US 2013 0230511A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0230511 A1 HEYMACH et al. (43) Pub. Date: Sep. 5, 2013

(54) BIOMARKERS FOR RESPONSE TO Publication Classification TYROSINE KNASE PATHWAY INHIBITORS IN CANCER (51) Int. Cl. GOIN33/74 (2006.01) (71) Applicant: Board of Regents, The University of A 6LX39/395 (2006.01) Texas System, (US) A613 L/506 (2006.01) A613 L/4439 (2006.01) (72) Inventors: John V. HEYMACH, Pearland, TX CI2O I/68 (2006.01) (US); Ignacio I. WISTUBA, Houston, A613 L/4045 (2006.01) TX (US); Monique B. NILSSON, Sugar (52) U.S. Cl. Land, TX (US); Lauren A. BYERS, CPC ...... G0IN33/74 (2013.01); C12O 1/6886 Houston, TX (US); Ximing TANG, (2013.01); A61 K3I/4045 (2013.01); A61 K Houston, TX (US); Fei YANG, Houston, 3 1/506 (2013.01); A61 K3I/4439 (2013.01): TX (US); Edward KIM, Houston, TX A6IK39/39558 (2013.01) (US); Anne S. TSAO, Houston, TX USPC ...... 424/133.1: 435/7.23; 435/6.11: 514/.414: (US); Scott M. LIPPMAN, Houston, 514/252.18; 514/338 TX (US); Waun Ki HONG, Houston, TX (US); Roy S. HERBST, Houston, TX (US) (57) ABSTRACT (73) Assignee: BOARD OF REGENTS, THE Copy number gains detected in tumors and associated with UNIVERSITY OF TEXAS SYSTEM, drug sensitivity and resistance in vivo and in vitro can be used Austin, TX (US) as biomarkers to select, predict and monitor drug treatment outcomes in cancer patients treated with (21) Appl. No.: 13/758,728 inhibitors. Methods to identify patients with NSCLC or other malignancies who are more likely to benefit from tyrosine (22) Filed: Feb. 4, 2013 kinase inhibitors Such as VEGF or VEGFR inhibitors when O O used either as monotherapy or in combination with other Related U.S. Application Data therapies such as or EGFR inhibitors, and who (60) Provisional application No. 61/594,800, filed on Feb. are in the advanced stages of disease and/or who have under 3, 2012. gone adjuvant therapy are also provided herein. Patent Application Publication Sep. 5, 2013 Sheet 1 of 14 US 2013/0230511 A1

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BOMARKERS FOR RESPONSE TO KIT pathway inhibitor, respectively. In a further aspect, a TYROSINE KNASE PATHWAY INHIBITORS patient determined to have elevated levels of two or three of IN CANCER KDR, PDGFR, and KIT may be treated with two or more inhibitors of the VEGF/VEGFR, PDGFR, or KIT pathways. BACKGROUND OF THE INVENTION In another aspect, a patient determined to have elevated levels 0001. This application claims the benefit of U.S. Provi of two or three of KDR, PDGFR, and KIT may be treated with sional Patent Application No. 61/594,800, filed Feb. 3, 2012, an inhibitor that inhibits two, or all three, of the VEGF/ the entirety of which is incorporated herein by reference. VEGFR, PDGFR, and KIT pathways, for example, 0002 This invention was made with government support or . under Prospect Grant W81XWH-07-1-0306 awarded by the 0007. In one aspect, an elevated KDR, PDGFR, or KIT U.S. Department of Defense, Grant W81XWH-06-1-0303 level may be a gain in the gene copy number of one or more awarded by the U.S. Department of Defense, and Grants 5P50 of the genes. In another aspect, an elevated KDR, PDGFR, or CA070907-14 and CA-16672 awarded by the National Insti KIT level may be an increased mRNA expression. In yet tutes of Health. The government has certain rights in the another aspect, an elevated KDR, PDGFR, or KIT level may invention. be an increased protein expression. In certain aspects, an 0003 Pursuant to 37 C.F.R. 1.821(c), a sequence listing is elevated KDR level may be an increased mRNA or protein submitted herewith as an ASCII compliant text file named expression level of a KDR-regulated gene, for example, HIF “UTSCP1203US.txt, created on Feb. 4, 2013 and having a 1C. size of 4 KB. The content of the aforementioned file is hereby 0008. In some preferred embodiments, a cancer patient for incorporated by reference in its entirety. treatment or assessment accordingly the embodiments may have a NSCLC or a glioblastoma. In some further aspects, the FIELD OF INVENTION cancer may be a metastatic cancer or a cancer that has devel oped resistance to one or more anti-cancer agent. In certain 0004. This invention relates generally to cancer treatments aspects, the cancer patient for treatment according to the with tyrosine kinase inhibitors and more particularly, to meth embodiments, may receive or have received a secondary ods of predicting cancer treatment outcome for a cancer therapy Such as a Surgery or radiotherapy. Thus, in some patient through copy number gain of the KDR, PDGFR, aspects, a treatment of the embodiments is used as an adjuvant and/or KIT genes. treatment. In other aspects, the cancer patient may be treated With a secondary therapy Such as a second drug (e.g., that is BACKGROUND OF THE INVENTION not a platinum-based chemotherapeutic agent) or an EGFR 0005 Successful treatment of cancer has remained elusive inhibitor. A secondary therapy for used according to the despite rapid advances in the field in recent years. One major embodiments may be applied before, after or essentially complicating factor in effective treatment is that conventional simultaneously with a treatment of the embodiments. diagnostics to characterize tumors offer limited insight as to 0009 Certain aspects of the embodiments concern what types of anti-cancer therapy may be successful for treat PDGFR, VEGF/VEGFR and/or KIT pathway inhibitors. For ing any given cancer. In fact, cancer cells exhibit a wide range example, VEGF/VEGFR pathway inhibitors may be, without of resistance/susceptibility to various anti-cancer therapies, limitation, , Sunitinib, , afliber thus it has been difficult to predict whether a particular cancer cept, BIBF1120, , , dovitinib, , will be resistant or susceptible to any given therapy. The , semaxanib, , PP-121, tellatinib, TSU-68, vascular endothelial receptor-2 (“VEGFR-2), Ki8751, , , , Vatalanib, or van for example, is known to be present on tumor vascular endot detanib. PDGFR pathway inhibitor include, without limita helial cells Inhibitors of VEGFR-2 (KDR) have been devel tion, imatinib, Sunitinib, axitinib, BIBF1120, paZopanib, oped with the goal of inhibiting tumorangiogenesis in cancer pnoatinib, MK-2461, dovitinib, , PP-121, tella patients. However, there are currently no validated markers tinib, CP 673451, TSU-68, Ki8751, tivoZanib, , for predicting which cancer patients are likely to respond to motesanib, MEDI-575, or regorafenib. KIT pathway inhibi inhibitors of the VEGF/VEGFR pathway. Likewise, powerful tors include, but are not limited to, imatinib, axitinib, paZo inhibitors of the PDGFR and KIT pathways are being devel panib, dovitinib, tellatinib, Ki8751, tivozanib, masitinib, oped for anti-cancer therapy, but it is unclear what types of motesanib, Sunitinib, IMG-3G3, , , rego cancers would be most responsive to such therapies. Methods rafenib, or Vatalanib. are needed to help select cancer patients who will experience 0010. In another embodiment, the present invention pro greater benefit from these inhibitors and who are potentially vides a method of predicting the sensitivity of a cancer in a spared the toxicities of these drugs if they are less likely to patient to a VEGF/VEGFR, PDGFR, and/or KIT pathway benefit. inhibitor comprising obtaining a sample of the cancer and determining the KDR, PDGFR, and/or KIT level in the cells SUMMARY OF THE INVENTION comprising the sample, wherein if the KDR, PDGFR, and/or 0006. In one embodiment, the present invention provides a KIT level is elevated, then the cancer is predicted to be sen method of treating a cancer patient comprising selecting a sitive to a corresponding VEGF/VEGFR, PDGFR, or KIT patient determined to have a cancer with an elevated KDR, pathway inhibitors. In certain aspects, a patient predicted to PDGFR, or KIT level, and then treating the patient with a be sensitive to VEGF/VEGFR, PDGFR, or KIT pathway VEGF/VEGFR, PDGFR, or KIT pathway inhibitor. In one inhibitor may be treated with at least one inhibitor of the aspect, a patient determined to have an elevated KDR level VEGF/VEGFR, PDGFR, or KIT pathways. In a further may be treated with a VEGF/VEGFR pathway inhibitor. In aspect, the method further provides for identifying the patient another aspect, a patient determined to have an elevated as having a cancer that is predicted to be sensitive to VEGF/ PDGFR or KIT level may be treated with either a PDGFR or VEGFR, PDGFR, or KIT pathway inhibitors, and reporting US 2013/023051 1 A1 Sep. 5, 2013

whether the cancer is predicted to be sensitive or resistant to KDR, PDGFR, or KIT level in the cells comprising the the inhibitor. (e.g., by providing written, oral or electronic sample, and selecting a VEGF/VEGFR, PDGFR, or KIT report). In some aspects, such a report can be provided to the pathway inhibitor for drug therapy if the level determined in patient, a doctor, a hospital, an insurance company, or a (b) is elevated or selecting an EGFR inhibitor platinum-based payee. chemotherapy if the level determined in (b) is not elevated. 0011. Another embodiment of the present invention pro 0018. The present invention also provides a method of vides a method of monitoring the efficacy of VEGF/VEGFR, determining a prognosis of a cancer patient comprising PDGFR, or KIT pathway inhibitor treatment on a cancer obtaining a sample of the patient’s cancer and determining comprising obtaining samples of the cancer from at least two the KDR level in the cells comprising the sample, wherein the time points during the course of treatment, determining the cancer is determined to have a worse prognosis if the KDR KDR, PDGFR, or KIT level in the cells comprising the level is determined to be elevated. samples, and comparing the KDR, PDGFR, or KIT levels, 0019. The present invention also provides a method of wherein the VEGF/VEGFR, PDGFR, or KIT pathway inhibi determining a prognosis of a cancer patient comprising tor treatment is efficacious if the KDR, PDGFR, or KIT level obtaining a sample of the patients cancer and detecting poly decreases over the course of treatment. morphisms at nucleotides -37 and 1416 in the KDR gene in 0012. In some aspects, the level of mRNA or protein of a the cells comprising the sample, wherein the cancer is deter gene regulated by a KDR-regulated gene may be used to mined to have a better prognosis if the -37AG/GG and 1416 represent the KDR level. In one aspect, the KDR-regulated AT/TT polymorphisms are present. In one aspect, if the poly gene is HIF-1C. and the gene regulated by HIF-1C. is EZH2 or morphisms are absent, then an aggressive anticancer therapy Met. may be applied. 0013. In another embodiment, the present invention pro 0020 Methods of predicting a treatment outcome for a vides a method of predicting the sensitivity of a cancer in a cancer patient, methods of monitoring responsiveness to drug patient to an EGFR inhibitor therapy or platinum-based che therapy, and methods of selecting drug therapy are provided motherapy comprising obtaining a sample of the cancer and herein. Also provided are methods to identifying cancer determining the KDR level in the sample, wherein if the KDR patients who are more likely to benefit from tyrosine kinase level is not elevated, then the cancer is predicted to be sensi inhibitors, such as VEGF or VEGFR inhibitors when used tive to EGFR inhibitors or platinum-based chemotherapy. In eitheras monotherapy or in combination with other therapies, a further aspect, the method provides for identifying the such as chemotherapy or EGFR inhibitors, and who are in the patient as having a cancer that is predicted to be sensitive to advanced stages of disease and/or who have undergone adju EGFR inhibitors or platinum-based chemotherapy, and vant therapy. Further provided are methods to identify which reporting whether the cancer is predicted to be sensitive to patients are more likely to be resistant to tyrosine kinase EGFR inhibitors or platinum-based chemotherapy. For inhibitors such as EGFR inhibitors. The methods described example, reporting can comprise providing a written, oral or herein are useful either as a predictive marker prior to starting electronic report, e.g., to the patient, a doctor, a hospital, an a drug therapy or as a marker of acquired resistance for insurance company, or a payee. patients more likely to benefit from treatment with tyrosine 0014. In certain aspects, a patient determined to have a kinase inhibitors, such as VEGF or VEGFR inhibitors, alone normal or decreased KDR level may be treated with an EGFR or in combination regimens. Moreover, methods are provided inhibitor or platinum-based chemotherapeutic agent. that identify patients who would benefit from targeting the Examples of EGFR inhibitors include, without limitation, PDGFR or KIT pathways, alone or in combination with erlotinib, , , PF299804, , panitu VEGFR pathway inhibitors, in NSCLC and other malignan mab, , , , OSI-420, cies with CNGs in the PDGFR or KIT genes. C1-1033, , WHI-P154, or . Platinum-based 0021. Each method described herein includes at least the chemotherapeutic agents for use according to the embodi steps of providing a biological sample from a cancer patient; ments include, without limitation, cisplatin or carboplatin. determining CNG of at least one of the following genes: 0015. In one aspect, the patient has not yet undergone an KDR, PDGFR, and KIT in the sample, wherein a gene copy anti-cancer therapy. In another aspect, the patient may have number of 4 or greater for the KDR, PDGFR, or KIT gene is received at least one dose of an anti-cancer therapy, such as an considered CNG and predictive of poor treatment outcome: EGFR inhibitor or platinum-based chemotherapeutic agent. and, when appropriate, administrating a drug or other therapy Accordingly, I Some aspects a method may be a method of to the cancer patient based on the CNG of one or more of these monitoring (acquired) resistance to said therapy comprising genes. In addition, other prognostic methods and/or method detecting an elevated KDR level. A patient determined to steps may be used together with these methods. have acquired resistance to an EGFR inhibitor or platinum 0022. Some aspects of the embodiments involve a subject, based chemotherapeutic agent may be treated with a VEGF/ Such as a cancer patient. As used herein a Subject or patient VEGFR pathway inhibitor. can be human or non-human animal Subject (e.g., a dog, cat, 0016. In a further embodiment, the present invention pro mouse, horse, etc). In certain aspects, the Subject has a cancer, vides a method of treating a cancer patient comprising deter Such as an oral cancer, oropharyngeal cancer, nasopharyngeal mining if the patient has a cancer that is sensitive to VEGF/ cancer, respiratory cancer, urogenital cancer, gastrointestinal VEGFR, PDGFR, or KIT pathway inhibitors and treating the cancer, central or peripheral nervous system tissue cancer, an patient determined to have a cancer that is sensitive to VEGF/ endocrine or neuroendocrine cancer or hematopoietic cancer, VEGFR, PDGFR, or KIT pathway inhibitors with VEGF/ glioma, sarcoma, carcinoma, , melanoma, VEGFR, PDGFR, or KIT pathways inhibitors. fibroma, meningioma, brain cancer, oropharyngeal cancer, 0017. In another embodiment, the present invention pro nasopharyngeal cancer, renal cancer, biliary cancer, pheo vides a method of selecting a drug therapy for a cancer patient chromocytoma, pancreatic islet cell cancer, Li-Fraumeni comprising obtaining a sample of the cancer, determining the tumors, thyroid cancer, parathyroid cancer, pituitary tumors, US 2013/023051 1 A1 Sep. 5, 2013

adrenal gland tumors, osteogenic sarcoma tumors, neuroen (mm) comparing 26 lung cancers having KDRCNG with 26 docrine tumors, breast cancer, lung cancer, head and neck lung cancers without CNG. In the box plots, bars indicate cancer, prostate cancer, esophageal cancer, tracheal cancer, median score, X indicates mean scores, and dashed lines indi liver cancer, bladder cancer, stomach cancer, pancreatic can cate standard deviation. FIG. 2B (adenocarcinoma) and 2C cer, ovarian cancer, uterine cancer, cervical cancer, testicular (squamous cell carcinoma) are representative examples of cancer, colon cancer, rectal cancer or skin cancer. immunohistochemical expression of CD34-positive vessels 0023. As used herein the specification, “a” or “an may (MVD) (magnification x200). mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising, the words “a” or 0030 FIG.3 shows KDR copy number gain (CNG) asso 'an' may mean one or more than one. ciated with outcome in NSCLC patients treated with adjuvant 0024. The use of the term “or” in the claims is used to chemotherapy. Kaplan-Meier curves for overall survival (OS) mean “and/or unless explicitly indicated to refer to alterna and recurrence-free survival (RFS) by KDRCNG in NSCLC tives only or the alternatives are mutually exclusive, although patients and two Subgroups of platinum adjuvant therapy and the disclosure supports a definition that refers to only alter without adjuvant therapy (E. event; N, total number of cases). natives and “and/or.” As used herein “another may mean at 0031 FIG. 4A-E show KDR copy number gain (CNG) least a second or more. and VEGFR-2 expression associated with resistance to cispl 0025 Throughout this application, the term “about is atin. FIG. 4A shows the correlation of KDR copy number used to indicate that a value includes the inherent variation of gain (CNG) with in vitro resistance to cisplatin. NSCLC cell error for the device, the method being employed to determine lines demonstrating CNG (26 gene copies) showed signifi the value, or the variation that exists among the study Sub cantly higher ICs compared with cell lines without CNG. jects. FIG. 4B shows the correlation between the concentrations of 0026. Other objects, features and advantages of the cisplatin required to inhibit NSCLC cell growth (ICs) and present invention will become apparent from the following VEGFR-2 protein expression levels by reverse phase protein detailed description. It should be understood, however, that array (RPPA). FIG. 4C shows that siRNA targeting KDR the detailed description and the specific examples, while indi (siKDR) in NSCLC cell line H23 significantly inhibited the cating preferred embodiments of the invention, are given by expression of VEGFR-2 by Western blot (WB) and KDR way of illustration only, since various changes and modifica mRNA by reverse transcriptase quantitative PCR (RT-qPCR) tions within the spirit and scope of the invention will become compared with basal and scrambled control siRNA (Bars: apparent to those skilled in the art from this detailed descrip s.d.; *, P-0.05). FIG. 4D shows that knocking down KDR tion. using siRNA decreased the viability of NSCLC cell line H23 exposed to cisplatin by MTS assay (data are graphed as mean BRIEF DESCRIPTION OF THE DRAWINGS percent increase-percents.d.). Knockdown of KDR in H23 0027. The following drawings form part of the present cells caused a 1.9-fold decrease in the cisplatin ICs (53 specification and are included to further demonstrate certain versus 97.9 mol/L in siKDR knockdown H23 cells versus aspects of the present invention. The invention may be better untransfected cells; P<0.05) and a 3.5-fold decrease in the understood by reference to one or more of these drawings in carboplatin ICs (27.9 versus 97 umol/L in siKDR knock combination with the detailed description of specific embodi down H23 cells versus non-transfected cells; P<0.05). FIG. ments presented herein. 4E shows the migration of NSCLC cell line H23 by Boyden 0028 FIG. 1A-E show that KDR copy number gain chamber assay (left) was inhibited by knocking down KDR (CNG) is correlated with VEGFR-2 protein expression in using KDR in cells with and without stimulation with VEGF non-small cell lung carcinomas (NSCLC) tumors. FIG. 1A (Bars: S.d.; *P<0.05: **P<0.003). The right panel shows the (copy number gain) and 1B (no copy number gain) are rep quantification of the migration assay of NSCLC cell lines resentative examples of KDR copy number examined by before and after knocking down KDR using siKDR in cells fluorescence in situ hybridization (FISH) in NSCLC tissue with and without stimulation with VEGF showed decreased specimens. Signals represent the KDR gene probe or the migration in H23 cells (6-9 KDR copies). internal control probe (magnification x 1000). FIG. 1C (ad 0032 FIG. 5A-E show KDR copy number gain (CNG) enocarcinoma) and 1D (squamous cell carcinoma) are repre correlated with HIF-1C. expression in NSCLC cell lines and sentative examples of immunohistochemical expression of tumor tissue specimens. FIG. 5A shows HIF-1C. protein VEGFR-2 in NSCLC tissue specimens. VEGFR-2 protein expression determined by ELISA correlated with KDRCNG expression was present both in the cytoplasm and membrane in a series of NSCLC cell lines (Bars: S.d.; cell lines with of tumor cells (magnification x200). FIG. 1E shows expres CNG 6-9 copies versus 3-5 copies and no CNG, *P<0.02). sion of VEGFR-2 in tumors with KDRCNG compared with FIG. 5B shows HIF-1C. expression by ELISA was markedly lung cancers without CNG. The box-plots depict scores of inhibited by knocking down KDR using siKDR in the immunohistochemical (IHC) expression of VEGFR-2 cyto NSCLC H23 cell line with and without Stimulation with plasm and VEGFR-2 membrane comparing 26 lung cancers VEGF (Bars: S.d.; *P<0.01). FIG. 5C shows expression of having KDR CNG with 26 lung cancers without CNG. In the nuclear HIF-1C. in tumors with KDR CNG compared with box plots, bars indicate median score, X indicates mean lung cancers without CNG. The box-plots depict scores of scores, and dashed lines indicate standard deviation. immunohistochemical (IHC) expression of nuclear HIF-1C. 0029 FIG. 2A-C show KDR copy number gain (CNG) comparing 22 lung cancers having KDR CNG with 25 lung correlated with microvascular density (MVD) in non-small cancers without CNG. In the box plots, bars indicate median cell lung carcinomas (NSCLC) tumors. FIG. 2A shows score, X indicates mean scores, and dashed lines indicate expression of MVD in tumors with KDR CNG compared standard deviation. FIG.5D (adenocarcinoma) and 5E (squa with lung cancers without CNG. The box-plots depict scores mous cell carcinoma) are representative examples of low of immunohistochemical assessment of MVD and vessel area (FIG. 5D) and high (FIG. 5E) IHC expression of HIF-1C. in US 2013/023051 1 A1 Sep. 5, 2013

NSCLC tissue specimens (magnification x200). Arrows, DETAILED DESCRIPTION OF THE INVENTION positive nuclear HIF-1C, immunostaining. 0042 Methods and compositions for predicting disease 0033 FIG. 6 shows VEGFR inhibitor, Sunitinib, inhibits outcome for cancer patients treated with tyrosine kinase cell migration in H23 cells which harbors VEGFR CNGs. inhibitors are provided herein. Copy number gain (“CNG”) of Imatinib, which targets BCL/ABL, Kit, and PDGFR, does not certain genes can serve as biomarkers for predicting cancer inhibit cellular migration. In contrast, the VEGFR inhibitor, treatment outcome of kinase inhibitors, especially inhibitors Sunitinib, has no effect on migration of A549 cells, which do of vascular endothelial (“VEGFR), not have amplification of VEGFR. receptor (“EGFR), platelet-derived 0034 FIG. 7A-C show that HIF-1C. levels are decreased growth factor receptor (“PDGFR), and kinase insert domain by VEGFR inhibition in VEGFR amplified cells. FIG. 7A receptor (“KIT). Specifically, the copy number gain of KDR, shows higher levels of HIF-1C. in cell lines with VEGFR PDGFR, and KIT genes, alone or in combination with each CNGs compared to those without. FIG. 7B shows a statisti other, can be used to predict whether a patient may benefit cally significant decrease in HIF-1C. levels in H23 cells (KDR from one or more tyrosine kinase inhibitor drug therapies. CNG+) treated with the VEGFR inhibitor Sunitinib. FIG. 7C 0043. As such, methods of predicting a treatment outcome shows no change in HIF-1C. levels was detected in A549 cells, for a cancer patient, methods of monitoring responsiveness to which do not contain VEGF CNGs. drug therapy, methods of selecting drug therapy and methods 0035 FIG. 8 shows that VEGFR pathway inhibition with of identifying patients with NSCLC or other malignancies bevacizumab decreases HIF-1C.-regulated proteins, includ who are more likely to benefit from VEGF, VEGFR, or EGFR ing EZH2, Met, and phosphorylated Met, in H23 and Calu1 inhibitors, and/or inhibitors of the PDGFR and/or KIT path cells, which have VEGFRCNGs. Two VEGFR amplified cell ways are provided herein. Each method includes at least the lines, H23 and Calul, were treated with the VEGFR pathway steps of: (a) providing a biological sample from a cancer inhibitor bevacizumab and evaluated for changes in proteins patient; (b) determining CNG, wherein a gene copy number regulated by HIF-1C. Multiple HIF-1C-regulated proteins of greater than 4 for either the KDR, PDGFR, or KIT gene is were decreased in the presence of bevacizumab, including considered CNG and predictive of poor treatment outcome: EZH2, Met, and phosphorylated Met. and (c) administrating a drug or other therapy to the cancer 0036 FIG. 9 shows the Kaplan-Meier curves for overall patient based on the CNG of one or more genes. survival (OS) by genotypes of two KDR single nucleotide 0044) Deregulated kinase activity is a frequent cause of polymorphisms in adenocarcinoma and squamous cell carci disease, particularly cancer, where kinases regulate many noma of lung (E. event, N, total number of cases). aspects that control cell growth, movement, and death. Many 0037 FIG. 10 A-C show that VEGFR TKIs inhibit cell of the genetic defects can identify the key components of migration in KDR amplified cell lines. Each cell line was signaling pathways responsible for proliferation and differ tested with or without VEGF (50 ng/mL) and with or without entiation. One class ofkinases that are frequently deregulated AZD2171, Sunitinib, and imatinib (bars: S.d.; *P<0.05 vs. in cancer are receptor tyrosine kinases (“RTKs) involved in control; #P-0.05 vs. VEGF alone). FIG.10A shows the quan signal transduction. In general, RTKs are monomeric Surface tification for the number of migrating cells relative to control receptors that dimerize upon activation. RTKs have an extra for the Calu-1 cell line. FIG. 10B shows the quantification for cellular binding domain, a transmembrane domain, and an the number of migrating cells relative to control for the intracellular kinase domain. Ligand binding to the extracel HCC461 cell line. FIG. 10C shows the quantification for the lular domain induces dimerization of the Surface receptor, number of migrating cells relative to control for the H1993 which in turn induces phosphorylation of tyrosine residues cell line. within an “activation loop of the intracellularkinase domain. 0038 FIG. 11 shows the effect of VEGFRTKIs on tumor 0045 Tumor growth is critically dependent on neovascu cell secretion of cytokines H23 tumor cells were treated with larization (Folkman, 1971). The ligand vascular endothelial control media or media containing the VEGFRTKI Sunitinib growth factor (“VEGF) is an endothelial cell mitogen that is (1 uM) for 24 hours. Conditioned media was collected and a specific mediator of angiogenesis and has two identified cytokine levels (VEGF, PDGF, IL-8, HGF, and FGF2) were tyrosine kinase receptors, VEGF receptor-1 and -2 (Fidler et assessed by ELISA assay. Imatinib was used as a negative al., 1994; Waltenberger et al., 1994: Ferrara et al., 1997: control. Hanahan et al., 2011). 0039 FIG. 12 shows that KDR copy number gain was 0046) VEGFR-2 coded by the gene FLK-I (located in associated with increased levels of EGFR and greater expres 4q12) is the predominant mediator of vascular endothelial sion of mTOR pathway components mTOR and p70S6K. growth factor-Stimulated endothelial cell functions, including KDR copy number was compared with expression of a broad cell migration, proliferation, Survival, and enhancement of panel of proteins screened by reverse phase protein array for vascular permeability. (Terman et al., 1991; Bernatchez et al., various cell lines. 1999). VEGFR-2 exhibits robust protein-tyrosine kinase 0040 FIG. 13 shows that VEGF increased tumor cell sur activity in response to the binding of vascular endothelial vival in the presence of erloninib and axitinib reversed the growth factor (“VEGF) ligand (Waltenberger et al., 1994). effect. HCC827 cells, which harbor the EGFR activating 0047. In human epithelial tumors, including lung, vascular mutation, were treated with VEGF and with or without the endothelial growth factor-2 (“VEGFR-2” or noted VEGFR TKI axitinib. After 24 hours, increasing concentra as'VEGFR2) is expressed in malignant cells as well as in the tions of erlotinib were added to the cells. endothelial cell of tumor vasculature. Furthermore, in non 0041 FIG. 14 shows that patients with EGFR-driven can small cell lung carcinoma (“NSCLC), VEGFR-2 is overex cer that were treated with erlotinib did worse when they had pressed in malignant cells of tumor tissues and associated high vs. low levels of KDR (P=0.001). This analysis was with a poor outcome (Ishii et al., 2004; Ludovini et al., 2004: performed on clinical specimens from the BATTLE clinical Seto et al., 2006; Carrillo de Santa Pau et al., 2009: Donnem trial. et al., 2009). Moreover, tumor cell expression of VEGFR-1 US 2013/023051 1 A1 Sep. 5, 2013

can drive tumor cell invasiveness and promote hypoxia-inde promote the Survival and invasiveness of cancer cells (Kijima pendent upregulation of hypoxia inducible factor-1C. (HIF et al., 2002). There are a number of drugs in clinical use or 1C) (Nilsson et al., 2010; Roybal et al., 2010). EGFR (“epi development that inhibit KIT, including imatinib and Suni dermal growth factor receptor) is a cell surface receptor tinib. activated by binding of its specific ligands, including epider 0051 Kinase insert domain receptor (“KDR), a VEGF mal growth factor and transforming growth factor C. receptor, is a type III and is also ("TGFC). Upon activation by its growth factor ligands, known as vascular endothelial growth factor receptor 2 EGFR undergoes a transition from an inactive monomeric (“VEGFR-2'). KDR also refers to the human gene encoding form to an active homodimer. In addition to forming the receptor. KDR has also been designated as CD309 (cluster homodimers, EGFR may pair with another member of the of differentiation 309). KDR is also known as Flk1 (Fetal ErbB receptor family, such as ErbB2/Her2/neu, to create an Liver Kinase 1). As described herein, VEGFR-2/KDR is a activated heterodimer. Mutations of EGFR or amplification known endothelial target also expressed in NSCLC tumor can lead to its constant activation, resulting in uncontrolled cells. As described in Example 1 below, the association cell division, a predisposition of cancer. Consequently, muta between alterations in the KDR gene and clinical outcome in tions and amplifications of EGFR have been identified in patients with resected NSCLC (n=248) was investigated. several types of cancer, including lung cancer, glioblastoma KDR copy number gains (CNGs), measured by quantitative multiforme, and renal cancer, and have been associated with PCR and fluorescence in situ hybridization, were detected in improved clinical benefit for patients receiving EGFR inhibi 32% of tumors and were associated with significantly higher tors, such as erlotinib or gefitinib (Paez et al., 2004; Lynch et KDR protein and higher microVessel density than tumors al., 2004; Mok et al., 2009). While these patients may have without CNGs. KDR CNGs were also associated with sig improved responses to EGFR inhibitors, tumors eventually nificantly increased risk of death (HR=5.16; P=0.003) in become resistant. One mechanism for developing resistance patients receiving adjuvant platinum-based chemotherapy, is through amplification of the MET receptor tyrosine kinase but no differences were observed in patients not receiving (Engelman et al., 2007), which provides a “bypass for acti adjuvant therapy. To investigate potential mechanisms for Vating signaling pathways in the cancer cell even when EGFR these associations, NSCLC cell lines were assessed and it was is blocked. There is a need to identify other potential "bypass” found that KDR CNGs were significantly associated with in pathways that can be blocked with drug treatment to prevent vitro resistance to platinum chemotherapy, as well as or overcome EGFR inhibitor drug resistance. increased levels of nuclear HIF-1C. in both NSCLC tumor 0048 Generally, growth factors are polypeptides involved specimens and cell lines (C. is also noted sometimes herein as in the regulation of cell growth and differentiation, such as, alpha and B as beta, etc). Furthermore, KDR knockdown during embryonal development, in wound healing, in experiments using Small interfering RNA reduced platinum hematopoiesis, in the immune response, as well as in several resistance, cell migration, and HIF-1C. levels in cells bearing adverse reactions, including malignancies. As such, platelet KDR CNGs, providing evidence for direct involvement of derived growth factor (“PDGF) was originally found to pro KDR. No KDR mutations were detected in exons 7, 11, and mote cell growth and division, particularly in fibroblasts and 21 by PCR-based sequencing; however, two variant geno smooth muscle cells. Subsequently, however, PDGF has been types SNPs were associated with favorable OS in patients shown to be synthesized by a large number of different nor with adenocarcinoma. Cells with KDR CNG were also more mal cells as well as transformed cell types. PDGF acts by sensitive to inhibition with drugs inhibiting VEGFR-2, such binding to the PDGF receptor tyrosine kinases (PDGFRs), as Sunitinib, and cells with KDRCNG became more resistant including PDGFR-alpha. PDGFRs are currently known to to EGFR inhibitors after treatment with VEGF. Based on this, play a significant role in blood vessel formation orangiogen KDRCNG can promote a more malignant phenotype, includ esis and have been implicated in promoting tumor growth in ing increased chemoresistance, angiogenesis, and HIF-1C. different types of cancers, including lung cancer (Ballas et al., levels. Furthermore, KDRCNG can be a useful biomarker for 2011). There are a number of drugs that block PDGFRs. identifying patients at high risk for recurrence after adjuvant including imatinib and Sunitinib. There are currently no vali therapy, or that are more likely to be resistant to chemo dated markers for identifying which patients are likely to therapy, two groups that may benefit from VEGF or benefit from these drugs. VEGFR-2 blockade. KDR CNG may also identify patients 0049. The c-Kit protein is an RTK and is often designated more likely to benefit from VEGF or VEGFR-2 blockade, or as KIT in the literature together with a variety of other pos that might be resistant to EGFR inhibitors. sible variations, including, but not limited to, c-, kit, KIT, 0052. The KDR gene is adjacent to PFGFR and KIT, c-Kit, and c-KIT. Likewise, the gene encoding c-Kit is often receptor tyrosine kinase (“RTK) genes that are often co designated in the literature as kit or c-kit. Moreover, as with amplified as part of an amplicon. Multiple RTKs can interact protein designations, the terms c-kit, c-KIT, KIT, kit, and to drive the malignant phenotype in different cancers (Nilsson c-Kit can be associated with the gene that encodes the protein et al., 2010; Xu et al., 2010). Hence, the assessment of CNGs and variations thereof. Therefore, as used herein, any one of a of one or more of the three RTKs in the amplicon (KDR, number of possible variations of the term designating the KIT PDGFR, and KIT) may be useful to predict whether a patient protein and the gene encoding this protein can and may be may benefit from drugs targeting one or more of these RTKs, used interchangeably herein. alone or in combination. 0050. Furthermore, the protein-tyrosine kinase KIT is also 0053 Selective inhibitors are defined as those that have an the transmembrane receptor for (SCF). SCF, ICso value against the target kinase that is less than about/10, also known as “steel factor.” “c-kit ligand, or “CD117 is a and preferably less than about /20 the ICso value against a polypeptide that activates bone marrow precursors of a num non-target enzyme. In addition, inhibitors that are selective ber of blood cells. However, SCF's receptor (c-Kit) is also for a specific target kinase are defined as having a selectivity present on tumor cells including lung cancer cells and can ratio of at least about 10, and more preferably at least about US 2013/023051 1 A1 Sep. 5, 2013

40, of target inhibition over off-target inhibition. Bevaci (FISH), having fluorescently labeled nucleic acid probes or Zumab is an example of a selective VEGF/VEGFR inhibitor fluorescently labeled probes comprising nucleic acid analogs used in the present invention. Dual inhibitors are defined as can be used to determine the CNGs. Alternatively, poly those that inhibit two or more targets in a selective manner merase chain reaction, a nucleic acid sequencing assay, or a relative to non-target enzymes. Imatinib is an example of a nucleic acid microarray assay may be used. dual inhibitor used in the present invention. 0058. In general, in situ hybridization includes the steps of 0054 As provided herein, copy number gain (“CNG. or fixing a biological sample, hybridizing one or more chromo as referred to in the plural, “CNGs”) of certain genes are somal probes to target DNA contained within the fixed associated with increased likelihood of relapse in cancer sample, washing to remove non-specifically bound probe, patients receiving adjuvant therapy and/or chemotherapy. and detecting the hybridized probe. The in situ hybridization Specifically, the CNG of genes, such as KDR, PDGFR, and can also be carried out with the specimen cells from the KIT, can serve as biomarkers (also referred to herein as biological sample in liquid Suspension, followed by detection “markers’) alone or in combination with other biomarkers. by flow cytometry. A FISH assay can be used to evaluate These biomarkers can be used to predict treatment outcomes chromosomal copy number abnormalities in a biological in cancer patients who have received adjuvant therapy and sample from a patient. FISH probes for use in the methods patients treated with different drugs. More specifically, CNG may comprise a pair of probes specific to gene or chromo of the KDR, PDGFR, and KIT genes can, each alone or in Somal locus, which may include any portion of the sequence combination, serve as markers for predicting treatment out encoding the gene. comes for patients being treated with drug therapies includ 0059. The term “patient’ means all mammals including ing, but not limited to, VEGFR2, EGFR, PDGFR, and KIT humans. Examples of patients include humans, cows, dogs, inhibitors and chemotherapy. As used herein, a CNG is a gene cats, goats, sheep, pigs, and rabbits. Preferably, the patient is copy number of 4 or greater. Patients with CNG will benefit a human. from treatments with tyrosine kinase inhibitors or other drugs 0060 A “disorder” or “disease' is any condition that targeting the VEGFR, PDGFR, or KIT pathways (e.g., an would benefit from treatment with a substance/molecule or antibody to VEGF). method of the invention. This includes chronic and acute 0.055 As noted herein, each of the methods described disorders or diseases including those pathological conditions comprises the step of: (a) providing a biological sample from that predispose the mammal to the disorder in question. Fur a cancer patient; (b) determining CNG for at least one of the thermore, non-limiting examples of disorders to be treated following genes: KDR, PDGFR, and KIT in the sample, herein include malignant and benign tumors; non- wherein a gene copy number of at least 4 for either of the and lymphoid malignancies; neuronal, glial, astrocytal, hypo KDR, PDGFR, or KIT genes is predictive of poor drug treat thalamic, and other glandular, macrophagal, epithelial, stro ment outcome; and, (c) if appropriate, administrating a drug mal, and blastocoelic disorders; and inflammatory, immuno or other therapy to the cancer patient based on the prediction logic, and other angiogenic disorders. obtained. In addition, other prognostic method steps may be 0061 The methods described herein are useful in treating used together with these methods. For example, protein cancer, particularly, metastatic disease and after adjuvant expression in the patient sample may also be determined, the therapy, such as Surgery or radiotherapy. Generally, the terms proteins including VEGFR2 and others, such as soluble “cancer and "cancerous” refer to or describe the physiologi VEGFR2 (a truncated version of VEGFR2), VEGFR1, cal condition in mammals that is typically characterized by VEGFR3, HIF-1C, EGFR, PDGFR, EZH2, and KIT. unregulated cell growth. More specifically, cancers that are 0056. For the methods provided herein, the term biologi treated using any one or more tyrosine kinase inhibitors, other cal samples refers to any biological sample obtained from an drugs blocking the receptors or their ligands, or variants individual, including body fluids, body tissue, cells, or other thereof, and in connection with the methods provided herein sources known to those skilled in the art. Also, the terms include, but are not limited to, carcinoma, lymphoma, blas “sample” and “biological sample' are used interchangeably toma, Sarcoma, , squamous cell cancer, lung cancer herein. For example, a sample can be a tissue sample, such as (including Small-cell lung cancer, non-Small cell lung cancer, a peripheral blood sample that contains circulating tumor adenocarcinoma of the lung, and squamous carcinoma of the cells, or a lung tumor tissue biopsy or resection. Other lung), cancer of the peritoneum, hepatocellular cancer, gas samples may include a thin layer cytological sample, a fine tric or stomach cancer (including gastrointestinal cancer and needle aspirate sample, a lung wash sample, a pleural effusion gastrointestinal Stromal cancer), pancreatic cancer, glioblas sample, a fresh frozen tissue sample, a paraffin embedded toma, cervical cancer, ovarian cancer, liver cancer, bladder tissue sample, or an extract or processed sample produced cancer, breast cancer, colon cancer, , from any of a peripheral blood sample. Body fluids, such as endometrial or uterine carcinoma, salivary gland carcinoma, lymph, sera, whole fresh blood, peripheral blood mono kidney or renal cancer, prostate cancer, Vulval cancer, thyroid nuclear cells, frozen whole blood, plasma (including fresh or cancer, various types of head and neck cancer, melanoma, frozen), urine, saliva, semen, synovial fluid, and spinal fluid Superficial spreading melanoma, lentigo maligna melanoma, are also suitable as biological samples. Samples can further acral lentiginous melanomas, nodular melanomas, as well as include breast tissue, renal tissue, colonic tissue, brain tissue, B-cell lymphoma (including low grade/follicular non muscle tissue, synovial tissue, skin, hair follicle, bone mar Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL: row, and tumor tissue. intermediate grade/follicular NHL; intermediate grade dif 0057 The genetic biomarkers (also referred to herein as a fuse NHL; high grade immunoblastic NHL; high grade lym “biomarker' or “marker) provided herein can be detected phoblastic NHL; high grade small non-cleaved cell NHL: using any method known in the art. For example, a biological bulky disease NHL; mantle cell lymphoma; AIDS-related sample obtained from the patient can be analyzed via in situ lymphoma; and Waldenstrom's Macroglobulinemia); hybridization, such as fluorescent in situ hybridization chronic lymphocytic leukemia (CLL); acute lymphoblastic US 2013/023051 1 A1 Sep. 5, 2013

leukemia (ALL); Hairy cell leukemia; chronic myeloblastic through cis-phosphorylation/autophosphorylation) and by leukemia; and post-transplant lymphoproliferative disorder binding to activator proteins, inhibitor proteins, or Small mol (PTLD), as well as abnormal vascular proliferation associ ecules. ated with phakomatoses, edema (Such as that associated with 006.6 Small molecules can inhibit the EGFR tyrosine brain tumors), and Meigs' syndrome. kinase, which is on the cytoplasmic side of the receptor. 0062 An effective response of a patient or a patients Without kinase activity, EGFR is unable to activate itself, “responsiveness” to treatment refers to the clinical or thera which is a prerequisite for binding of downstream adaptor peutic benefit imparted to a patient at risk for, or suffering proteins. Ostensibly, by halting the signaling cascade in cells from, a disease or disorder. Such benefit may include cellular that rely on this pathway for growth, tumor proliferation and or biological responses, a complete response, a partial migration is diminished. Gefitinib, erlotinib, lapatinib (mixed response, a stable disease (without progression or relapse), or EGFR and ERBB2 inhibitor), afatinib, and PF299804 are a response with a later relapse. For example, an effective examples of small molecule kinase inhibitors. Patients have response can be reduced tumor size or progression-free Sur been divided into EGFR-positive and EGFR-negative based vival in a patient diagnosed with cancer. upon whether a tissue test shows a mutation. EGFR-positive 0063 Treatment outcomes can be predicted, monitored patients have shown an impressive 60% response rate, which and selected and/or patients benefiting from Such treatments exceeds the response rate for conventional chemotherapy. can be identified via the methods described herein for the 0067 PDGFR inhibitors useful in connection with the tyrosine kinase inhibitors of the VEGF/VEGFR pathway or methods described herein include, but are not limited to, related pathways, including VEGFR inhibitors, drugs target imatinib, Sunitinib, axitinib, BIBF1120 (Vargatef), pazo ing VEGF or other VEGF family ligands, such as VEGF-C, panib, ponatinib, MK-2461, dovitinib, crenolanib, PP-121, EGFR inhibitors, PDGFR inhibitors, and KIT inhibitors. As telatinib, CP 673451, TSU-68, Ki8751, tivoZanib, masitinib, such, VEGFR2 inhibitors useful in identifying patients and motesanib, regorafenib, and MEDI-575. Preferred inhibitors predicting, monitoring, or selecting treatments include, but are imatinib and Sunitinib. are not limited to Sunitinib, Sorafenib, axitinib, , 0068 KIT inhibitors useful in the methods described cediranib, bevacizumab, ramucirumab, BIBF1120, afliber herein include, but are not limited to imatinib, Sunitinib, dasa cept, tivoZanib, semaxanib, dovitinib, PP-121, tellatinib, tanib, IMC-3G3, pazopanib, dovitinib, tellatinib, Ki8751, TSU-68, Ki8751, motesanib, regorafenib, Vatalanib, pona tivOZanib, masitinib, motesanib, regorafenib, Vatalanib, and tinib, and pazopanib. Preferred inhibitors are sunitinib and nilotinib. Preferred inhibitors are imatinib and Sunitinib. bevacizumab. 0064. Likewise, many therapeutic approaches are aimed at 0069 A. Detection of Copy Number Gain the EGFR. Cetuximab and panitumab are examples of mono 0070. As applied herein, CNG is when the gene copy clonal antibodies. However, the former is of the IgG1 type, number is 4 or greater. Hybridization-based assays include, the latter of the IgG2 type. Other monoclonal antibodies but are not limited to, traditional “direct probe' methods, such directed towards blocking EGFR are Zalutumumab, nimotu as Southern blots or in situ hybridization (e.g., FISH), and Zumab, and matuZumab. These monoclonal antibodies block comparative probe methods, such as Comparative Genomic the extracellular ligand binding domain. With the binding site Hybridization (CGH). The methods can be used in a wide blocked, signal molecules can no longer attach there and variety of formats including, but not limited to Substrate (e.g., activate the tyrosine kinase. Furthermore, additional EGFR membrane or glass)-bound methods or array-based inhibitors useful in connection with the methods described approaches as described below. herein include, but are not limited to, erlotinib gefitinib, afa 0071 Generally, in situ hybridization includes the steps tinib, lapatinib, neratinib, WHI-P154, OSI-420, C1-1033, and of: (1) fixation of tissue or biological structure to be analyzed: PF299804. Currently, the identification of EGFR as an (2) prehybridization treatment of the biological structure to oncongene has led to the development of anticancer thera increase accessibility of target DNA, and to reduce nonspe peutics directed against EGFR, including, but not limited to, cific binding; (3) hybridization of the mixture of nucleic acids gefitinib and erlotinib for lung cancer, and cetuximab for to the nucleic acid in the biological structure or tissue; (4) colon cancer. post-hybridization washes to remove nucleic acid fragments 0065 Tyrosine kinases area subgroup of the larger class of not bound in the hybridization; and (5) detection of the protein kinases. Fundamentally, a protein kinase is an enzyme hybridized nucleic acid fragments. The reagents used in each that modifies a protein by chemically adding phosphate of these steps and the conditions for use vary depending on groups via phosphorylation. Such modification often results the particular application. The probes are typically labeled, in a functional change to the target protein or Substrate by e.g., with radioisotopes or fluorescent reporters. The pre changing the enzyme activity, cellular location, or association ferred size range is from about 200 bp to about 1000 bp, more with other proteins. Chemically, the kinase removes a phos preferably between about 400 and about 800 bp for double phate group from ATP and covalently attaches it to one of Stranded, nick translated nucleic acids. three amino acids (serine, threonine, or tyrosine) that have a 0072. In comparative genomic hybridization methods, a free hydroxyl group. Most kinases act on both serine and first collection of (sample) nucleic acids (e.g., from a possible threonine, and certain others, tyrosine. There are also a num tumor) is labeled with a first label, while a second collection ber of kinases that act on all three of these amino acids. of (control) nucleic acids (e.g., from a healthy cell/tissue) is Generally, kinases are enzymes known to regulate the major labeled with a second label. The ratio of hybridization of the ity of cellular pathways, especially pathways involved in nucleic acids is determined by the ratio of the two (first and signal transduction or the transmission of signals within a second) labels binding to each fiber in the array. Where there cell. Because protein kinases have profound effects on a cell, are chromosomal deletions or multiplications, differences in kinase activity is highly regulated. Kinases can be turned on the ratio of the signals from the two labels will be detected and or off by phosphorylation (sometimes by the kinase itself the ratio will provide a measure of the copy number. US 2013/023051 1 A1 Sep. 5, 2013

0073. A variety of other nucleic acid hybridization for including increased chemoresistance, angiogenesis, and HIF mats are known to those skilled in the art. For example, 1.C. levels. Furthermore, KDRCNG in malignant cells repre common formats include Sandwich assays and competition or sents a predictive marker of worse outcome in patients with displacement assays. The sensitivity of the hybridization surgically resected NSCLC treated with platinum adjuvant assays may be enhanced through use of a nucleic acid ampli chemotherapy. fication system that multiplies the target nucleic acid being detected. Examples of Such systems include the polymerase (0079 Also described in Example 1, tumors with KDR chain reaction (PCR) system and the ligase chain reaction CNG in the malignant cells showed significantly higher (LCR) system. Other methods include the nucleic acid VEGFR-2 protein expression in the cytoplasm and mem sequence based amplification. brane of those cells, as well as higher MVD and larger vessel areas in the tumor stroma, compared with tumors lacking the 0074 Amplification-Based Assays KDR CNG. One possible explanation for this association is 0075 Amplification-based assays could be used to mea that tumor cell VEGFR-2 binds circulating VEGF, increasing Sure CNGs. In Such amplification-based assays, the nucleic local concentrations of the ligand which turn increases angio acid sequences act as a template in an amplification reaction genesis through effects on tumor endothelium. Another pos (e.g., Polymerase Chain Reaction (“PCR)). In a quantitative sible explanation is that VEGFR-2-overexpressing lung can amplification, the amount of amplification product will be cer cells may express increased levels of VEGF and other proportional to the amount oftemplate in the original sample. pro-angiogenic factors via upregulation of HIF-1C, which in Comparison to appropriate (e.g., healthy tissue) controls pro turn could promote autocrine or paracrine signaling that fur vides a measure of the copy number of the desired target ther increases expression. However, these mechanisms are nucleic acid sequence. Methods of "quantitative' amplifica not mutually exclusive. Furthermore, correlations between tion are well known to those of skill in the art. For example, KDRCNG and higher expression of HIF-1C. in NSCLC cell quantitative PCR involves simultaneously co-amplifying a lines and tumor specimens Support the latter hypothesis. known quantity of a control sequence using the same primers. Moreover, it has been demonstrated that activation of several This provides an internal standard that may be used to cali receptor tyrosine kinases (RTKs), including RET, VEGFR-1, brate the PCR reaction. Detailed protocols for quantitative EGFR, and PDGFR, increases HIF-1C. levels in a cell-spe PCR are provided in Innis et al. (1990). Other suitable ampli cific manner in tumors (Nilsson et al., 2010: Hirami et al., fication methods include, but are not limited to, ligase chain 2004: Phillipset al., 2005). Therefore, these data represent the reaction (LCR), transcription amplification, and self-sus first evidence suggesting that VEGFR-2 may be another RTK tained sequence replication. that plays a role in increasing the levels of HIF-1C. expression 0076 B. Detection of Expressed Protein in cancer. 0077. A polypeptide can be detected and quantified by any of a number of means known to those of skill in the art, 0080. As further provided in the study, KDR CNG in including analytic biochemical methods, such as electro malignant cells predicted a worse outcome of NSCLC phoresis, capillary electrophoresis, high performance liquid patients receiving platinum adjuvant chemotherapy after Sur chromatography (“HPLC), thin layer chromatography gical resection with curative intent, but was not predictive in (“TLC), hyperdiffusion chromatography, and the like, or patients without adjuvant therapy. As such, KDR CNG rep various immunological methods, such as fluid or gel precipi resents a biomarker for predicting resistance to adjuvant plati tation reactions, immunodiffusion (single or double), immu num-based chemotherapy in NSCLC patients and other can noelectrophoresis, radioimmunoassay (“RIA), enzyme cer patients. In the study, VEGFR-2 knockdown reduced linked immunosorbent assay (“ELISA), immunofluorescent chemoresistance and cell migration, and lowered HIF-1C. assays, western blotting, and the like. levels, using in vitro NSCLC models. Hence, the VEGFR-2 0078. As provided in Example 1 below, a high frequency blockade may sensitize tumors bearing KDR CNGs to che of KDR CNG (32%) in both major histology types of motherapy through direct effects on the tumor cells them NSCLC, adenocarcinoma and squamous cell carcinoma, by selves, in addition to its effect on tumor endothelial cells. qPCR, has been confirmed in a subset of cases by FISH in KDR CNGs can, therefore, identify a group of NSCLC lung cancer. Conversely, mutations of KDR were rarely patients that would receive greater relative benefit from com detected in NSCLC cell lines and not detected in tumor speci binations of VEGF pathway inhibitors with chemotherapy, or mens; however, two variant genotype SNPs (1416 AT/TT and VEGF pathway inhibitors alone, than patients lacking KDR -37 AG/GG) were associated with favorable OS in patients CNGS. with adenocarcinoma. KDRCNGs in tumors were associated I0081. That KDR CNG by SNP array and higher levels of with significantly higher KDR protein expression and higher VEGFR-2 expression by RPPA in a large series of NSCLC microvessel density than tumors without CNGs. Notably, cell lines correlated significantly with in vitro resistance to KDR CNG predicted worse overall survival in patients who platinum dugs (cisplatin for KDR CNG, and cisplatin and received platinum adjuvant therapy but not in untreated carboplatin for VEGFR-2 expression) provides support to the patients. To investigate potential mechanisms for these asso reported clinical observation. The increased sensitivity of the ciations NSCLC cell lines were assessed and it was found that NSCLC cell lines having KDRCNG to invitro treatment with KDRCNGs were significantly associated with in vitro resis cisplatin or carboplatin after inhibition of KDR mRNA and tance to platinum chemotherapy, as well as increased levels of protein expressions further supports the concept that KDR nuclear HIF-1C. in both NSCLC tumor specimens and cell CNG may promote platinum resistance in NSCLC. Although lines. Furthermore, KDR knockdown experiments using the exact mechanism needs to be elucidated, it is postulated small interfering RNA reduced platinum resistance, cell that the increased expression of HIF-1C. may be induced by migration, and HIF-1C. levels in cells bearing KDR CNGs, KDR CNG, and subsequent VEGFR-2 expression, in malig providing evidence for direct involvement of KDR. Tumor nant NSCLC cells may explain increased platinum resistance cell KDR CNGs promote more malignant phenotypes, in NSCLC. Interestingly, HIF-1C. has been previously asso US 2013/023051 1 A1 Sep. 5, 2013

ciated with chemoresistance in NSCLC and other solid discovered by the inventor to function well in the practice of tumors (Mi et al., 2008: Koukourakis et al., 2002; Tan et al., the invention, and thus can be considered to constitute pre 2009). ferred modes for its practice. However, those of skill in the art 0082 In NSCLC, chemoresistance to doxorubicin in cell should, in light of the present disclosure, appreciate that many lines A549 has been shown to be partially mediated by changes can be made in the specific embodiments which are enhancement of HIF-1C. mediated angiogenesis (Mi et al., disclosed and still obtain a like or similar result without 2008). In addition, in the same NSCLC cell line, HIF-1C. departing from the spirit and scope of the invention. overexpression-associated chemoresistance might be due to the negative regulation of cyclin D1, leading to the decrease Example I of the cells in S phase and Subsequent resistance of cancer cells to antimetabolic cell cycle-specific agents (Wen et al., I0085. The objective of this study was to characterize the 2010). molecular abnormalities of VEGFR-2 in epithelial malignant I0083. The variant genotypes of KDRSNPs 1416 (AT/TT) cells of NSCLC major histology types, adenocarcinoma and and -37 (AG/GG) associated with a favorable OS in the squamous cell carcinoma, and correlate with patients clini multivariate analysis. This is the first report showing associa cal characteristics. The inventors studied KDR copy number tion between KDR SNP genotypes and prognosis in lung gain (“CNG), mutation, and genetic variations in malignant cancer. In breast cancer patients the KDR SNP 1416 A/T cells of surgically resected NSCLC tumor tissues and corre genotypic variant was associated with the expression of lated the results with pathological features in NSCLC progesterone receptors, and its presence Suggested a better patients tumors and with their platinum adjuvant treatments prognosis for carriers of the Tallele (Forsti et al., 2007). and outcomes. In addition, using a series of NSCLC cell lines Interestingly, the KDR SNP 1416A/T (Q472H), a non-syn and tissue specimens, the inventors investigated molecular onymous coding polymorphism, is located in the fifth immu mechanisms associated with KDRCNG in resistance to plati noglobulin-like domain within the extracellular region of num, particularly the potential role of HIF-1, a key regulator VEGFR-2 and is important for preventing VEGF-indepen of angiogenesis in malignant tumors. dent receptor dimerization and signal transduction (Tao et al., 0086 Material and Methods 2001). The other prognostic KDR SNP in lung adenocarci I0087 NSCLCTumor Specimens and Cell Lines. noma patients, SNP-37AG/GG is located in intron 11 within I0088 Archived frozen and formalin-fixed and paraffin the protein kinase domain and has not been associated with embedded (FFPE) tissues from NSCLC patients who were any specific protein functional effect. These findings indicate surgically resected with curative intent were obtained. Tis that KDR CNG was frequently detected in NSCLC tumors sues were selected from the Lung Cancer Specialized Pro and associated with platinum resistance in vivo and in vitro, gram of Research Excellence (SPORE) tissue bank at The and may be a useful biomarker for identifying patients at high University of Texas M. D. Anderson Cancer Center (Houston, risk for recurrence after adjuvant therapy, a group that may Tex.). The tissue banking and the study were approved by the benefit from VEGFR-2 blockade. In addition, KDR SNP Institutional Review Board. The tumors were classified using genotypes correlate with outcome in patients with Surgically the 2004 World Health Organization (WHO) classification resected NSCLC tumors. This is the first report to demon system (Mountain, 1997). Two hundred forty-eight NSCLC strate the clinical importance of CNG and genetic variations specimens (159 adenocarcinomas and 89 squamous cell car of KDR in NSCLC. cinomas) were randomly selected to test KDR abnormalities. 0084. The following examples are included to demon Detailed clinical and pathologic information of the cases is strate preferred embodiments of the invention. It should be presented in Table 1. The median follow-up of the patients appreciated by those of skill in the art that the techniques was 3.53 years for those who were censored. All NSCLC cell disclosed in the examples which follow represent techniques lines utilized were authenticated by DNA-fingerprinting. TABLE 1 Clinicopathologic characteristics of non-Small lung carcinoma examined for KDR abnormalities.

All Cases Cases Tested For Cases Tested For Tested Copy Gain SNPs (N = 248) (N = 139) (N = 200) Characteristic Number (%) Number (%) Number (%) Mean Age in Years 64.6 (26.4-86.9) 64.9 (32.2-84) 63.97 (26.4-86.9) (range) Gender

Female 110 (44) 57 (41) 88 (44) Male 138 (56) 82 (59) 112 (56) Tumor Histology Adenocarcinoma 159 (64) 85 (61) 127 (64) Squamous cell 89 (36) 54 (39) 73 (36) carcinoma TNM Pathology Stage

I 120 (49) 70 (51) 86 (43) II 50 (20) 28 (20) 40 (20) III 72 (29) 39 (28) 63 (34) IV 6 (2) 2 (1) 6 (3) US 2013/023051 1 A1 Sep. 5, 2013 10

TABLE 1-continued Clinicopathologic characteristics of non-Small lung carcinoma examined for KDR abnormalities.

All Cases Cases Tested For Cases Tested For Tested Copy Gain SNPs (N = 248) (N = 139) (N = 200) Characteristic Number (%) Number (%) Number (%) Smoking status+ Current 102 (41) 52 (37) 89 (45) Former 108 (44) 64 (46) 82 (41) Never 38 (15) 23 (17) 29 (14) Neoadjuvant therapy+ No 181 (73) 115 (83) 133 (67) Yes 62 (27) 24 (17) 67 (33) Adjuvant therapy No 138 (56) 69 (50 90 (45) Yes 110 (44) 70 (50) 110 (55 * SNP, Single Nucleotide Polymorphism. +Patients who had smoked at least 100 cigarettes in their lifetime were defined as ever smokers, and smokers who quit smoking at least 12 months before lung cancer diagnosis were defined as former smokers, +All patients who received neoadjuvant and adjuvant chemotherapy received platinum (cisplatin or carboplatin), and the chemotherapy regimen most frequently administered was carboplatin-taxol combination,

0089 KDR Copy Number Analysis in Tumor Specimens. aliquots by nick translation (Vysis Nick Translation Kit, Des 0090. Two methodologies were utilized to test KDRCNG Plaines, Ill.) with SpectrumRed (SR) conjugated duTPs. in NSCLC tumor specimens: real-time quantitative PCR ethanol precipitated with herring sperm and human Cot-1, (qPCR) and fluorescence in situ hybridization (FISH). To and the pellet resuspended int-DenHyb (Insitus Biotechnolo enrich for malignant cell content for qPCR analysis, tumor gies, Albuquerque, N. Mex.). The KDR probe was validated tissues were manually microdissected from optimal cutting in normal specimens for chromosomal mapping and appro temperature (OCT) compound-embedded frozen tissue sec priate specificity and sensitivity. A similarly constructed tions for subsequent DNA extraction. Tumor DNA was probe mapping to 6p21 (VEGFA) and labeled in Spec extracted using Pico Pure DNA Extraction Kit (Arcturus, trumGreen was used as an internal control. The four-micron Mountain View, Calif.) according to the manufacturers thick sections were incubated for two hours to overnight at instructions. DNA samples with proportions of microdis 56°C., deparaffinized in Citri-Solv (Fisher, Waltham, Mass.), sected tumor cell greater than 70% were qualified for qPCR and washed in 100% ethanol. The slides were sequentially analysis. KDR gene copy number was detected by real-time incubated in 2x saline-sodium citrate buffer (SSC) at 75° C. quantitative PCR (qPCR) using the ABI 7300 real time PCR for 18-23 min, digested in 0.5 mg/mL proteinase K/2xSSC at system (Applied Biosystems, Foster City, Calif.). The prim 45° C. for 18-23 min, washed in 2XSSC for 5 min, and ers used to amplify KDR were KF-5'-GACACACCCTCAG dehydrated in ethanol. Probe was applied to the selected GCTCTTG-3' (SEQ ID NO: 1) and KR-5'-ACTTTTCAC hybridization area using 25-100 ng of KDR per 113 mm CGCCTGTTCTC-3' (SEQ ID NO:2). Each PCR was area, which was covered with a glass coverslip and sealed performed using Power SYBR Green PCR Master Mix (Ap with rubber cement. DNA denaturation was performed for 15 plied Biosystems, Foster City, Calif.) at 50° C. for 2 min and min at 85°C. and hybridization was allowed to occurat 37°C. 95°C. for 10 min followed by 40 cycles at 95°C. for 15s and for 36-48 hours. Post-hybridization washes were performed 60° C. for 1 min. B-Actin was introduced as the endogenous sequentially with 2xSSC/0.3% Nonidet P-40 (NP40) (pH reference gene and TaqMan Control Human Genomic DNA 7.0–7.5) at 72° C. for 2 minand 2xSSC for 2 min, followed by (Applied Biosystems, Foster City, Calif.) was amplified as a dehydration in ethanol. Chromatin was counterstained with standard control for calibration. All sample and standard DAPI (0.3 g/mL in Vectashield mounting medium, Vector DNA reactions were set in triplicate to gauge reaction accu Laboratories). Gene copy number analysis was done in racy. The target gene copy number was quantified using the approximately 50 nuclei per tumor in at least four areas, and comparative C, method. Gene copy number of greater than 4 the selection of the area was guided by images captured in the was considered as CNG, as previously reported. H&E-stained section. Greater than two gene copies per cell 0091 KDR copy number analysis in NSCLC malignant on average was considered as CNG. tumor cells was also performed using a dual-color FISH 0092. KDR Copy Number and VEGFR-2 and HIF-1C. assay. The KDR probe was prepared from the BAC clone Expression Analyses in Cell Lines. RP11-21A18 obtained from CHORI (Oakland, Calif.). The 0093. Whole-genome SNP array profiling was performed following set of primers was used to confirm the inclusion of in 75 NSCLC cell lines using the Illumina Human1 M-Duo the sequences of interest by touchdown PCR: 5'-TGAGACT DNA Analysis BeadChip (Illumina, Inc., San Diego, Calif.). TGAGCAATCACTAGGCT-3' (SEQ ID NO:3) and Prior to analysis, SNP data were normalized to the regional 5'-TAACCAAGGTACTTCGCAGGGATT-3' (SEQ ID baseline copy number to account for aneuploidy. For NO:4). DNA was purified from a single-cell colony (Qiagen VEGFR-2 reverse phase protein array (RPPA) analysis per QIAamp DNA Mini Kit) and amplified (Qiagen repli-G kit) formed in 63 NSCLC cell lines, protein lysate was collected per the manufacturer's instructions. DNA was labeled in 1 Jug from sub-confluent cultures after 24 hours growth in media US 2013/023051 1 A1 Sep. 5, 2013

with 10% fetal bovine serum (FBS) and assayed by RPPA as from 0 to 120 Lumol/L for cisplatin and 0 to 200 umol/L for previously described (Cheng et al., 2005; Byers et al., 2009). carboplatin. After 72 h of drug exposure, 20 uL of MTS Cisplatin and carboplatin sensitivity was determined by MTS solution was added per well. Cells were incubated for 1-4 (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphe hours at 37° C. and read at a wavelength of 490 nm. nyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) assay for 0100. The cell migration assay using NSCLC cell lines each cell line and the concentration required for 50% growth was performed (Nilsson et al., 2010). A total of 700 mL of inhibition (ICs) was determined. MTS assays were repeated serum-free RPMI with or without VEGF-A (50 ng/mL) was at least three times for each cell line and the mean ICso value added to the lower compartment of the 24-well transwell used for analysis. For HIF-1 expression analysis, the cells migration inserts (8.0 um pore size: BD Biosciences, NJ, were serum-starved for 24 h and stimulated with 50 ng/mL USA). Cells (5x10) were added to the upper chambers and VEGF-A (R&D Systems, MN, USA). Cells were incubated incubated for 24 h. Cells in the upper compartment were in normoxia and protein lysates were collected after 8 h. removed by mechanical scraping, and cells that migrated to HIF-1C, ELISA (R&D Systems, MN, USA) was performed the underside of the membrane were stained and counted in a according to the manufacturers instructions. light microscope using a 40x magnification, as previously 0094) Microvascular Density (MVD), VEGFR-2 and HIF described. 1.C. Expression Analyses in Tumors. 0101 KDR Mutation and SNPs Genotyping Analyses. 0095. Histology sections were incubated at room tempera ture with primary antibodies against VEGFR-2 (dilution 0102) For KDR mutation analysis in NSCLC cell lines, 1:50, Abcam, Cambridge, Mass.) for 90 min, CD34 (dilution exons 7, 11, 21, 26, 27 and 30 were examined using PCR 1:100, Lab Vision, Fremont, Calif.) for 35 min, and HIF-1C. based sequencing and intron-based PCR primers. Primer (dilution 1:100, Novus Biologicals, Littleton, Colo.) for 65 sequence for KDR mutation detection were as follows: Ex7F, min. Tissue sections were then incubated with the secondary 5'-TTTGGAAGTTCAGTCAACTC-3' (SEQ ID NO:5), antibody (EnVision Dual Link+; DAKO, Carpinteria, Calif.) Ex7R, 5-ATCTCACTTGTCAAGGCACAG-3 (SEQ ID for 30 min, after which diaminobenzidine chromogen was NO:6); Ex11F, 5'-TGCGCTGTTATCTCTTTCTT-3' (SEQ applied for 5 min. ID NO:7), Ex11R, 5'-AATCTCCAATATGCCTCACA-3' 0096 Protein expression was quantified by immunohis (SEQID NO:8); Ex21F,5'-TTGATGTCCTCCTTGTCTGC tochemistry using light microscopy with a 200x magnifica 3' (SEQ ID NO:9), Ex21R, 5'-CATGCAGGAAGCAC tion by two observers who were blinded to the clinical and TAGCC-3' (SEQ ID NO:10); Ex26F, 5'-CAGCATTCAG other molecular variables. Tissue samples were analyzed for GAAGAAAGAGG-3' (SEQ ID NO:11); Ex26R, VEGFR-2 expression in the cytoplasm and membrane of 5'-GCTTCTTGGATGGAGGTGAC-3 (SEQ ID NO:12); malignant cells, and for HIF-1C. in the nucleus. A 4-value Ex27F, 5'-AAGCCATAACAACAGTCTTCTGTG-3 (SEQ intensity score (0, 1+, 2+, 3+) was used and the percentage ID NO:13), Ex27R, 5'-GAGATGGCCTTGAAGTCACC-3' (0% to 100%) of the extent of reactivity. The final score was (SEQ ID NO:14); Ex30-1F, 5'-CTGCCAACTC obtained by multiplying the intensity and extent-of-reactivity CTTTGTTTGC-3' (SEQ ID NO:15); Ex30-1R, 5'-CG values (range, 0 to 300). MVD was assessed by AriolR 2.0 GTTTGCACTCCAATCTCT-3' (SEQ ID NO:16); Ex30-2F, Image System (AriolR, Genetix, San Jose, Calif.) using the 5'-AAGGCTCAAACCAGACAAGC-3' (SEQ ID NO:17), criteria of Weidner et al. (1991). The areas of highest neovas Ex30-2R, 5-TCATGTGATGTCCAGGAGTTG-3' (SEQ ID cularization were identified as the regions of invasive carci NO:18). noma with the highest numbers of discrete microVessels (0103). Each PCR was done using HotStar Taq Master Mix stained for CD34. Any brown-stained endothelial cell or (Qiagen, Valencia, Calif.) for 40 cycles at 94° C. for 30s, 59° endothelial cell cluster that was clearly separate from adja C. for 30s, and 72°C. for 30s, followed by a 7-min extension cent microVessels, tumor cells, and other connective tissue at 72° C. Mutation and SNP genotyping were performed elements was considered a single, countable microVessel. As using the ABI Prism 7900 Sequence Detection System (Ap previously published (Weidner et al., 1991), the numbers of plied Biosystems, Foster City, Calif.). SNP genotyping was CD34-positive vessels were counted in three selected performed by laboratory personnel blinded to patient status, hotspots consisting of a 200x magnification field (0.6 mm and all procedures were repeated on a randomly selected 5% field area), and the MVD and vessel areas were defined as the of the samples in order to validate the genotyping accuracy. mean count of microVessels or vessel area (mm) per 0.6- 0104 Statistical Analysis. mm field area. 0105 Demographic and clinical information were com 0097 Small Interfering RNA (siRNA) Transfection, Plati pared using the Chi-square or Fisher exact tests for category num Cytotoxicity, and Cell Migration Assays in Cell Lines. variables, and Wilcoxon rank-Sum or Kruskal-Wallis tests for 0098 NSCLC cells were transfected with siRNA target continuous variables. The distributions of overall survival ing KDR and control siRNA (OriCiene Technology, Md., (OS) and recurrence-free survival (RFS) were estimated by USA) at a final concentration of 10 nM using Lipofectamine the Kaplan-Meier method and compared between groups RNAiMAX (Invitrogen, CA, USA) according to the manu using the log-rank test. Cox proportional hazard models were facturers instructions. Medium was replaced after 24 h. To used for regression analyses of Survival data and conducted verify the knockdown efficiency, mRNA and protein of trans on OS defined as time from Surgery to death or last contact, fected cells were collected for real-time RT-PCR and Western and on RFS defined as time from surgery to recurrence or last blot analyses. contact. Follow-up time was censored at 5 years. For the 0099. The assessment of invitro resistance to cisplatin and correlation analysis of KDR CNG in NSCLC cell lines using carboplatin was determined by the MTS assay. NSCLC cell the whole-genome SNP arrays data with cisplatin sensitivity, lines were seeded in octuplicate at a density of 2,000 per well the Wilcoxon rank sum test was used. The NSCLC cell lines in 96-well plates. The following day, cells were treated with RPPA data was quantified using the SuperCurve method, cisplatin and carboplatin at various concentrations ranging which detects changes in protein level as previously reported. US 2013/023051 1 A1 Sep. 5, 2013

0106 Results 0114. These data suggest that KDR CNG in malignant 0107 KDR Gene CNG Analysis. cells may represent a predictive marker of worse outcome in 0108. In epithelial malignant NSCLC cells microdis patients with surgically resected NSCLC treated with plati sected from tumor tissues, KDR CNG was detected in 45 num-based adjuvant chemotherapy. (32%) of 139 tumors examined. Similar frequency of KDR 0115 The impact of neoadjuvant chemotherapy on KDR CNG was found in adenocarcinoma (26/85, 31%) and squa CNGs was also examined. The platinum neoadjuvant-treated mous cell carcinoma (19/54, 35%) histologies (P=0.572). tumors (33%, 8/24) had similar frequency of KDR CNGs The range of increased KDR copy numbers was from 4 to 11 than cases without neoadjuvant therapy (32%, 37/115). gene copies. None of 15 normal tissue samples adjacent to the 0116 KDR CNG and VEGFR-2 Protein Levels and Cor NSCLC tested showed KDR CNG. To confirm KDR CNG relation with Platinum Resistance in Cell Lines. results by qPCR, 20 tumor specimens with KDR CNG by 0117. The association detected between KDR CNG and qPCR were examined by FISH. KDR copy gains in the malig worse outcome in patients treated with platinum adjuvant nant cells were confirmed by FISH in all 20 NSCLC speci therapy prompted us to examine the correlation between mens detected by qPCR (FIG. 1A). KDR gain and VEGFR-2 protein levels in NSCLC cell lines 0109 Correlation Between KDR CNG and VEGFR-2 with in vitro resistance to platinum drugs. KDR CNG was Protein Expression and MVD. assessed by SNP array analysis in 75 NSCLC cell lines. Cell 0110. To assess the immunohistochemical expression of lines with KDR copy gains of 6-9 copies or 210 copies above VEGFR-2 in NSCLC malignant cells and the MVD (CD34) the regional baseline copy number were identified. Nineteen in lung tumor tissue stroma, 52 lung tumor specimens with (25%) cell lines showed KDRCNG defined as 26 copies. Of whole histologic sections from FFPE tissues were selected. these, three (4%) cell lines contained high-level gains (210 Of these, 26 cases had KDR CNG and 26 cases did not. copies), and 16 (21%) had CNG where gene copy number was VEGFR-2 protein expression was present both in the cyto between 6 and 9. Of interest, cisplatin sensitivity in cell lines plasm and membrane of malignant cells as well as in vessel with 26 KDR copies demonstrated significantly more resis endothelial cells (FIG. 1B). 0111 Levels of VEGFR-2 expression in cytoplasm and in tance to cisplatin (P=0.0179) (FIG. 4A). membrane were associated with KDR CNG in malignant 0118. Then, the expression of VEGFR-2 protein in a panel cells of NSCLC. Tumors with KDR CNG showed signifi of 63 untreated NSCLC cell lines was correlated by RPPA cantly higher cytoplasmic (P=0.013) and membrane (P=0. with each cell line's sensitivity to cisplatin or carboplatin. 009) VEGFR-2 protein expression in the malignant cells Higher VEGFR-2 expression levels were significantly asso ciated with resistance to both cisplatin (FIG. 4B) and carbo (FIG.1C), and higher MVD (P=0.018) and larger vessel areas platin by Pearson correlation. The correlation coefficient (r) (P=0.033) in the tumor stroma than cases without KDRCNG between VEGFR-2 expression and the concentration of cis (FIGS. 2A and 2B). platin and carboplatin required to inhibit cell growth by 50% 0112 Association Between Tumor KDR CNG, Clinico (ICs) were 0.346 (P=0.005) and 0.319 (P=0.011), respec pathologic Features, and Clinical Outcome. tively. 0113. When KDR CNG was correlated with patients clinicopathologic features, no correlation with tumor histol 0119) Effect of KDR Knockdown on Platinum Sensitivity ogy, Smoking status, and tumor stage was found. Interest and Cell Migration in Cell Lines. ingly, in the multivariate analysis after adjusting for stage and I0120) To investigate the role of KDRCNG and VEGFR-2 adjuvant therapy, KDR CNG was associated with poor OS overexpression in resistance to both cisplatin and carboplatin, (HR=4.0; 95% CI, 1.76 to 9.07; P=0.001) and shortened RFS siRNA was utilized to knockdown KDR expression in H23 (HR=1.83, 95% CI, 1.02 to 3.29; P=0.044) in 115 NSCLC and H461 NSCLC cell lines, which contain 6-9 KDR gene patients who underwent Surgical resection. Strikingly, KDR copies. In both cell lines, siRNA targeting KDR significantly CNG was associated with a significantly worse OS (HR=5. decreased KDR mRNA expression by real-time RT-PCR, and 16,95%CI, 1.75 to 15.2, P=0.003) in NSCLC patients receiv VEGFR-2 expression by Western blot, compared with control ing platinum adjuvant therapy, but not in patients without cells transfected with scrambled siRNA and nontransfected adjuvant therapy (P=0.349) (FIG.3 and Table 2). cells (P<0.05; FIG. 4C). To evaluate the effect of KDR over expression on sensitivity to cisplatin and carboplatin, the TABLE 2 expression of KDR was inhibited by transfecting H23 and H461 cells with control siRNA or siRNA targeting KDR and Multivariate analysis for outcome by KDR copy gain in non-Small cell then treating the cells with increasing concentrations of the lung carcinoma (NSCLC) patients by adiuvant chemotherapy. chemotherapy drugs. Cell viability was evaluated by MTS Adjusted assay. The sensitivity of H23 cells to cisplatin (FIG. 4D) or Hazard Ratio carboplatin treatment was increased in siKDR transfected Cases N Comparison Outcome (HR)* (95% CI) P cells compared with control siRNA-transfected or untrans All 115 Gain vs. no OS 4.00 (1.76, 9.07) O.OO1 fected cells, suggesting that VEGFR-2 is contributing to patients gain chemoresistance in this model. RFS .83 (1.02, 3.29) O.044 Adjuvant 61 Gain vs. no OS 5.16 (1.75, 15.2) O.OO3 I0121 Whether VEGFR-2 could promote tumor cell therapy gain migration was investigated next. Using the Boyden chamber RFS .87 (0.9, 3.92) O.1 assay, we observed that knockdown or reduction of VEGFR-2 No 54 Gain vs. no OS .99 (0.47, 8.4) O.349 adjuvant gain expression induced by siKDR transfection significantly therapy inhibited the migration of H23 cells compared with siRNA RFT .83 (0.66, 5.05) O.243 control-transfected or untransfected cells (FIG. 4E). Cells with KDRCNGs were also more sensitive to inhibition with drugs targeting KDR, PDGFR, and KIT, such as Sunitinib. US 2013/023051 1 A1 Sep. 5, 2013

0.122 Correlation Between KDR CNG and HIF-1C. TABLE 3 Expression in Cell Lines and Tumors. Distribution of genotypes in three KDR single nucleotide (0123. The observations that KDR CNGs were associated polymorphisms (SNP) in non-Small cell lung carcinoma (NSCLC). with increased angiogenesis, chemoresistance, and migration suggested that VEGFR-2 may be impacting the HIF-1C. path KDRSNP ID in NCBI Genotype Type Case (%) way, which is known to impact each of these cellular proper 889 rS2305948 GG Wild type 155 (78) GA Variant type 41 (20) ties (Nilsson et al., 2010; Roybal et al., 2010). AA Variant type 4 (2) 1416 rS1870377 AA Wild type 128 (64) 0.124. To investigate this further, HIF-1C. levels were AT Variant type 63 (32) evaluated by ELISA in a panel of NSCLC cell lines with a TT Variant type 9 (4) range of KDR copy numbers and expression of VEGFR-2. -37 rS2219471 AA Wild type 124 (62) HIF-1C. levels were higher in cell lines with KDR CNG, and AG Variant type 68 (34) significantly (P=0.02) higher in cells with 6-9 gene copies, GG Variant type 8 (4) compared to cells with no CNG (FIG. 5A). In H23 cells, NCBI, National Center for Biotechnology Information. which have KDRCNG, stimulation with 50 ng/mL VEGF-A for 8 h induced a rise in HIF-1C. expression. Furthermore, knockdown of KDR with siRNA significantly (P=0.01) TABLE 4 reduced HIF-1C. levels (FIG. 5B). These data indicated that Multivariate analysis for overall survival in three KDR single nucleotide VEGFR-2 can regulate HIF-1C. in a ligand-dependent, but polymorphisms (SNP) in non-small cell lung carcinoma (NSCLC). hypoxia-independent, manner in NSCLC cells. Adjusted Hazard 0.125. The association between KDR CNG and HIF-1C. in KDR Ration (HR)* NSCLC clinical specimens was investigated next. Similarly Cases SNP Genotype (95% CI) P to the results in the NSCLC cell lines, tumor tissue specimens NSCLC 889 GAVA vs GG 0.92 (0.51 to 1.66) O.78 1416 ATTT vs. AA 0.59 (0.34 to 1.01) O.OS6 with KDR CNG (n=25) demonstrated a significantly (P=0. –37 AG/GG vs. AA 0.6 (0.35 to 1.03) O.62 037) higher expression of nuclear HIF-1C. expression by Adenocarcinoma 889 GAAA vs. GG 0.63 (0.24 to 1.65) O.348 immunohistochemistry than tumors without CNG (n=22) 1416 ATTT vs. AA 0.45 (0.2 to 0.99) O.048 –37 AG/GG vs. AA 0.43 (0.2 to 0.92) O.O31 (FIGS. 5C and 5D). Squamouns cell 889 GAAA vs. GG 1.16 (0.53 to 2.51) 0.713 0126 KDR Mutation and SNP Analyses. carcinoma 1416 ATTT vs. AA 0.76 (0.36 to 1.61) O468 0127. To investigate whether alterations in the KDR gene –37 AG/GG vs. AA 0.84 (0.4 to 1.78) O649 other than CNGs may impact NSCLC tumors, the inventors Adjusting for tumor stage; follow-up is censored at 5 years, assessed the KDR gene for mutations and SNPs. For KDR I0129. Furthermore, among NSCLC patients with the KDR mutation analysis in NSCLC cell lines, the inventors exam 889 GA/AA variant genotypes, those who received platinum ined 6 KDR exons shown to be mutant in adenocarcinoma neoadjuvant and/or adjuvant chemotherapy showed a signifi tumors (Ding et al., 2008; Bernatchez et al., 1999; Carrillo de cantly better OS (HR=0.22; 95% CI, 0.05 to 0.94; P=0.041) Santa Pau et al., 2009; Weidner et al., 1991: Koukourakis et than patients who did not receive chemotherapy in the mul al., 2002; Tan et al., 2009; Qi et al., 2001). In 37 tested tivariate analysis after adjusting for histology and tumor NSCLC cell lines, only two mutations in the KDR gene were stage. However, no survival benefit was found in NSCLC found, an intronic T+2A exon 11 mutation in HCC2450 and patients with KDR 889 GG wild genotype (HR=1.23; 95% a CGT946CAT point mutation in exon 21 in HCC2279. No CI, 0.64 to 2.35; P=0.538). mutation affecting exons 11 or 21 was detected in 200 I0130 Finally, all KDR SNP genotypes were compared with primary tumor expression for VEGFR-2 and MVD in 52 NSCLC tissues specimens examined. NSCLC specimens. However, no genotypes correlated with 0128. In addition, three KDR SNPs (889G/A, 1416A/T, the expression of any of these markers in NSCLC tumors. and -37A/G) were genotyped in DNA extracted from 200 NSCLC tumors and correlated with patients clinicopatho Example II logic features, including outcome (Table 3). No correlation I0131 The inventors observed that in KDR amplified cell was found between the SNP genotypes distribution and OS or lines, inhibition of the VEGFR pathway using the multitar RFS of all NSCLC patients examined. When the data were geting TKI Sunitinib (which has activity against VEGFR, analyzed by tumor histology, among the 127 lung adenocar PDGFR, and Kit) results in a decrease in cellular migration. cinoma patients examined, both KDR 1416 AT/TT (HR=0. However, imatinib, which targets BCL/ABL, Kit, and 45; 95%CI, 0.2 to 0.99; P=0.048) and -37AG/GG (HR=0.43; PDGFR, does not inhibit cellular migration, Suggesting a role for VEGFR in migration. In contrast, the VEGFR inhibitor, 95% CI, 0.2 to 0.92; P=0.031) variant genotypes were asso Sunitinib, has no effect on migration of A549 cells which do ciated with a favorable OS in the multivariate analysis after not have amplification of VEGFR. Representative data are adjusting for tumor stage and neoadjuvant therapy (FIG. 9 shown in FIG. 6. and Table 4). However, no KDR SNP genotype was associ 0.132. In lung cancer as well as in neuroblastoma cells, ated with OS in lung squamous cell carcinoma patients (FIG. multiple receptor tyrosine kinases, including VEGFR1, 9). Moreover, no genotype in the three KDRSNPs was asso EGFR, PDGFR, and RET, can drive HIF-1C. levels. There ciated with RFS in NSCLC patients divided by histology fore, whether VEGFR drives HIF-1C. expression in NSCLC type. cells with VEGFR amplification was investigated. Higher US 2013/023051 1 A1 Sep. 5, 2013

levels of HIF-1C. were observed in cell lines with VEGFR More specifically, it will be apparent that certainagents which CNGs compared to those without (FIG. 7A). H23 cells (KDR are both chemically and physiologically related may be Sub CNG+) were treated with the VEGFR inhibitor Sunitinib and stituted for the agents described herein while the same or a statistically significant decrease in HIF-1C. levels was similar results would beachieved. All such similar substitutes observed as determined by ELISA assay (FIG.7B). Imatinib, and modifications apparent to those skilled in the art are which does not inhibit VEGFR, did not affect HIF-1C. levels. deemed to be within the spirit, scope and concept of the No change in HIF-1C. levels were detected in A549 cells, invention as defined by the appended claims. which do not contain VEGF CNGs (FIG. 7C). In addition, two VEGFR amplified cell lines, H23 and Calul, were treated REFERENCES with the VEGFR pathway inhibitor bevacizumab and changes in proteins regulated by HIF-1C. were evaluated. As shown in 0.136 The following references, to the extent that they FIG. 8, multiple HIF-1C.-regulated proteins were decreased in provide exemplary procedural or other details Supplementary the presence of bevacizumab, including EZH2, Met, and to those set forth herein, are specifically incorporated herein phosphorylated Met. by reference. I0137 Ballas et al., Rationale for targeting VEGF, FGF, Example III and PDGF for the treatment of NSCLC, Onco. Targets Ther., 4:43-58, 2011. 0133. The inventors further evaluated the effect of VEGF 0.138 Bernatchez et al., Vascular Endothelial Growth Fac and VEGFR TKIs on tumor cell migration using additional tor Effect On Endothelial Cell Proliferation, Migration, NSCLC cell lines with KDR CNGs (Calu1, HCC461, and and Platelet-Activating Factor Synthesis is Flk-1-Depen H1993). Similar to the previous observations, VEGFRTKIs dent, J. Biol. Chem., 274:31047-31054, 1999. decreased tumor cell migration (FIG.10). Because the inven 0.139. Byers et al., Reciprocal Regulation of c-Src and tors found VEGFR TKIS to decrease HIF-1C. levels in STAT3 in Non-Small Cell Lung Cancer, Clin. Cancer Res., NSCLC cells with KDRCNGs, and HIF-1C. is a key regulator 15:6852-6861, 2009. of many angiogenic factors, the inventors next investigated 0140 Carrillo de Santa Pau et al., Prognostic Significance the effect of VEGFRTKIs on tumor cell secretion of cytok of the Expression of Vascular Endothelial Growth Factors ines including VEGF, PDGF, IL-8, HGF, and FGF2. H23 A, B, C, and D and their Receptors R1, R2 and R3 in tumor cells were treated with control media or media con Patients with Nonsmall Cell Lung, Cancer, 115:1701 taining the VEGFRTKI Sunitinib (1 uM) for 24 hours. Con 1712, 2009. ditioned media was collected and cytokine levels were 0141 Cheng et al., Assay of Rab25 Function in Ovarian assessed by ELISA assay. VEGFR inhibition resulted in sig and Breast Cancers, Methods Enzymol., 403:202-215, nificantly decreased levels of tumor-derived PDGF-AB/BB, 2005. IL-8, and HGF (FIG. 11). Imatinib was used as a negative 0142. Ding etal. Somatic Mutations Affect Key Pathways control as it does not inhibit VEGFR. in Lung Adenocarcinoma, Nature, 455: 1069-1075, 2008. 0.143 Donnem et al., Inverse Prognostic Impact of Angio Example IV genic Marker Expression in Tumor Cells Versus Stromal 0134. To investigate signaling pathways that may be dif Cells in Non Small Cell Lung Cancer, Clin. Cancer Res., ferentially expressed between tumor cells with or without 13:6649-6657, 2009. KDRCNGs, the inventors compared KDR copy number with 0144 Engelman et al., MET amplification leads to gefi expression of a broad panel of proteins screened by reverse tinib resistance in lung cancer by activating ERBB3 sig phase protein array (RPPA). Cell lines with high copy num naling, Science, 316:1039-1043, 2007. bers of KDR had significantly greater expression of mTOR 0.145) Ferrara et al., The Biology of Vascular Endothelial pathway components (mTOR and p70S6K). In addition, KDR Growth Factor, Endocr: Rev., 18:4-25, 1997. CNG was associated with increased levels of EGFR (FIG. 0146 Fidler et al., The Implications of Angiogenesis for 12). The inventors next evaluated whether VEGFR might the Biology and Therapy of Cancer Metastasis, Cell, promote erlotinib resistance. The inventors treated HCC827 79:185-188, 1994. cells, which harbor the EGFR activating mutation, with 0147 Folkman, Tumor Angiogenesis: Therapeutic Impli VEGF with or without the VEGFR TKI axitinib. After 24 cations, N. Engl. J. Med., 285:1182-1186, 1971. hours, increasing concentrations of erlotinib were added to 0.148. Forsti et al., Polymorphisms in the KDR and the cells. VEGF increased tumor cell survival in the presence POSTN Genes: Association with Breast Cancer Suscepti of erloninib, whereas axitinib reversed the effect (FIG. 13). bility and Prognosis, Breast Cancer Res. Treat, 101:83-93, Furthermore, inclinical specimens from the BATTLE clinical 2007. trial, patients who had EGFR-driven disease and were treated 0.149 Hanahan et al., Hallmarks of Cancer: the Next Gen with erlotinib did worse when they had high levels of eration, Cell, 144:646-674, 2011. VEGFR2, in comparison with those with low levels of 0150. Innis et al., PCR Protocols, A Guide to Methods and VEGFR2 (P=0.001; FIG. 14). Applications, Academic Press, Inc. N.Y., 1990. 0135 All of the methods disclosed and claimed hereincan 0151 Hirami et al., Relation of Epidermal Growth Factor be made and executed without undue experimentation in light Receptor, Phosphorylated-Akt, and Hypoxia-Inducible of the present disclosure. While the compositions and meth Factor-1alpha in Non-Small Cell Lung Cancers, Cancer ods of this invention have been described interms of preferred Lett., 214:157-164, 2004. embodiments, it will be apparent to those of skill in the art that 0152 Ishii et al., Enhancement of Pleural Dissemination variations may be applied to the methods and in the steps or in and Lymph Node Metastasis of Intrathoracic Lung Cancer the sequence of steps of the method described herein without Cells by Vascular Endothelial Growth Factors (VEGFs), departing from the concept, spirit and scope of the invention. Lung Cancer, 45:325-337, 2004. US 2013/023051 1 A1 Sep. 5, 2013 15

0153 Kijima et al., Regulation of cellular proliferation, lian Target of Rapamycin Signaling Pathway and Activa cytoskeletal function, and signal transduction through tion of Hypoxia Inducible Factor-1alpha, J. Biol. Chem., CXCR4 and c-Kit in small cell lung cancer cells, Cancer 280:22473-22481, 2005. Res., 62:6304-6311, 2002. (0163 Qi et al., VEGF-Induced Activation of Phosphoi nositide 3-Kinase is Dependent on Focal Adhesion Kinase, 0154 Koukourakis et al., Hypoxia-Inducible Factor Exp. Cell Res., 263:173-182, 2001. (HIF1A and HIF2A), Angiogenesis, and Chemoradio 0164 Roybal et al., miR-200 Inhibits Lung Adenocarci therapy Outcome of Squamous Cell Head-And-Neck Can noma Cell Invasion and Metastasis by Targeting Flt1/ cer, Int. J. Radiat. Oncol. Biol. Phys., 53:1192-1202, 2002. VEGFR1, Mol. Cancer. Res., 9:25-35, 2010. O155 Ludovini et al., Vascular Endothelial Growth Factor, 0.165. Seto et al., Prognostic Value of Expression of Vas P53, Rb, Bcl-2 Expression and Response To Chemo cular Endothelial Growth Factor and Its Flt-1 and KDR therapy In Advanced Non-Small Cell Lung Cancer, Lung Receptors. In Stage I Non-Small-Cell Lung Cancer, Lung Cancer; 46:77-85, 2004. Cancer, 53:91-96, 2006. 0156 Lynch et al., Activating mutations in the epidermal 0166 Tan et al., The Key Hypoxia Regulated Gene CAIX growth factor receptor underlying responsiveness of non is Upregulated In Basal-Like Breast Tumours and is Asso small-cell lung cancer to gefitinib, N. Engl. J. Med., 350: ciated with Resistance to Chemotherapy, Br. J. Cancer, 100:405-411, 2009. 2129-2139, 2004. 0.167 Tao et al., Kinase Insert Domain Receptor (KDR) (O157 Miet al., RNAAptamer-Targeted Inhibition of NF Extracellular Immunoglobulin-Like Domains 4-7 Contain kappa B Suppresses Non-Small Cell Lung Cancer Resis Structural Features that Block Receptor Dimerization and tance to Doxorubicin, Mol. Ther:, 16:66-73, 2008. Vascular Endothelial Growth Factor-Induced Signaling, J. 0158 Mok et al., Gefitinib or carboplatin-paclitaxel in Biol. Chem. 276:21916-21923, 2001. pulmonary adenocarcinomas, N. Engl. J. Med., 361:947 (0168 Terman et al., Identification of a New Endothelial 957, 2009. Cell Growth Factor Receptor Tyrosine Kinase. Oncogene, 6:1677-1683, 1991. 0159 Mountain, Revisions in the International System for 0169 Waltenberger et al., Different Signal Transduction Staging Lung Cancer, Chest, 111:1710-1717, 1997. Properties of KDR and Flt1. Two Receptors for Vascular 0160 Nilsson et al., Multiple Receptor Tyrosine Kinases Endothelial Growth Factor, J. Biol. Chem., 269:26988 Regulate HIF-1alpha and HIF-2alpha in Normoxia and 26995, 1994. Hypoxia in Neuroblastoma: Implications for AntiAngio 0170 Weidner et al., Tumor Angiogenesis and Metasta genic Mechanisms of Multikinase Inhibitors. Oncogene, sis Correlation in Invasive Breast Carcinoma, N. Engl. J. 29:2938-2949, 2010. Med., 324:1-8, 1991. 0171 Wen et al., Suppression of Cyclin D1 by Hypoxia 0161 Paez et al., EGFR mutations in lung cancer: corre Inducible Factor-1 Via Direct Mechanism Inhibits the Pro lation with clinical response to gefitinib therapy, Science, liferation and 5-fluorouracil-induced of A549 304:1497-1500, 2004. Cells, Cancer Res., 70:2010-2019, 2010. 0162 Phillips et al., Epidermal Growth Factor and 0172 Xu et al., Epidermal Growth Factor Receptor Regu Hypoxia-Induced Expression of CXC Chemokine Recep lates MET Levels and Invasiveness Through Hypoxia-In tor 4 on Non-Small Cell Lung Cancer Cells is Regulated by ducible Factor-1alpha In Non-Small Cell Lung Cancer The Phosphatidylinositol 3-Kinase/PTEN/AKT/Mamma Cells, Oncogene, 29:2616-2627, 2010.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 18

SEO ID NO 1 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Synthetic Primer <4 OOs SEQUENCE: 1

gacacac cct caggct cittg

SEO ID NO 2 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Synthetic Primer

<4 OOs SEQUENCE: 2

acttitt cacc gcctgttctic US 2013/023051 1 A1 Sep. 5, 2013 16

- Continued

<210s, SEQ ID NO 3 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 3 tgagacittga gcaatcacta ggct 24

<210s, SEQ ID NO 4 &211s LENGTH: 24 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 4 talaccalaggt actitcgcagg gatt 24

<210s, SEQ ID NO 5 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 5 tittggaagtt cagt caactic

<210s, SEQ ID NO 6 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 6 atct cacttg tdaaggcaca g 21

<210s, SEQ ID NO 7 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OO > SEQUENCE: 7 tgcgctgtta t ct ctitt citt

<210s, SEQ ID NO 8 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 8 aatcto caat atgcct caca

<210s, SEQ ID NO 9 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence US 2013/023051 1 A1 Sep. 5, 2013 17

- Continued

FEATURE: OTHER INFORMATION: Synthetic Primer

SEQUENCE: 9 ttgatgtc.ct c cttgtctgc

SEQ ID NO 10 LENGTH 19 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer

SEQUENCE: 1.O

Catgcaggaa gCactago C 19

SEQ ID NO 11 LENGTH: 21 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer

SEQUENCE: 11

Cagcattcag gaagaaagag g 21

SEQ ID NO 12 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer

SEQUENCE: 12 gctt Cttgga tiggaggtgac

SEQ ID NO 13 LENGTH: 24 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer

SEQUENCE: 13 aagc cataac aacagt ctitc tdtg 24

SEQ ID NO 14 LENGTH: 2O TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic Primer

SEQUENCE: 14 gagatggcct taagt cacc US 2013/023051 1 A1 Sep. 5, 2013

- Continued <210s, SEQ ID NO 15 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 15 ctgccaactic citttgtttgc

<210s, SEQ ID NO 16 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 16 cggtttgcac tocaat ct ct

<210s, SEQ ID NO 17 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 17 aaggct caaa C cagacaagc

<210s, SEQ ID NO 18 &211s LENGTH: 21 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic Primer <4 OOs, SEQUENCE: 18 t catgtgatgtcCaggagtt g 21

1. A method of treating a cancer patient comprising: 9. The method of claim 1, further comprising treating the (a) selecting a patient determined to have a cancer with an patient with a second anti-cancer therapy. elevated KDR, PDGFR, or KIT level; and 10. The method of claim 9, wherein the second anti-cancer (b) treating the patient with a VEGF/VEGFR, PDGFR, or therapy is not a platinum-based chemotherapeutic agent oran KIT pathway inhibitor. EGFR inhibitor. 2. The method of claim 1, wherein the elevated KDR, 11. The method of claim 1, wherein the patient has under PDGFR, or KIT level is further defined as again in gene copy gone Surgery or radiotherapy and the treatment is an adjuvant number, increased mRNA expression, or increased protein treatment. expression. 12. The method of claim 1, wherein the VEGF/VEGFR 3. The method of claim 1, wherein the elevated KDR level pathway inhibitor is ramucirumab, Sunitinib, bevacizumab, is further defined as an increased mRNA or protein level of a , BIBF1120, Sorafenib, cediranib, dovitinib, pazo KDR-regulated gene. panib, ponatinib, semaxanib, axitinib, PP-121, tellatinib, 4. The method of claim3, wherein the KDR-regulated gene TSU-68. Ki8751, tivozanib, motesanib, regorafenib, vata is HIF-1C. lanib, or vandetanib. 13. The method of claim 1, wherein the PDGFR pathway 5. The method of claim 1, wherein the cancer patient has a inhibitor is imatinib, Sunitinib, axitinib, BIBF1120, pazo NSCLC or glioblastoma. panib, pnoatinib, MK-2461, dovitinib, crenolanib, PP-121, 6. The method of claim 1, wherein the cancer is metastatic. telatinib, CP 673451, TSU-68, Ki8751, tivoZanib, masitinib, 7. The method of claim 1, wherein the patient is treated motesanib, MEDI-575, or regorafenib. with a VEGF/VEGFR pathway inhibitor. 14. The method of claim 1, wherein the KIT pathway 8. The method of claim 1, wherein the patient is treated inhibitor is imatinib, axitinib, pazopanib, dovitinib, tellatinib, with a combination of two or more VEGF/VEGFR, PDGFR, Ki8751, tivo Zanib, masitinib, motesanib, Sunitinib, 3G3, or KIT pathway inhibitors. nilotinib, dasatinib, regorafenib, or Vatalanib. US 2013/023051 1 A1 Sep. 5, 2013

15. A method of predicting sensitivity of a cancer in a (b) treating the patient determined to have a cancer that is patient to VEGF/VEGFR, PDGFR, or KIT pathway inhibi sensitive to VEGF/VEGFR, PDGFR, or KIT pathway tors comprising: inhibitors with VEGF/VEGFR, PDGFR, or KIT path (a) obtaining a sample of the cancer; and ways inhibitors. 41-42. (canceled) (b) determining the KDR, PDGFR, and KIT level in the 43. A method of determining a prognosis of a cancer sample, wherein if the KDR, PDGFR, or KIT level is patient comprising: elevated, then the cancer is predicted to be sensitive to (a) obtaining a sample of the patient's cancer; and VEGF/VEGFR, PDGFR, or KIT pathway inhibitors. (b) detecting polymorphisms at nucleotides-37 and 1416 16-28. (canceled) in the KDR gene in the cells comprising the sample, wherein the cancer is determined to have a better prog 29. A method of predicting sensitivity of a cancer in a nosis if the -37 AG/GG and (or?) 1416 AT/TT polymor patient to EGFR inhibitors or platinum-based chemotherapy phisms are present. comprising: 44. A method of treating a cancer patient comprising: (a) obtaining a sample of the cancer; and (a) determining the cancer patient’s prognosis according to (b) determining the KDR level in the sample, wherein if the claim 43; and KDR level is not elevated, then the cancer is predicted to (b) applying an aggressive anticancer therapy if the poly be sensitive to EGFR inhibitors or platinum-based che morphisms are absent. motherapy. 45. (canceled) 46. The method of claim 1 further comprising the step of 30-39. (canceled) determining the expression levels of VEGFR-2 in the biologi 40. A method of treating a cancer patient comprising: cal sample wherein the presence of VEGFR-2 is further pre (a) determining if the patient has a cancer that is sensitive dictive of poor treatment outcome. to VEGF/VEGFR, PDGFR, or KIT pathway inhibitors 47-48. (canceled) according to claim 15; and