ' 37 .
  • keloid fibroblasts derived from non-tumorous "scarred skin” show many of the same characteristics as Cav-1 deficient tumor stroma, with activation of HIF1 -alpha and NFkB, as well as a shift towards aerobic
  • GSEA gene set enrichment analysis
  • one aspect of the present invention provides a method of therapeutic stratification of cancer patients, said method comprising: (a) providing a stromal material, wherein first stromal material is derived from a biological sample obtained from patients having a cancer tumor; (b) determining a genome -wide transcriptional profile of the stromal material, wherein the transcriptional profile compries at least a set of one or more gene transcripts up- regulated and/or at least a set of one or more gene transcripts down-regulated in the stromal material; (c) comparing the genome-wide transcriptional profile (e.g., the set of at least one or more gene transcripts up-regulated and/or at least one or more gene transcripts down-regulated in the stromal material) of said stromal material with at least one or more Cav-1 -deficient tumor stromal signatures, wherein the the at least one or more Cav-1 -deficient tumor stromal signatures comprise at least a set of one or more gene transcripts selected from the group consisting of gene
  • the Cav-1 -deficient tumor stromal signature comprises gene transcripts up-regulated in the Cav-1 deficient tumor stroma, wherein the gene transcripts up-regulated in the Cav-1 deficient stroma comprise a set of at least one or more up- regulated gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combintinations thereof.
  • the Cav-1 -deficient tumor stromal signature comprises gene transcripts down-regulated in the Cav-1 deficient tumor stroma, wherein the gene transcripts down-regulated in the Cav-1 deficient stroma comprise a set of at least one or more down-regulated gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combintinations thereof. [0053] .
  • the Cav-1 -deficient tumor stromal signatures comprise gene transcripts up-regulated in the Cav-1 deficient tumor stroma and gene trascripts down- regulated in the Cav-1 deficient tumor stroma, wherein the gene transcripts up-regulated in the Cav-1 deficient stroma and the gene transcripts down-regulated in the Cav-1 deficient stroma comprise a set of at least one or more up-regulated gene transcripts and down-regulated gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combintinations thereof.
  • the method of therapeutic stratification of cancer patients comprises: (a) providing a biological sample obtained from patients having a cancer tumor; (b) determining a genome-wide transcriptional profile of the biological sample, wherein the transcriptional profile compries at least a set of one or more gene transcripts up-regulated and/or at least a set of one or more gene transcripts down-regulated in the biological sample; (c) comparing the genome-wide transcriptional profile (e.g., the set of at least one or more gene transcripts up-regulated and/or at least one or more gene transcripts down-regulated in the biological sample ) of said biological sample with Cav-1 -deficient tumor stromal signature, wherein the Cav-1 -deficient tumor stromal signature comprises at least a set of one or more sets of gene transcripts, wherein the at least one or more sets of gene transcripts comprise gene transcripts selected from the group consisting of gene transcripts having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combintinations thereof; and (d) strat
  • the comparison of transcriptional profiles reveals stromal reactive oxygen species (ROS) production, as the driver of stromal DNA damage and tumor progression and metastasis.
  • the comprison results are used to identify cancer patients more likely to benefit from treatment with certain thearpeutic agents, including DNA damaging agents and/or Poly(ADP-ribose) polymerase (PARP) inhibitors.
  • the comprision of the transcriptional profiles can be used to identify cancer patients more likely to benefit from treatment with Monocarboxylate Transporter (MCTl) inhibitors and/or a combination of PARP inhibitors and MCTl inhibitors.
  • MCTl Monocarboxylate Transporter
  • PARP inhibitors and MCTl inhibitors Monocarboxylate Transporter
  • PARP inhibitors have been proposed as potential therapeutic agents for treatment of various cancers as well as neuroprotective agents.
  • PARP inhibitors are known in the art and may also be elucidated using various techniques.
  • Non-limiting examples of PARP inhibitors include such as Iniparib (BSI-201), olaparib (AZD-2281), and veliparib (ABT-888), 3- aminobenzamide, 4-aminobenzamide, 3-aminophtalhydrazide, 1,5-dihydroxyisoquinoline.
  • Preferred PARP inhibitors include benzamide, 3-aminophtalhydrazide, 1,5- dihydroxyisoquinoline, 6(5H)-Phenanthridinone, 4-Amino-l,8-naphthalimide, 1,5- Isoquinolinediol, 3-Aminobenzamide, and N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-N,N- dimethylacetamide hydrochloride (PJ34).
  • Inhibition of PARP may be effected by a number of approaches.
  • a preferred method of the present invention is to employ specific inhibitors of PARP.
  • benzamide, 3-aminobenzamide, 4- aminobenzamide, 3-aminophtalhydrazide and 1,5-dihydroxyisoquinoline may be used to inhibit apoptosis induced by PARP activity.
  • U.S. Pat. No. 5,756, 510 (Griffin et al.) discloses benzamide analogs having PARP inhibitory efficacy and those compounds may be used in the present invention methods and compositions; the entire contents of which are incorporated herein by reference.
  • U.S. Pat. No. Re. 36,397 discloses PARP inhibitors useful for vascular stroke and neurodegenerative disorders and those compounds may be used in the present invention methods and compositions.
  • the PARP inhibitors for use in conjunction with the biological markers and methods of the present invention may also be determined by various assays described in the literature.
  • the following publications teach various methods which may be employed to elucidate PARP inhibitors: (1) U.S. Pat. No. 5,756,510 (Griffin et al); (2) Banasik et al. Specific inhibitors of poly( ADP-ribose) synthetase and mono( ADP-ribose) transferase., J Biol Chem, volume 267, pages 1569-1575 (1992); and (3) Schanraufstatter et al. Oxidant injury of cells.
  • Suitable therapeutic agents for use with the transcriptional gene signatures and methods of the present invention include metformin and Monocarboxylate Transporter (MCT1) inhibitors, including AR-C117977 and AR-C155858 and pharmaceutically acceptable salts thereof.
  • MCT1 Monocarboxylate Transporter
  • AR-C155858 has the following structural formula.
  • MCT1 inhibitors examples include WO 2010/089580 (published 12 August 2010).
  • the comparison of a set of two or more transcriptional profiles derived from biological samples obtained from the same or different biological sources (e.g., individuals), as described herein, includes identification of relative changes in the levels of gene transcripts in the respective samples.
  • the comparison at least a set of two or more trascriptional profiles are used to identify a subject or a group of subjects with a significant probability of a poor clincial outcome.
  • comparisons of transcriptional gene profiles are used to identify cancer patients that are more like to benefit from one or more thereaputic agents.
  • the comparison of the set of two or more trascriptional profiles is used to identify a subject or a group of subjects with a significant probability of benefiting from treatment with existing or new thereapeutic agents, including PARP inhibitors and/or MCTI inhibitors.
  • the probability that a patient or a group of patients are expected to benefit from treatment with PARP inhibitors alone or MCTI inhibitors alone or in combinations with PARP inhibitors is from about 25% to about 30%, from about 25% to about 35%, from about 25% to about 45%, from about 25% to about 50%, from about 25% to about 55%, from about 25% to about 60%, from about 25%, to about 65%, from about 25% to about 70%, from about 25%, to about 75%, from about 30% to about 35%, from about 30% to about 45%, from about 30% to about 50%, from about 30% to about 55%, from about 30% to about 60%, from about 30%, to about 65%, from about 30% to about 70%, from about 30%, to about 75%, from about 35% to about 45%, from about 35% to about 50%, from about 35% to about 55%, from about 35% to about 60%, from about 35%, to about 65%, from about 35% to about 70%, from about 35%, to about 75%, from about 40% to about 45%, from about 40% to about 45%, from about 40%
  • comparision of trascriptional profiles in accordance with the present invention identifies a cancer patents or a group of cancer patients having a probability of from about 25% to about75% of a poor clinical outcome, wherein the poor clinical outcome is selcted from the group consisting of cancer recurrence, progression of a cancer, overall survival of cancer, cancer metastasis and combinations thereof.
  • the probability of a poor clinical outcome is from about 25% to about 30%, from about 25% to about 35%, from about 25%o to about 45%, from about 25% to about 50%, from about 25% to about 55%, from about 25%o to about 60%, from about 25%, to about 65%, from about 25% to about 70%, from about 25%o, to about 75%, from about 30% to about 35%, from about 30% to about 45%, from about 30%) to about 50%, from about 30% to about 55%, from about 30% to about 60%, from about 30%), to about 65%, from about 30% to about 70%, from about 30%, to about 75%, from about 35%) to about 45%, from about 35% to about 50%, from about 35% to about 55%, from about 35% to about 60%, from about 35%, to about 65%, from about 35% to about 70%, from about 35%), to about 75%, from about 40% to about 45%, from about 40% to about 50%, from about 40%) to about 55%, from about 40% to about 60%, from about 40%, to about 65%, from about 40%
  • markers of autophagy or oxidative stress in the tumor stroma are associated with increased lympho-vascular invasion, increased lymph-node metastasis, or poor clinical outcome, in a variety of different epithelial cancer subtypes.
  • markers include ATG16L1 and the cathepsins (K and D) for autophagy, as well as carbonic anhydrase IX (CAIX) and HIF1 -alpha for oxidative stress 49-56 .
  • the present inventors have shown that simple immuno-staining with antibodies directed against Cav-1 can be used effectively to detect two different populations of breast cancer patients (high-risk vs. low-risk), based on their stromal Cav-1 status. Genome-wide transcriptional profiling of these two different stromal populations shows that they are dramatically different, each with a unique transcriptional profile.
  • the present inventors focused on the stromal Cav-1 -deficient population, because they are associated with early tumor recurrence, and metastasis.
  • GSEA Gene set enrichment analysis
  • the transcriptional gene signatures of the present invention comprise a set of at least one or more gene transcripts up-regulated in cancer tumor stroma. In another embodiment the transcriptional gene signatures of the present invention comprise a set of at least one or more gene transcripts down-regulated in cancer tumor stroma.
  • the cancer tumor stroma is derived from derived from a Cav-1 -deficient cancer tumor stromal material. In another embodiment, the cancer tumor stroma is derived from derived from a Cav-1 -positive cancer tumor stromal material.
  • any type of cell in the body may be a source of cancer, any suitable types of cancer cells or cellular materials may be used in the present invention.
  • a suitable cancer type includes carcinoma (cancer of the epithelial cells), sarcoma (cancer of the bone, muscle or other connective tissues), lymphoma (cancer of the lymphatic system), leukemia (cancer of blood cells or blood precursor cells) and melanoma (cancer of the pigment-providing cells).
  • leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias, such as, myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia leukemias and myelodysplasia syndrome
  • chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera
  • lymphomas such as but not limited to Hodgkin's disease, non- Hodgkin's disease
  • multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma
  • chondrosarcoma Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lympho
  • choriocarcinoma (yolk-sac tumor), prostate cancers such as but not limited to, prostatic intraepithelial neoplasia, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers such as but not limited to renal cell carcinoma, a
  • cancers include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B.
  • the transcriptional gene signatures in accordance with the present invention are drived from a genome-wide transcriptional profiling of a tumor stromal material which is derived from a Cav-1 -deficient breast cancer tumor stroma, wherein the tumor stromal signatures comprise (a) a first set of up-regulated gene transcripts, wherein the first set of up- regulated gene transcripts comprises at least a set of one or more up-regulated gene transcritps having gene symbols listed Supplemental Tables 1, 3, or combintinations thereof; and (b) a second set of down-regulated gene transcripts, wherein the second set of down-regulated gene transcripts comprises at least a set of one or more gene transcripts having gene names and/or gene symbols listed Supplemental Tables 1, 3, or combintinations thereof.
  • the tumor stromal signatures comprise (a) a first set of up-regulated gene transcripts, wherein the first set of up- regulated gene transcripts comprises at least a set of one or more up-regulated gene transcritps having gene symbols listed Supplemental Tables
  • LOC80054 SLC8A1, VMOl, IL28RA, LOC100288109, APITD1, TRIP6, TMEM165,
  • down-regulated gene transcritps having gene symbols listed Supplemental Tables 1, 3, or combintinations thereof are selected from the group consisting of HNRNPA0, ST3GAL3, LOC100288446, RGNEF, NTRK3, KIFAP3, FAM3A, FAM169A,
  • LOC80154 NLK, SNORD20, THRA, XGPY2, SIGLEC6, DHCR24, LOC100129198, PTPN14, MAP7, LOCI 00128292, LOC646976, ZNF20, TMEM9B, IL17RB, TUBD1, TRIM66,
  • the tumor stromal signatures comprise (a) a first set of up- regulated gene transcripts, wherein the first set of up-regulated gene transcripts comprises at least a set of one or more up-regulated gene transcritps having gene names and/or gene symbols listed in Supplemental Tables 1, 3, or combintinations thereof, wherein the up-regulated gene transcripts are selected from the group consisting of LOCI 00293390, RNF217, PGBD2, LACTB, TRITl, RNF217, LOC654433, DFFA, DNAJC5, ZNF365, ZNF365, MDN1, ERG, CTSB, SGSM3, MYL5, HLA-Z, IKZF1, C15orf23, CD84, C21orf7, VPS26B, SUFU, MSH6, S1PR3, FCGR2B, BCL2L11, RFC5, RAB3D, LOC100128943, PSMB9, GALNT11, APOC2, ISM2,
  • LOC100131109 PLD1, , SIK3, FAM55B, PTGR1, UBE3B, PTPRS, DLG5, ZHX3, SOX5,
  • LOC388152 KCNK1, ITIH5, RARG, ABHD3, IRAK1BP1, TRIM38, SLC19A3, XP07, PDZD4, ESR1, LOC285147, ZNF193, MPP7, PKD1L2, PDZD4, , KCNQIOTI, RAD1, KCMF1, LOC100293798, IGF1R, ZFP3, C21orf62, TLR6, CTBP2, , DDX46, FAM156B, RUNDC3B, TMEM138, LOC100131938, UNC13C, ZNF641, OBSCN, C18orfl5, UBN2, MPHOSPH9, CRIM1, , SLC25A11, VWC2, DEAF1, DNAJC1, ADORA2B, PARK7,
  • LOC80154 NLK, SNORD20, THRA, XGPY2, SIGLEC6, DHCR24, LOC100129198, PTPNl 4, MAP7, LOCI 00128292, LOC646976, ZNF20, TMEM9B, IL17RB, TUBD1, TRIM66,
  • MGC16121 SLC16A6, OAF, SETD4, C8orf84, ZFP90, C7orf63, GAB1, TRIM66, PCSK1N, SNORD121A, GFRA1, GAB1, MS4A2, NHEDC2, HPSE2, PGR, PGR, SLC18A2, AUTS2, AUTS2, KBTBD4, ERBB4, ADCYAP1, ADCYAP1, and combinations thereof.
  • the transcriptional gene signature in accordance with the present invention is drived from a genome-wide transcriptional profiling of a tumor stromal material which is derived from a Cav-1 -deficient breast cancer tumor stroma, and wherein the tumor stromal signature comprises at least a set of one or more gene transcripts up-regulated in the tumore stromal material, wherein the at least set of one or more gene transcritps comprise gene transcripts having gene names and/or gene symbols selected from the group consisting of: LOC100293390, RNF217, PGBD2, LACTB, TRIT1, RNF217, LOC654433, DFFA, DNAJC5, ZNF365, ZNF365, MDN1, ERG, CTSB, SGSM3, MYL5, HLA-Z, IKZF1, C15orf23, CD84, C21orf7, VPS26B, SUFU, MSH6, S1PR3, FCGR2B, BCL2L11, RFC5, RAB3
  • LOC100288109 APITD1, TRIP6, TMEM165, TBXASl, KLHL12, LY86, KLF3, WHSC1, ZBTB46, SNRK, MLL2, KCNJ13, THGIL, CYP4V2, SLC23A2, OGFRLl, SLC02B1, PTPNl, ALOX5AP, MORC2, ABCD1P4, UCK2, TMED5, FCGR2C, FAM185A, PHF20L1, EPB41L3, LARP4B, DHRSX, PDPKl, APEX2, CTSB, MRPL9, LOCI 00293165, Clorfl04, STX7, GFIl, AP4E1, ARHGAP5, C14orfl06, SUDS3, BAT2L1, BCAS3, LMF2, INTS4, FCGR2A,
  • the transcriptional gene signature in accordance with the present invention is drived from a genome-wide transcriptional profiling of a tumor stromal material which is derived from a Cav-1 -deficient breast cancer tumor stroma, and wherein the tumor stromal signature comprises at least a set of one or more gene transcripts down-regulated in the tumore stromal material, wherein the at least set of one or more gene transcritps comprise gene transcripts having gene names and/or gene symbols selected from the group consisting of: at least a set of one or more genes having gene symbols selected from the group consisting of: HNRNPA0, ST3GAL3, LOC100288446, RGNEF, NTRK3, KIFAP3, FAM3A, FAM169A,
  • LOC80154 NLK, SNORD20, THRA, XGPY2, SIGLEC6, DHCR24, LOC100129198, PTPN14, MAP7, LOCI 00128292, LOC646976, ZNF20, TMEM9B, IL17RB, TUBD1, TRIM66,
  • RNA extracted from these samples was subjected to genome-wide transcriptional profiling.
  • the present inventors identified 238 gene transcripts that were specifically up-regulated and 232 gene transcripts that were down-regulated in the stroma of tumors showing a loss of Cav-1 expression (p ⁇ 0.01 and fold-change (f.c.) > 1.5) (summarized in Supplemental Table 1). Using these stringent criteria, the present inventors were able to transcriptionally separate these two patient populations, in accordance with their IHC stromal Cav-1 status (negative versus positive). A HeatMap of the total patient cohort is shown in
  • GSEA Gene Set Enrichment Analysis
  • GSEA gene set enrichment analysis
  • Results of the GSEA are summarized in Table 1.
  • the GSEA results show up- regulation of cellular processes normally associated with "sternness", inflammation, DNA damage, oxidative stress, hypoxia, apoptotic signaling, autophagy, and mitochondrial dysfunction in the tumor stroma of patients lacking stromal Cav-1.
  • Neurogenesis was associated with increased DNA damage in the tumor stroma ' .
  • GOLPH3 also known as MIDAS/GPP34
  • mtDNA mitochondrial DNA
  • MIDAS stands for mitochondrial DNA absence sensitive factor u .
  • PRKDC is a component of the machinery required for the proper repair and maintenance of the fidelity of mitochondrial
  • Cav-1 status 15 More specifically, the gene transcripts up-regulated in Cav-1 -deficient tumor stroma (1,819 transcripts encoding 1,297 unique genes; p ⁇ 0.05 and fold-change (f.c.) > 1.15) (Supplemental Table 3) were compared with (1) tumor stroma and (2) recurrence stroma gene lists, as defined below:
  • Tumor Stroma vs. Normal Stroma List compares the transcriptional profiles of tumor stroma obtained from 53 patients to normal stroma obtained from 38 patients. Genes transcripts that were consistently upregulated in the tumor stroma were selected and assigned a p-value, with a cut-off of p ⁇ 0.05 (this set contains 6,777 unique genes) 16 .
  • Recurrence Stroma List compares the transcriptional profiles of tumor stroma obtained from 11 patients with tumor recurrence to the tumor stroma of 42 patients without tumor recurrence. Genes transcripts that were consistently upregulated in the tumor stroma of patients with recurrence were selected and assigned a p-value, with a cut-off of p ⁇ 0.05 (this set contains 3,354 unique genes) 16 .
  • recurrence stroma a 214 transcript overlap; p ⁇ 0.001
  • the gene transcripts up-regulated in Cav-1 -deficient tumor stroma show a significant association with tumor recurrence, and are in accordance with the results of Morag Park and colleagues 15 .
  • Figure 10A and B show that the Cav-1 -deficient tumor stromal signature is up- regulated in breast cancer(s), both ER(+) and ER(-) sub-types. However, a more significant association was observed with ER(-) breast cancers. In addition, the Cav-1 -deficient stromal signature was also associated with increased recurrence in breast cancer patients (Figure IOC).
  • FIG. 11 shows that the Cav-1 -deficient stromal signature is clearly associated with increased recurrence and decreased overall survival, despite the fact that these breast cancer-derived tumors were not subjected to laser capture microdissection.
  • cancer cells induce oxidative stress (pseudo-hypoxia) in adjacent fibroblasts, which activate certain key transcription factors (namely HIF1 -alpha and NFkB), that then drive the onset of autophagy,
  • HIF1 -alpha and NFkB are the master regulator(s) of aerobic glycolysis (the response to hypoxia and oxidative stress) and
  • proteomics can be used to discover suitable biomarkers for use in accordance with the present invention.
  • Proteomics is the study of proteome, the protein complement of the genome.
  • proteome also used to refer to the totality of the proteins present in a sample (e.g. tissue, organism, or cell culture) at a certain point of time.
  • proteomics includes, among other things, study of the global changes of protein expression in a sample (also referred to as "expression proteomics").
  • Proteomics typically includes the following steps: (1) separation of individual proteins in a sample by 2-D gel electrophoresis (2-D PAGE); (2) identification of the individual proteins recovered from the gel, e.g.
  • Proteomics methods are valuable supplements to other methods of gene expression profiling, and can be used, alone or in combination with other methods of the present invention, to detect the biomarkers of the present invention.
  • one aspect of the present invention is to provide a prognostic method for and/or treatment of cancer.
  • cancers having a stromal component/cells can be diagnosed and/or treated.
  • Stromal cells are connective tissue cells of an organ found in the loose connective tissue, including uterine mucosa (endometrium), prostate, bone marrow, bone marrow precursor cells, and the ovary and the hematopoietic system.
  • the most common types of stromal cells include fibroblasts, immune cells, pericytes, endothelial cells, and inflammatory cells.
  • cancers having a stromal component can occur in any organ or tissue with stromal component, including uterine mucosa (endometrium), prostate, bone marrow, bone marrow, the ovary and the hematopoietic system.
  • the present invention provides a method of stratifying a subject or a group of subjects having a cancer tumor for a clinical trial.
  • the method comprises: (a) roviding a stromal material, wherein first stromal material is derived from a biological sample obtained from a subject having a cancer tumor; (b) determining a genome-wide transcriptional profile of the stromal material; (c) comparing the genome-wide transcriptional profile (or a subgenome transcriptional profile derived therefrom) of said stromal material a Cav- 1 -deficient tumor stromal signature, wherein the Cav-1 -deficient tumor stromal signature comprises a set of at least one or more genes selected from the genes in Supplemental Table 1 ; and (d) stratifying the subject for a clinical trial based on the results of the comparison step (c).
  • Example 1 Laser Capture Microdissection and Genome- Wide Expression
  • Tumor stroma was laser capture microdissected using a Leica LCM system from 4 cases showing high stromal Cav-1 expression and 7 cases with loss of stromal Cav-1.
  • Total RNA was amplified using the NuGENTM WT-OvationTM FFPE RNA Amplification System V2 and cDNA was hybridized to Affymetrix GeneChip® arrays.
  • One-way ANOVA was setup to extract differentially expressed genes between Cav-1 positive and negative stromal samples.
  • Stromal tissue was laser capture microdissected (LCM) from fresh frozen (FF) tumor tissue samples collected from the patients pre-treatment, and samples included were closely matched for age, race, stage and grade.
  • RNA was amplified using the NuGENTM OvationTM Pico WTA System.
  • First-strand synthesis of cDNA was performed using a unique first-strand DNA/RNA chimeric primer mix, resulting in cDNA/mRNA hybrid molecules.
  • second-strand synthesis was performed and double-stranded cDNA was formed with a unique DNA/RNA heteroduplex at one end.
  • RNA within the heteroduplex was degraded using RNaseH, and replication of the resultant single-stranded cDNA was achieved through DNA/RNA chimeric primer binding and DNA polymerase enzymatic activity.
  • the amplified single-stranded cDNA was purified for accurate quantitation of the cDNA and to ensure optimal performance during the fragmentation and labeling process.
  • the single stranded cDNA was assessed using spectrophotometric methods in combination with the Agilent Bioanalyzer.
  • the appropriate amount of amplified single-stranded cDNA was fragmented and labeled using the EncoreTM Biotin Module.
  • the enzymatically and chemically fragmented product (50-100 nt) was labeled via the attachment of biotinylated nucleotides onto the 3 '-end of the fragmented cDNA.
  • the resultant fragmented and labeled cDNA was added to the hybridization cocktail in accordance with the NuGENTM guidelines for hybridization onto Affymetrix GeneChip® arrays. Following the hybridization for 18 hours at 45°C in an Affymetrix GeneChip® Hybridization Oven 640, the array was washed and stained on the GeneChip® Fluidics Station 450 using the appropriate fluidics script, before being inserted into the Affymetrix autoloader carousel and scanned using the GeneChip® Scanner 3000.
  • MSigDB is a database of gene sets:
  • the enrichment analysis consisted of computing p-values for the intersections between a gene list of interest X and each gene set Y in MSigDB. First, we computed the overlap between X and Y. Then, we computed the probability (p-value) that the observed overlap between sets X and Y is less than or equal to the overlap between set X and a randomly-chosen set of size equal to the size of set Y. This probability was calculated by applying the cumulative density function of the hypergeometric distribution on the size of set X, the size of set Y, the observed overlap between X and Y, and the total number of available genes.
  • Example 3 Analysis of Clinical Outcome in Human Breast Cancer Patients
  • Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo/
  • Samples were first analyzed in subsets based on their ER status. Additional subsets were defined by classifying samples among five canonical breast cancer subtypes, including luminal A, luminal B, normal-like, basal, and Her-2- overexpressing disease. Samples were classified by computing their correlation against five expression profile centroids representing these breast cancer subtypes and assigning them to the subtype with the highest corresponding correlation coefficient 71. Samples with a maximum correlation coefficient below 0.3 were considered unclassified. Differential expression of the averaged gene signature magnitude among these sample subsets was evaluated using a two-tailed t-test. Kaplan-meier analysis was used to evaluate survival trends within sample subsets.
  • the Log-rank test was used to evaluate differences in survival curves for high vs. low signature- expressing populations.
  • samples with ER immuno- histochemistry (IHC) data were selected for analysis, including 959 ER-positive and 323 ER- negative, for a total of 1,282 samples; in addition, 102 normal healthy breast tissue controls were used for comparison purposes.
  • samples with information on ESR1 mRNA transcript levels were selected for analysis, including 1,674 ESR1 -positive and 478 ESR1 -negative, for a total of 2,152 samples; in addition, 102 normal healthy breast tissue controls were used for comparison purposes.
  • the ESR1 high/low expression cutoff was defined at the RMA-normalized expression value of 7.5. This was determined from the bimodal
  • the fold-change data are represented as stromal Cav-1 (negative/positive), resulting in a positive fold-change for the transcripts that are up-regulated in Cav-1 negative stromal patients.
  • Supplemental Table 1 contains 238 gene transcripts that were specifically up- regulated and 232 gene transcripts that were down-regulated in the stroma of tumors showing a loss of stromal Cav-1 expression (p ⁇ 0.01 and fold-change (f.c.) > 1.5).
  • the two patient populations are transcriptionally different.
  • One-way ANOVA was setup to extract differentially expressed genes between Cav-1 positive and Cav-1 negative samples.
  • the resultant p-values were further adjusted by multi-test correction (MTC) method of FDR step-up.
  • MTC multi-test correction
  • the standardized intensity data from the stringent gene list (p-value ⁇ 0.01 and fold change >_1.5) were used in generating the hierarchical clustering HeatMap.
  • the Cav-1 -deficient stromal gene signature is associated with tumor recurrence and poor survival in ER(+) and luminal A breast cancer patients.
  • Supplemental Table 2 contains 5,424 transcripts encoding 3,459 unique genes (p ⁇ 0.1 and fold-change (f.c.) > 1.15) that were up-regulated in the stroma of tumors showing a loss of stromal Cav-1 expression.
  • Supplemental Table 3 contains 1,819 transcripts encoding 1,297 unique genes (p ⁇ 0.05 and fold-change (f.c.) > 1.15) that were up-regulated in the stroma of tumors showing a loss of stromal Cav-1 expression.
  • Cav-1 -deficient stroma showed the up-regulation of myofibroblast differentiation (15 transcripts), autophagy (22 transcripts), lysosomal proteases (5 transcripts), lysosomal proteins (29 transcripts), and glycolysis/pyruvate metabolism (15 transcripts), autophagy (22 transcripts), lysosomal proteases (5 transcripts), lysosomal proteins (29 transcripts), and glycolysis/pyruvate metabolism (15
  • Supplemental Tables 4-14 contain the data shown in HeatMaps, as Figures 2-8.
  • Cav-1 -deficient stroma showed the up-regulation of hypoxia target genes (65 transcripts) and mitochondrial-associated proteins (41 transcripts).
  • Cav- 1 -deficient stroma showed the up- regulation of the TNF/NFkB signaling (11 transcripts), the immune response (31 transcripts), and redox/stress signaling (19 transcripts). [00111] As provided in Supplemental Table 11 : Cav- 1 -deficient stroma showed the up- regulation of the DNA damage response (67 transcripts).
  • the Cav-1 -deficient stromal gene signature is up-regulated in breast cancer and is associated with tumor recurrence.
  • panels A and B box-plots show that the Cav-1 -deficient stromal signature is up-regulated in all breast cancers, both ER(+) and ER(-) sub-types, relative to normal healthy breast tissue.
  • ER status was determined by immuno-histochemistry
  • panel B ER status was inferred from ESR1 transcript expression.
  • the Cav-1 -deficient stromal signature was associated with increased recurrence in breast cancer patients.
  • Figure 9 illustrates Venn diagrams for the intersection of the cav-1 -deficient stromal gene signature with other breast cancer tumor stromal gene sets.
  • recurrence stroma a 214 transcript overlap; p ⁇ 0.001
  • the gene transcripts up-regulated in Cav-1 -deficient tumor stroma show a significant association with tumor recurrence, and are in accordance with the results of Morag Park and colleagues 15 .
  • the present inventors compared the gene transcripts up-regulated in Cav-1 -deficient tumor stroma (1,819 transcripts encoding 1,297 unique genes; p ⁇ 0.05 and fold- change (fc.) > 1.15; Supplemental Table 3) with ⁇ Upper) tumor stromal and ⁇ Lower) recurrence stromal gene lists, as defined in the text of the manuscript.
  • Supplemental Table 15 contains the 3,459 unique genes that were up-regulated in the stroma of tumors showing a loss of stromal Cav-1 expression.
  • Supplemental Table 16 contains the 1,297 unique genes that were up-regulated in the stroma of tumors showing a loss of stromal Cav-1 expression.
  • Figure 12 illustrates understanding of the hierarchy of the cellular processes associated with a Cav-1 -deficient tumor microenvironment. Oxidative stress in fibroblasts is known to be sufficient to induce 1) myo-fibroblast differentiation, 2) DNA damage, and 3) a pseudo-hypoxic
  • Example 1 Transcriptional Profiling of a Cav-1 Deficient Tumor
  • GSEA gene set enrichment analysis
  • HeatMaps illustrate the upregulation of gene transcripts associated with myofibroblast differentiation, autophagy, lysosomal degradation, glycolysis, hypoxia, mitochondria, inflammation and redox signaling, DNA damage and repair, aging, BRCA1- mutation positive and ER-negative breast cancer patients, apoptosis, and neural stem cells (See Supplemental Tables 4-14).
  • Neurogenesis was associated with increased DNA damage in the tumor stroma ' .
  • GOLPH3 also known as MIDAS/GPP34
  • mtDNA mitochondrial DNA
  • MIDAS stands for mitochondrial DNA absence sensitive factor u .
  • PRKDC is a component of the machinery required for the proper repair and maintenance of the fidelity of mitochondrial
  • mtDNA DNA (mtDNA) , so its up-regulation is suggestive of increase mtDNA damage.
  • Arsenic treatment is associated with increased ROS production, mtDNA damage, and mitochondrial dysfunction, with reduced ATP production, driving the onset of aerobic glycolysis 13 ' 14 .
  • Normal Stroma List Compares the transcriptional profiles of tumor stroma obtained from 53 patients to normal stroma obtained from 38 patients. Genes transcripts that were consistently upregulated in the tumor stroma were selected and assigned a p- value, with a cut-off of p ⁇ 0.05 (this set contains 6,777 unique genes) 16 .
  • recurrence stroma a 214 transcript overlap; p ⁇ 0.001
  • the gene transcripts up-regulated in Cav-1 -deficient tumor stroma show a significant association with tumor recurrence, and are in accordance with the results of Morag Park and colleagues 15 .
  • Example 4 Comparison with the Transcriptional Profiles of Breast Cancers that were Not Subjected to Laser Capture Microdissection
  • Figure ⁇ , ⁇ shows that the Cav-1 -deficient tumor stromal signature is up-regulated in breast cancer(s), both ER(+) and ER(-) sub-types. However, a more significant association was observed with ER(-) breast cancers. In addition, the Cav-1 -deficient stromal signature was also associated with increased recurrence in breast cancer patients ( Figure IOC). [00137] Similarly, we also looked at the prognostic value of the Cav-1 -deficient stromal signature in ER(+) and luminal A breast cancer patients, which account for nearly 60% of all breast cancer patients 17 ' 18. Interestingly, Figure 11 shows that the Cav-1 -deficient stromal signature is clearly associated with increased recurrence and decreased overall survival, despite the fact that these breast cancer-derived tumors were not subjected to laser capture
  • GSEA Gene set enrichment analysis
  • GSEA has identified significant differentially expressed sets of genes, even where the average difference in expression between two phenotypes is only 20% for genes in the gene set.
  • the gene set identified in the first GSEA analysis (oxidative
  • Witkiewicz AK Casimiro MC, Dasgupta A, Mercier I, Wang C, Bonuccelli G, Jasmin JF, Frank PG, Pestell RG, Kleer CG, Sotgia F, Lisanti MP. Towards a new "stromal-based" classification system for human breast cancer prognosis and therapy. Cell Cycle 2009; 8: 1654-8. 3. Witkiewicz AK, Dasgupta A, Nguyen KH, Liu C, Kovatich AJ, Schwartz GF, Pestell RG, Sotgia F, Rui H, Lisanti MP. Stromal caveolin-1 levels predict early DCIS progression to invasive breast cancer. Cancer Biol Ther 2009; 8: 1167-75.
  • Ertel A Bimodal gene expression and biomarker discovery. Cancer Inform 2010; 9: 11-4. 18. Ertel A, Dean JL, Rui H, Liu C, Witkiewicz AK, Knudsen KE, Knudsen ES. RB-pathway disruption in breast cancer: differential association with disease subtypes, disease-specific prognosis and therapeutic response. Cell Cycle 2010; 9:4153-63.
  • Tumor-Stroma Co-Evolution A New Paradigm for Understanding Tumor Metabolism, the Field Effect, and Genomic Instability in Cancer Cells. Cell Cycle 2010; 9:3256-76.
  • microenvironment a new genetically tractable model for human cancer associated fibroblasts. Cancer Biol Ther 2011; 11 :383-94.
  • TGF-beta/SMAD signaling through an interaction with the TGF-beta type I receptor. J Biol Chem 2001; 276:6727-38.
  • PEGylated catalase prevents metastatic tumor growth aggravated by tumor removal. Free Radic Biol Med 2006; 41 : 1449-58.
  • DAZAP2 35 jADXEC.11581.Cl_at iHomo sapiens DAZ associated protein 2 (DAZAP2) transcript variant
  • NCF1 neutrophil cytosolic factor 1
  • ADXECAD.7829 s at iHomo sapiens apolipoprotein C-ll (APOC2) mRNA.
  • RDBP RD RNA binding protein
  • DNASE2 deoxyribonuclease II lysosomal
  • MORC2 466 iADXEC.2672.Cl_s _at
  • MORC2 466 iADXEC.2672.Cl_s _at
  • CD68 CD68 transcript variant 2 mRNA.
  • HMG20B high-mobility group 20B
  • KIF3C kinesin family member 3C
  • NUP210 nucleoporin 210kDa
  • NARF nuclear prelamin A recognition factor
  • ZNF407 iADXEC.19326.Cl_ . at iHomo sapiens zinc finger protein 407 (ZNF407) transcript variant
  • ERP44 Homo sapiens endoplasmic reticulum protein 44
  • LOC285965 0.0114s
  • G protein-coupled receptor 65 G protein-coupled receptor 65
  • TTC12 iiHomo sapiens tetratricopeptide repeat protein 12
  • CD8A CD8a molecule
  • GTPBP2 GTP binding protein 2
  • ADXECRS.6935_x_at PREDICTED Homo sapiens hypothetical LOC730235 (LOC730235)
  • CTF1 cardiotrophin 1
  • ADXECRS.530_s_at PREDICTED Homo sapiens hypothetical protein LOC100129677
  • ADCY7 Homo sapiens adenylate cyclase 7
  • TATA box binding protein (TBP)-associated factor H AF10 0.0239
  • TEM90A transmembrane protein 90A

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Abstract

La présente invention concerne des marqueurs biologiques utiles relatifs à des cancers ayant des éléments du stroma. Ces marqueurs biologiques nouvellement identifiés sont utiles en tant que signatures de pronostic pour évaluer un pronostic de cancer chez un sujet ayant un cancer (par exemple, risque de récurrence de cancer, progression d'un cancer chez un sujet ayant un cancer, survie globale du cancer par un patient ayant un cancer). Ils peuvent également être mis en œuvre en tant que procédé de surveillance de l'évolution d'un cancer du sein chez des sujets subissant un traitement pour le cancer ou en tant que procédé de stratification d'un sujet ou d'un groupe de sujets ayant une tumeur cancéreuse pour un essai clinique.
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