US 201302743 15A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2013/0274315 A1 Birrer et al. (43) Pub. Date: Oct. 17, 2013
(54) PRO-ANGIOGENC GENES IN OVARAN (60) Provisional application No. 60/901,455, filed on Feb. TUMORENDOTHELIAL CELL, SOLATES 14, 2007. (71) Applicants: The University of Texas MD Anderson Publication Classification Cancer Center, Houston, TX (US); The Government of the U.S.A as (51) Int. Cl. represented by the Secretary of the CI2O I/68 (2006.01) Department of He, Rockville, MD (US) (52) U.S. Cl. CPC ...... CI2O I/6886 (2013.01) (72) Inventors: Michael J. Birrer, Mt. Airy, MD (US); USPC ...... 514/44A: 435/6.12: 506/9: 435/7.1 Tomas A. Bonome, Washington, DC (US); Anil Sood, Pearland, TX (US); (57) ABSTRACT Chunhua Lu, Missouri City, TX (US) A gene profiling signature for ovarian tumor endothelial cells is disclosed herein. The gene signature can be used to diag nosis or prognosis an ovarian tumor, identify agents to treat an (21) Appl. No.: 13/863,219 ovariantumor, to predict the metastatic potential of an ovarian tumor and to determine the effectiveness of ovarian tumor (22) Filed: Apr. 15, 2013 treatments. Thus, methods are provided for identifying agents that can be used to treat ovarian cancer, for determining the effectiveness of an ovarian tumor treatment, or to diagnose or Related U.S. Application Data prognose an ovarian tumor. Methods of treatment are also (60) Division of application No. 12/541,729, filed on Aug. disclosed which include administering a composition that 14, 2009, now Pat. No. 8,440,393, which is a continu includes a specific binding agent that specifically binds to one ation-in-part of application No. PCT/US2008/054014, of the disclosed ovarian endothelial cell tumor-associated filed on Feb. 14, 2008. molecules and inhibits ovarian tumor in the Subject. Patent Application Publication Oct. 17, 2013 Sheet 1 of 20 US 2013/02743 15 A1
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PRO-ANGOGENC GENES IN OVARAN develop chemoresistance and die as a result of their cancer. TUMORENDOTHELIAL CELL, SOLATES Thus, a need exists to identify alternative treatments for ova rian cancer. CROSS REFERENCE TO RELATED APPLICATIONS SUMMARY OF THE DISCLOSURE 0001. This is a divisional of U.S. patent application Ser. 0008. A gene profiling signature is disclosed herein that No. 12/541,729, filed Aug. 14, 2009, which is a continuation can be used to predict clinical outcome and develop therapeu in-part application of International Patent Application PCT/ tics for treating ovarian cancer in a subject. For example, the US2008/054014, filed Feb. 14, 2008, designating the United ovarian endothelial cell tumor-associated molecules identi States and published in English as WO 2008/101118, which fied by the gene profile signature can serve as prognostic claims the benefit of U.S. Provisional Application No. indicators as well as targets for specific therapeutic molecules 60/901,455, filed on Feb. 14, 2007. The entire contents of that can reduce or eliminate ovarian cancer. Thus, methods of these prior applications are incorporated herein by reference. identifying an agent for treating an ovarian tumor are pro vided. In some examples, the methods include contacting a ACKNOWLEDGMENT OF GOVERNMENT cell. Such as an ovarian tumor cell or an ovarian tumor endot SUPPORT helial cell, with one or more test agents under conditions sufficient for the one or more test agents to alter the activity of 0002 This invention was made with government support at least one ovarian endothelial cell tumor-associated mol under contract CA083639 awarded by the National Institutes ecule listed in any of Tables 1-5. The method includes detect of Health. The government has certain rights in the invention. ing the activity of the at least one ovarian endothelial cell tumor-associated molecule in the presence and absence of the FIELD OF THE DISCLOSURE one or more test agents. The activity of the at least one ovarian 0003. This disclosure relates to the field of ovarian cancer endothelial cell tumor-associated molecule in the presence of and in particular, to methods for treating ovarian cancer by the one or more test agents is then compared to the activity in targeting ovarian endothelial cell tumor-associated molecules the absence of such agents to determine if there is differential identified by an ovarian tumor endothelial cell gene expres expression of the at least one ovarian endothelial cell tumor sion profile and methods for identifying therapeutic agents. associated molecule. Differential expression of the ovarian endothelial cell tumor-associated molecule in the presence of BACKGROUND the test agent(s) indicates that the one or more test agents can be used to treat an ovarian tumor. 0004 Ovarian cancer is the fifth most common form of 0009 Methods are also provided for treating an ovarian cancer in women in the United States, accounting for three tumor. In some examples, the method includes administering percent of the total number of cancer cases and twenty-six to the subject a therapeutically effective treatment to inhibit percent of those occurring in the female genital tract. The ovarian tumor growth. In an example, the treatment includes American Cancer Society estimated that 15,310 deaths would administering a therapeutically effective amount of a specific be caused in women living in the United States in 2006. A binding agent that binds with high specificity to one of the large majority of women who die of ovarian cancer will have ovarian endothelial cell tumor-associated molecules listed in had serous carcinoma of the ovarian epithelium, a condition Tables 1, 2, 4 or 5 and alters expression or activity of the which occurs in sixty percent of all cases of ovarian cancer molecules, thereby treating the ovarian tumor in the Subject (Boring et al., Cancer J. Clin. 44: 7-26, 1994). (for example, by decreasing tumor vascular growth, tumor 0005 Women with ovarian cancer are typically asymp growth or tumor volume). In particular examples, the specific tomatic until the cancer has metastasized. As a result, most binding agent preferentially binds to and inhibits expression women with ovarian cancer are not diagnosed until the cancer or activity of one of the ovarian endothelial cell tumor-asso has progressed to an advanced and usually incurable stage ciated molecules that is upregulated in an ovarian tumor (Boente et al., Curr: Probl. Cancer 20: 83-137, 1996). Sur endothelial cell, such as Zeste homologue 2 (EZH2), EGF vival rates are much better in women diagnosed with early like domain, multiple 6 (EGFL6), tumor necrosis factor, stage ovarian cancers, about ninety percent of these women alpha-induced protein 6 (TNFAIP6), Twist homologue 1 are still alive five years after diagnosis. (TWIST1), stanniocalcin 1 (STC1), homeodomain-only pro 0006 Treatment of ovarian cancer typically involves a tein (HOP), chondroitin sulfate proteoglycan 2 (CSPG2), and variety of treatment modalities. Generally, Surgical interven plexin domain containing 1 (PLXDC1). In other particular tion serves as the basis for treatment (Dennis S. Chi & Will examples, ovarian tumor growth is inhibited by the specific iam J. Hoskins, Primary Surgical Management of Advanced binding agent preferentially binding to and inhibiting expres Epithelial Ovarian Cancer, in Ovarian Cancer 241, Stephen sion of one of the ovarian endothelial cell tumor-associated C. Rubin & Gregory P. Sutton eds., 2d ed. 2001). Treatment of molecules listed in any of Tables 1, 2, 4 or 5 which are serous carcinoma often involves cytoreductive Surgery (hys involved in angiogenesis, such as molecules involved in cell terectomy, bilateral salpingo-oophorectomy, omentectomy, proliferation, tube formation or cell motility and are upregu and lymphadenectomy) followed by adjuvant chemotherapy lated in ovarian tumor endothelial cells. with paclitaxel and either cisplatin or carboplatin (Eltabbakh, 0010 Methods are also provided for determining the G. H. & Awtrey, C. S., Expert Op. Pharmacother. 2(10): effectiveness of an agent for the treatment of an ovarian tumor 109-24, 2001). in a Subject with the ovarian tumor. In an example, the method 0007. Despite a clinical response rate of 80% to primary includes detecting expression of an ovarian endothelial cell treatment with Surgery and chemotherapy, most Subjects tumor-associated molecule in a sample from the Subject fol experience tumor recurrence within two years of treatment. lowing administration of the agent. The expression of the The overwhelming majority of subjects will eventually ovarian endothelial cell tumor-associated molecule following US 2013/02743 15 A1 Oct. 17, 2013
administration is compared to a control. Such as specific 0013 The foregoing and other features of the disclosure binding agents that bind to and inhibit one of the ovarian will become more apparent from the following detailed endothelial cell tumor-associated molecules listed in any of description of several embodiments which proceeds with ref Tables 1, 2, 4 or 5 that is upregulated in ovarian endothelial erence to the accompanying figures. tumor cells. An alteration in the expression of the ovarian endothelial cell tumor-associated molecule (such as a BRIEF DESCRIPTION OF THE FIGURES decrease in expression of a molecule upregulated in ovarian 0014 FIG. 1 is a graph illustrating the comparative fold tumor endothelial cells or an increase in expression of a change in relative expression levels between microarray data molecule downregulated in Such cells) following treatment and real-time quantitative RT-PCR data of selected genes indicates that the agent is effective for the treatment of the from the pro-angiogenic gene signature provided in Table 1. ovarian cancer in the Subject. In a specific example, the 0015 FIG. 2 is a graph illustrating protein expression in method includes detecting and comparing the protein expres ovarian endothelial cells for a subset of the proteins detected sion levels of the ovarian endothelial cell tumor-associated following staining of samples with immunofluorescently-la molecules. In other examples, the method includes detecting beled PTK2, Fyn, MMP-9, 32-arrestin, Jagged 1 and and comparing the mRNA expression levels of the ovarian PLXDC1. endothelial cell tumor-associated molecules. (0016 FIGS. 3A, 3B and 3C are digital images of siRNA 0.011 Methods of diagnosing and prognosing an ovarian mediated silencing of (FIG. 3A) EZH2. (FIG. 3B) Jagged 1 tumor (such as a tumor that overexpresses at least one of the and (FIG. 3C) protein tyrosine kinase 2 (PTK2), as assessed disclosed ovarian endothelial cell tumor-associated mol using Western blot analyses. ecules) are provided. In some examples, such methods are (0017 FIG. 3D is a graph illustrating the effect of EZH2, performed prior to the treatment methods described herein. Jagged 1 or PTK2 silencing on human umbilical vein endot However, such methods can also be used independently of the helial cell (HUVEC) tube formation. disclosed treatment methods. In particular examples, the (0018 FIG. 3E is a graph illustrating the effect of EZH2 method includes determining the metastatic potential of an silencing on HUVEC migration. ovarian tumor in a subject by detecting expression of at least 0019 FIG. 3F is a graph illustrating the effect of Jagged 1 one ovarian endothelial cell tumor-associated molecule in a silencing on HUVEC migration. sample obtained from a subject with an ovarian tumor. The at (0020 FIG. 3G is a graph illustrating the effect of PTK2 least one ovarian endothelial cell tumor-associated molecule silencing on HUVEC migration. is involved in promoting angiogenesis, such as cell prolifera 0021 FIG. 4 is a digital image illustrating down regulation tion, cell motility or tube formation, such as EZH2. The of EZH2 by mouse EZH2 siRNA in mouse ovarian endothe method further includes comparing expression of the at least lial cells. one ovarian endothelial cell tumor-associated molecule in the (0022 FIGS.5A, 5B and 6 provide graphs illustrating the sample obtained from the subject with the ovarian tumor to a therapeutic effects of siRNA-mediated EZH2 down regula control. An alteration in the expression of the at least one tion on HeyA8 (FIG. 5A) and SKOV3ip1 (FIGS. 5B and 6) ovarian endothelial cell tumor-associated molecule involved ovarian tumors. in promoting angiogenesis indicates that the Subject has an (0023 FIGS. 7A-7H illustrate EZH2 expression in human ovarian tumor with increased metastatic potential. ovarian carcinoma. FIG. 7A provides digital images repre 0012. In additional examples, methods are disclosed for sentative of human tumors with low and high EZH2 expres predicting a clinical outcome in a Subject with an ovarian sion based on immunohistochemical staining FIG. 7B pro tumor, such as advanced stage epithelial ovarian cancer. In an vides Kaplan-Meier curves of disease-specific mortality for example, the methods include detecting expression of at least patients whose ovarian tumors expressed high and low levels one ovarian endothelial cell tumor-associated molecules ofEZH2 (EZH2-T). The log-rank test (two-sided) was used to listed in Tables 1-5 or combinations thereof (such as at least 1, compare differences between the two groups. Increased at least 3, at least 5 or at least 10 of such molecules) in a EZH2-T was significantly associated with decreased overall sample obtained from the subject with the ovarian tumor. The survival (p<0.001). FIG. 7C provides digital images repre methods also can include comparing expression of the at least sentative of human ovarian tumor vasculature (arrowheads one ovarian endothelial cell tumor-associated molecule in the point to endothelial cells) with low and high immunohis sample obtained from the subject with the ovarian tumor to a tochemical staining for EZH2. FIG. 7D provides Kaplan control (Such as a normal sample or value representing Such Meier curves of disease-specific mortality of patients whose expression expected in a normal sample), wherein an alter ovarian vasculature expressed low versus high EZH2 (EZH2 ation in the expression of the at least one ovarian endothelial Endo). EZH2-Endo was predictive of poor overall survival. cell tumor-associated molecule indicates that the Subject has FIG. 7E provides digital images representative of human a decreased chance of Survival. For example, an alteration in ovarian tumors with low or high immunohistochemical stain the expression, such as an increase in the expression of EZH2 ing for VEGF. FIG.7F provides a bar graphVEGF expression indicates a poor prognosis, such as a decreased chance of was strongly associated with high EZH2-Endo (p<0.01). Survival. In one example, a decreased chance of Survival FIG. 7G provides digital images representative of human includes a Survival time of equal to or less than a year. Alter ovarian tumors with low or high immunohistochemical stain ations in the expression can be measured using methods ing for microvessel density (MVD). FIG. 7H provides a bar known in the art, and this disclosure is not limited to particular graph illustrating high MVD counts in a tumor were signifi methods. For example, expression can be measured at the cantly associated with high EZH2-Endo expression (p<0. nucleic acid level (Such as by real time quantitative poly 001). Images in panels A, C, and E were taken at original merase chain reaction or microarray analysis) or at the protein magnification x200, and in panelg at original magnification level (such as by Western blot analysis). X200. US 2013/02743 15 A1 Oct. 17, 2013
0024 FIGS. 8A-8C are graphs illustrating VEGF-in silencing decreases EZH2 expression levels. EZH2 expres creased EZH2 expression in endothelial cells. Results in sion levels were analyzed in E2F1, E2F3 and E2F5 silenced FIGS. 8A and 8B are in response to 6-hour treatments with samples using Q-RT-PCR. The fold difference in levels of EGF (25 ng/uL), VEGF (50 mg/LL), conditioned medium mRNA expression represents the mean of triplicate experi (CM) from the non-cancerous ovarian epithelial cell line ments compared to cells (A) and VEGF treated cells (B). IOSE120, two ovarian cancer cell lines OVCA420 and Error bars represents.e.m. *p-0.01. FIG. 10C is a bar graph SKOV3, and complete medium with either 10% serum (A) or illustrating the effect of VASH1 gene silencing on tube for 2% serum (B). Percent fold changes represent the mean+/-s. mation in endothelial cells. HUVECs were plated on Matrigel d. of triplicate experiments compared to untreated control after transfecting the cells with either control or human cells. *p-0.05; **p-0.01: ***p<0.001. FIG. 8A illustrates VASH1 siRNA. Vascular tube formation was evaluated by that EZH2 promoteractivity is increased in an endothelial cell microscopic observation. line in response to EGF, VEGF, and conditioned media from (0027 FIGS. 11A-11E illustrate the physical characteris ovarian cancer cell lines. EAhy926 hybridoma endothelial tics of siRNA/CH nanoparticles. FIG. 11A is a table provid cell line was cotransfected with the Renilla luciferase plasmid ing the composition of CH/TPP/siRNAs. FIG. 11B is a graph and firefly luciferase plasmid either with or without the EZH2 illustrating the mean particle size of siRNA/CH particles as promoter construct followed by treatment with EGF, VEGF measured using light scattering with a particle analyzer, and conditioned medium and promoter activity was deter showing that nanoparticles maintained 100-200 nm size up to mined. FIG. 8B illustrates that EZH2 mRNA levels are 7:1 ratio (CH:TPP). FIG. 11C is a graph illustrating that Zeta increased in HUVEC in response to EGF, VEGF, and condi potential of siRNA/CH nanoparticles showed slight positive tioned media from ovarian cancer cell lines. Cells were charge. FIG. 11D is a graph illustrating incorporation effi treated as indicated and purified RNA was used in real-time ciency of siRNA into CH nanoparticles with 3:1 ratio of quantitative RT-PCR. Control values were normalized using CH:TPP resulting in >75% incorporation efficiency. FIG. 3 housekeeping genes. FIG. 8C Pearsons analysis shows 11E is a digital image following atomic force microscopy significant correlation between EZH2 and VEGF expression (AFM) demonstrating that siRNA/CH nanoparticles were values (Log) from 29 microdissected high-grade serous pap spherical and <150 nm in size. illary ovarian adenocarcinomas. (0028 FIGS. 12A-12E illustrate incorporation, stability 0025 FIGS. 9A-9E show EZH2 gene silencing increases and intracellular uptake of siRNA/CH nanoparticles. FIG. VASH1 mRNA expression in endothelial cells. FIG.9A is a 12A is a digital image illustrating electrophoretic migration digital image of a polyacrylamide gel illustrating PCR prod of naked siRNA and siRNA/CH nanoparticles. SiRNA/CH ucts generated by as ChIP assay of EZH2 binding to human nanoparticles (open arrow) remained at top of the gel com VASH1 promoter in response to VEGF in HUVEC. Cross pared to naked siRNA (solid arrow), which migrated down linked chromatin from HUVEC was treated with (+) or with ward. FIG. 12B is a digital image illustrating electrophoretic out (-) VEGF and immunoprecipitated (IP) using EZH2 or migration of siRNA/CH nanoparticles in the presence of 50% mouse IgG antibodies. The input and immunoprecipitated serum. SiRNA/CH nanoparticles were collected at different DNA were subjected to PCR using primers corresponding to time points of incubation at 37° C. (Lane 1; naked siRNA, the 3800 to 3584 base pairs upstream of VASH1 transcription Lanes 2 to 5; siRNA/CH nanoparticles). Naked siRNA (solid start site. PCR products were examined on ethidium bromide arrow) was degraded over 12 to 24 hours in serum containing stained agarose gel. FIG.9B is a bar graph illustrating EZH2 media; whereas CH nanoparticles (open arrow) protected the mRNA levels in cells transfected with control or mouse EZH2 siRNA from degradation in serum. Increased binding effi siRNA and harvested after 72 hours. RNA was isolated and ciency of siRNA/CH nanoparticles was noted compared to subjected to real-time quantitative RT-PCR. The fold differ naked siRNA. FIG. 12C is a fluorescence microscopy digital ence in levels of EZH2 mRNA represents the mean of tripli image of HeyA8 cells after incubating either with siRNA cate experiments compared to control siRNA treated cells. alone or with siRNA/CH nanoparticles at 4°C. for 20 minutes Error bars represents.e.m. *p-0.05. FIG. 9C is a bar graph in PBS. FIG. 12D is a series of tracings from a flow cytometry illustrating the fold difference in levels of VASH1 mRNA as analysis demonstrating that uptake efficiency of nanopar compared to control siRNA treated cells. Error bars represent ticles into cells was increased by 72-fold after incubating cells s.e.m. *p-0.01. FIG.9D illustrates the effect of EZH2 gene in PBS at 4°C. for 20 minutes. FIG. 12E is graphical repre silencing on methylation status of VASH1 in VEGF-treated sentation of percentage of uptake of Alexa-555 siRNA by MOECs as detected by methylation specific PCR. The inhibi cells by flow cytometry analysis. tory units of methylated VASH1 were normalized by that of (0029 FIGS. 13 A-13E illustrate in vivo siRNA delivery the un-methylated VASH1 and represent the mean of tripli using CH nanoparticles and the distribution of siRNA follow cate experiments. FIG.9E is a digital image of a Western blot ing single intravenous injection of Alexa-555 siRNA/CH of lysate collected 48 hours after transfection of MOEC with nanoparticles in orthotopic HeyA8 tumor bearing nude mice. control, VEGF treated and mouse EZH2 siRNA treated cells. FIG. 13A provides a pair of digital images illustrating fluo 0026 FIGS. 10A-10C show E2F transcription factors rescent siRNA distribution in tumor tissue of hematoxylin increases upon VEGF treatment in MOEC. FIG. 10A is a bar and eosin, original magnification x200 (left); stained with graph illustrating expression levels of E2F transcription fac anti-CD31 (green) antibody to detect endothelial cells (right). tors in MOEC. Cells were treated with VEGF for 6 hours and FIG. 13B provides a pair of digital images of 50-um sections subjected to Q-RT-PCR. FIG. 10B provides a pair of bar stained with Cytox Green and examined with confocal graphs illustrating silencing of E2F1, E2F3 and E2F5 tran microscopy (original magnification x400) (left); lateral view scription factors by targeted siRNA in MOEC. Cells were (right). Images taken every 1 um were stacked and examined transfected with corresponding siRNAs. After 24 hours and from the lateral view. Nuclei were labeled green and fluores 48 hours, cells were collected; RNA was isolated and was cent siRNA (red) was seen throughout the section. At all time subjected to real-time Q-RT-PCR. E2F3 and E2F5 gene points, punctated emissions of the siRNA were noted in the US 2013/02743 15 A1 Oct. 17, 2013
perinuclear regions of individual cells, and siRNA was seen SEQUENCE LISTING in >80% of fields examined. (c) Western blot of lysates from orthotopic tumors collected 24, 48, 72 and 96 hours after a 0034. The nucleic and amino acid sequences listed in the single injection of control siRNA/CH or human (EZH2 Hs accompanying sequence listing are shown using standard siRNA/CH). FIG.13D provides multiple digital images illus letter abbreviations for nucleotide bases, and three letter code trating EZH2 gene silencing in HeyA8 tumor as well as tumor for amino acids, as defined in 37 C.F.R. 1.822. Only one endothelial cells. Tumors were collected after 48 hours of Strand of each nucleic acid sequence is shown, but the single injection of control siRNA/CH, or EZH2 Hs siRNA/ complementary strand is understood as included by any ref CH, or EZH2 Mm siRNA/CH and stained for EZH2 (green) erence to the displayed strand. In the accompanying sequence and CD31 (red). Images were taken at original magnification, listing: x200. FIG. 13E is a pair of graphs illustrating the effects of 0035) SEQ ID NOS: 1, 6-27, and 44-47 are nucleic acid EZH2 HS SiRNA/CH or EZH2 Mm SiRNA/CH on tumor sequences of exemplary primers. weight in mouse orthotopic tumor models. Nude mice were 0036 SEQ ID NOS: 2-5 and 28-43 are nucleic acid injected with HeyA8 or SKOV3ip 1 ovarian cancer cells and sequences of exemplary siRNAS. 1 week later, were randomly assigned (10 mice per group) to receive therapy: (1) control siRNA/CH, (2) EZH2 Hs siRNA/ DETAILED DESCRIPTION OF SEVERAL CH, (3) EZH2Mm siRNA/CH, and (4) combination of EZH2 EMBODIMENTS Hs siRNA/CH plus EZH2 Mm siRNA/CH. Mice were sacri 0037. Despite improvements in surgery and chemo ficed when any animals in control or a treatment group therapy, mortality rates in women with advanced ovarian became moribund (after 3 to 4 weeks of therapy) and mouse carcinoma have remained largely unchanged (Cannistra, N. weight, tumor weight and tumor location were recorded. Engl.J.Med. 329: 1550-1559, 1993). Therefore, novel thera Error bars represents.e.m. *p-0.05: **p<0.001. peutic strategies are needed. Growth of tumors, both at the 0030 FIG. 14 provides a series of digital images of West primary and metastatic sites, requires a blood Supply for ern blots of lysate collected 72 hours after transfection of expansion beyond 1-2 mm (Folkman, J. Nat. Canc. Inst. 82: HeyA8 cells or MOEC with control, human EZH2, or mouse 4-6, 1990). Targeting tumorangiogenesis by inhibiting endot EZH2 siRNA. helial cells that Support tumor growth is particularly promis ing because of their presumed genetic stability. The recent 0031 FIG. 15 is a pair of graphs illustrating the weight Success of a humanized monoclonal antibody bevacizumab distribution of HeyA8 and SKOV3ip1 tumors. Seven days (trade name Avastin R) against vascular endothelial growth following tumor cell injection, mice were randomly divided factor in prolonging the lives of patients with advanced colon into 4 groups (10 mice per group) to receive therapy: (1) and breast carcinoma demonstrates the promise of Such control siRNA/CH, (2) EZH2 Hs siRNA/CH, (3) EZH2 Mm approaches (Hurwitz et al., N. Engl.J. Med. 350: 2335-2342, siRNA/CH, and (4) combination of EZH2Hs siRNA/CH plus 2004 and Jain et al., Nat. Clin. Pract. Oncol. 3: 24-40, 2006). EZH2 Mm siRNA/CH. Mice were sacrificed when any ani However, the full spectrum of differences in the tumor vas mals in control or a treatment group became moribund (after culature compared to its normal counterpart is not known. 3 to 4 weeks of therapy) and tumor weight was recorded. Identification of additional targets on tumor endothelium may 0032 FIGS. 16A-16B (A) Effect of tumor (EZH2 Hs allow opportunities for developing new therapeutic siRNA/CH) or endothelial (EZH2 Mm siRNA/CH) targeted approaches to inhibit angiogenesis in a tumor-specific man EZH2 siRNA on MVD and pericyte coverage. Tumors har vested following 3 to 4 weeks of therapy were stained for 0038. In recent years, whole genome expression profiling CD31 (MVD; red) and desmin (pericyte coverage; green). All of cancer using methods such as microarray and serial analy pictures were taken at original magnification X200. The bars sis of gene expression (SAGE) have provided insight into the in the graphs correspond sequentially to the labeled columns molecular pathways involved in cancer onset and progres of images at left. Error bars represents.e.m. *p-0.05: **p<0. Sion. While selected genes in ovarian cancer vasculature have 001. (B) Effects of VASH1 gene silencing on tumor growth in been characterized, there is little information regarding glo vivo. Nude mice were injected with SKOV3ip1 ovarian can bal gene expression alterations in ovarian cancer endothe cer cells and 1 week later, were randomly divided into 5 lium. groups (10 mice per group): (1) control siRNA/CH, (2) EZH2 0039 Disclosed herein is a gene expression signature Mm siRNA1/CH, (3) EZH2 Mm siRNA2/CH (4) EZH2 Mm identifying endothelial cell tumor-associated molecules in siRNA3/CH (5) VASH1 Mm siRNA1/CH and (6) combina ovariantumor endothelial cell isolates. Endothelial cells were tion of EZH2 Mm siRNA1/CH plus VASH1 Mm siRNA/CH. purified from human ovarian tissues and invasive ovarian FIG. 16B is a bar graph illustrating the number of cells that epithelial cancers, and a gene expression profile was estab migrated in the presence and absence of VASH1 siRNA. Mice lished for ovarian tumor endothelial cells using microarray were sacrificed when any animals in control or a treatment analyses. The gene expression profile disclosed herein iden group became moribund (after 3 to 4 weeks of therapy) and tifies genes whose expression is differentially regulated in mouse weight, tumor weight and tumor location were tumor versus normal endothelial cells. This profile reveals recorded. Error bars represents.e.m. *p-0.05. distinct expression profiles for tumor endothelial cell isolates 0033 FIG. 17 illustrates the effects of EZH2 His siRNA/ as compared to non-tumor endothelial isolates. CH or EZH2 Mm siRNA/CH on proliferation. Tumors were 0040. The disclosed gene expression profile also reveals harvested following 3-4 weeks oftherapy and then stained for genes and collections or sets of genes that serve as effective proliferating cell nuclear antigen (PCNA). All images were molecular markers for angiogenesis in ovarian cancer, predict taken at original magnification x 100. The bars in the graphs clinical outcome as well as Such genes or gene sets that can correspond sequentially to the labeled columns of images at provide clinically effective therapeutic targets for ovarian left. Error bars represents.e.m. *p-0.05. cancer. This has significant implications for the treatment of US 2013/02743 15 A1 Oct. 17, 2013
ovarian cancer. For example, methods are disclosed for treat limited to, injection (Such as Subcutaneous, intramuscular, ing ovarian cancer (for example, reducing or inhibiting ova intradermal, intraperitoneal, and intravenous), oral, Sublin rian cancer growth by targeting ovarian endothelial cell gual, rectal, transdermal, intranasal, vaginal and inhalation tumor-associated molecules, such as molecules believed to be rOuteS. involved in angiogenesis). For example, molecules involved 0044 Agent: Any protein, nucleic acid molecule, com in cell motility, tube formation or cell proliferation can be pound, Small molecule, organic compound, inorganic com identified by the gene profile signature. In an example, a pound, or other molecule of interest. Agent can include a therapeutically effective amount of a specific binding agent is therapeutic agent, a diagnostic agent or a pharmaceutical administered to a subject. For example, the specific binding agent. A therapeutic orpharmaceutical agent is one that alone agent preferentially binds to one or more of the identified or together with an additional compound induces the desired ovarian endothelial cell tumor-associated molecules listed in response (such as inducing a therapeutic or prophylactic any of Tables 1, 2, 4, 5 or a combination thereof to alter the effect when administered to a subject). In a particular expression or activity of such molecule (e.g., increase expres example, a pharmaceutical agent (Such as a siRNA to any of sion or activity of a molecule that is downregulated in ovarian the genes listed in Tables 2 and Table 4) significantly reduces endothelial tumor cells or decrease expression or activity of a angiogenesis. A test agent is any Substance, including, but not molecule that is upregulated in Such cells). In one example, limited to, a protein (such as an antibody), nucleic acid mol the specific binding agent preferentially binds to one or more ecule (such as a siRNA), organic compound, inorganic com of the identified ovarian endothelial cell tumor-associated pound, or other molecule of interest. In particular examples, molecules listed in any of Tables 1, 2, 4, 5 or a combination a test agent can permeate a cell membrane (alone or in the thereofthat are upregulated in ovarian endothelial tumor cells presence of a carrier). (as indicated by a positive fold change in Table 1) to decrease 0045 Amplifying a nucleic acid molecule: To increase the expression or activity of the one or more molecules. As a number of copies of a nucleic acid molecule, such as a gene or result, ovarian cancer in the subject is thereby reduced or fragment of a gene, for example a region of an ovarian endot eliminated. In a particular example, the specific binding agent helial cell tumor-associated gene. The resulting products are is an inhibitor, such as a siRNA, of one or more of the dis called amplification products. closed ovarian endothelial cell tumor-associated molecules 0046. An example of in vitro amplification is the poly described in any of Table 1, 2, 4 or 5 whose expression is merase chain reaction (PCR), in which a biological sample upregulated in ovarian endothelial tumor cells, such as EZH2. obtained from a Subject (such as a sample containing ovarian In some examples, the specific binding agent preferentially cancer cells) is contacted with a pair of oligonucleotide prim binds to one or more of the identified ovarian endothelial cell ers, under conditions that allow for hybridization of the prim tumor-associated molecules listed in any of Tables 1,2,4, 5 or ers to a nucleic acid molecule in the sample. The primers are a combination thereof that are downregulated in ovarian extended under suitable conditions, dissociated from the tem endothelial tumor cells (as indicated by a negative fold plate, and then re-annealed, extended, and dissociated to change in Table 1) to increase expression or activity of the one amplify the number of copies of the nucleic acid molecule. or more molecules. As a result, ovarian cancer in the Subject Other examples of in vitro amplification techniques include is thereby reduced or eliminated. quantitative real-time PCR, strand displacement amplifica tion (see U.S. Pat. No. 5,744,311); transcription-free isother TERMS mal amplification (see U.S. Pat. No. 6,033,881); repair chain 0041. The following explanations of terms and methods reaction amplification (see WO 90/01069); ligase chain reac are provided to better describe the present disclosure and to tion amplification (see EP-A-320 308); gap filling ligase guide those of ordinary skill in the art in the practice of the chain reaction amplification (see U.S. Pat. No. 5,427.930); present disclosure. The singular forms “a” “an and “the coupled ligase detection and PCR (see U.S. Pat. No. 6,027, refer to one or more than one, unless the context clearly 889); and NASBATM RNA transcription-free amplification dictates otherwise. For example, the term "comprising a (see U.S. Pat. No. 6,025,134). nucleic acid molecule' includes single or plural nucleic acid 0047. A commonly used method for real-time quantitative molecules and is considered equivalent to the phrase “com polymerase chain reaction involves the use of a double prising at least one nucleic acid molecule.” The term 'or' stranded DNA dye (such as SYBR Green I dye). For example, refers to a single element of stated alternative elements or a as the amount of PCR product increases, more SYBR Green combination of two or more elements, unless the context I dye binds to DNA, resulting in a steady increase in fluores clearly indicates otherwise. As used herein, “comprises' cence. Another commonly used method is real-time quanti means “includes.” Thus, “comprising A or B.’ means “includ tative TaqMan PCR (Applied Biosystems). The 5' nuclease ing A, B, or A and B, without excluding additional elements. assay provides a real-time method for detecting only specific 0042 Unless explained otherwise, all technical and scien amplification products. The use of fluorogenic probes makes tific terms used herein have the same meaning as commonly it possible to eliminate post-PCR processing for the analysis understood to one of ordinary skill in the art to which this of probe degradation. The probe is an oligonucleotide with disclosure belongs. Although methods and materials similar both a reporter fluorescent dye and a quencher dye attached. or equivalent to those described herein can be used in the While the probe is intact, the proximity of the quencher practice or testing of the present disclosure, Suitable methods greatly reduces the fluorescence emitted by the reporter dye and materials are described below. The materials, methods, by Förster resonance energy transfer (FRET) through space. and examples are illustrative only and not intended to be Probe design and synthesis has been simplified by the finding limiting. that adequate quenching is observed for probes with the 0043 Administration: To provide or give a subject an reporter at the 5' end and the quencher at the 3' end. agent, such as a chemotherapeutic agent, by any effective 0048 Angiogenesis: A physiological process involving route. Exemplary routes of administration include, but are not the growth of new blood vessels from pre-existing vessels. US 2013/02743 15 A1 Oct. 17, 2013
Angiogenesis can occur under normal physiological condi framework regions of different light or heavy chains are rela tions such as during growth and development or wound heal tively conserved within a species. The framework region of an ing (known as physiological angiogenesis) as well as patho antibody, that is the combined framework regions of the con logical conditions such as in the transition of tumors from a stituent light and heavy chains, serves to position and align dormant state to a malignant state (known as pathological the CDRs in three-dimensional space. angiogenesis). As used herein, pro-angiogenic genes are 0054 The CDRs are primarily responsible for binding to genes that facilitate angiogenesis. Such as angiogenesis in an an epitope of an antigen. The CDRS of each chain are typi ovarian tumor. cally referred to as CDR1, CDR2, and CDR3, numbered 0049. The complex phenomenon of angiogenesis begins sequentially starting from the N-terminus, and are also typi with degradation of the basement membrane by cellular pro cally identified by the chain in which the particular CDR is teases. This allows endothelial cells to penetrate and migrate located. Thus, a VCDR3 is located in the variable domain of (process known as cell motility) into the extracellular matrix the heavy chain of the antibody in which it is found, whereas and then proliferate. In the final stages of this process, the aV, CDR1 is the CDR1 from the variable domain of the light endothelial cells align themselves to form capillary or tube chain of the antibody in which it is found. An antibody that like structures (process known as tube formation). These new binds RET will have a specific V region and the V, region structures then form a network that undergoes significant sequence, and thus specific CDR sequences. Antibodies with remodeling and rearrangement before fully functioning cap different specificities (such as different combining sites for illaries exist. Therefore, angiogenesis can be studied or iden different antigens) have different CDRs. Although it is the tified by monitoring tube formation, cell motility, and/or cell CDRs that vary from antibody to antibody, only a limited proliferation. number of amino acid positions within the CDRs are directly 0050 Antibody: A polypeptide ligand including at least a involved in antigenbinding. These positions within the CDRs light chain or heavy chain immunoglobulin variable region are called specificity determining residues (SDRs). which specifically recognizes and binds an epitope of an 0055 References to “V” or “VH’ refer to the variable antigen, Such as an ovarian endothelial cell tumor-associated region of an immunoglobulin heavy chain, including that of molecule or a fragment thereof. Antibodies are composed of an Fv, scFv, dsFv or Fab. References to “V,” or “VL refer to a heavy and a light chain, each of which has a variable region, the variable region of an immunoglobulin light chain, includ termed the variable heavy (V) region and the variable light ing that of an Fv, scFv, dsFv or Fab. (V) region. Together, the V, region and the V, region are 0056. A "monoclonal antibody' is an antibody produced responsible for binding the antigen recognized by the anti by a single clone of B-lymphocytes or by a cell into which the body. In one example, an antibody specifically binds to one of light and heavy chain genes of a single antibody have been the proteins listed in Tables 8 and 9. transfected. Monoclonal antibodies are produced by methods 0051. This includes intact immunoglobulins and the vari known to those of skill in the art, for instance by making ants and portions of them well known in the art, such as Fab' hybrid antibody-forming cells from a fusion of myeloma cells fragments, F(ab)' fragments, single chain Fv proteins with immune spleen cells. Monoclonal antibodies include (“scFv”), and disulfide stabilized Fv proteins (“dsFv'). A humanized monoclonal antibodies. schv protein is a fusion protein in which a light chain variable 0057. A "polyclonal antibody' is an antibody that is region of an immunoglobulin and a heavy chain variable derived from different B-cell lines. Polyclonal antibodies are region of an immunoglobulin are bound by a linker, while in a mixture of immunoglobulin molecules Secreted against a dsFVs, the chains have been mutated to introduce a disulfide specific antigen, each recognizing a different epitope. These bond to stabilize the association of the chains. The term also antibodies are produced by methods known to those of skill in includes genetically engineered forms such as chimeric anti the art, for instance, by injection of an antigen into a Suitable bodies (for example, humanized murine antibodies), hetero mammal (such as a mouse, rabbit or goat) that induces the conjugate antibodies (such as, bispecific antibodies). See B-lymphocytes to produce IgG immunoglobulins specific for also, Pierce Catalog and Handbook, 1994-1995 (Pierce the antigen which are then purified from the mammals Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3" Ed., S. W.H. Freeman & Co., New York, 1997. 0.058 A "chimeric antibody' has framework residues 0052 Typically, a naturally occurring immunoglobulin from one species, such as human, and CDRS (which generally has heavy (H) chains and light (L) chains interconnected by confer antigen binding) from another species, such as a disulfide bonds. There are two types of light chain, lambda murine antibody that specifically binds an ovarian endothelial and kappa. There are five main heavy chain classes (or iso cell tumor-associated molecule. types) which determine the functional activity of an antibody 0059 A “humanized' immunoglobulin is an immunoglo molecule: IgM, Ig|D, IgG, IgA and IgE. bulin including a human framework region and one or more 0053. Each heavy and light chain contains a constant CDRS from a non-human (for example a mouse, rat, or Syn region and a variable region, (the regions are also known as thetic) immunoglobulin. The non-human immunoglobulin “domains'). In combination, the heavy and the light chain providing the CDRs is termed a “donor,” and the human variable regions specifically bind the antigen. Light and immunoglobulin providing the framework is termed an heavy chain variable regions contain a "framework” region “acceptor.” In one embodiment, all the CDRs are from the interrupted by three hypervariable regions, also called donor immunoglobulin in a humanized immunoglobulin. “complementarity-determining regions' or “CDRs. The Constant regions need not be present, but if they are, they extent of the framework region and CDRs have been defined must be substantially identical to human immunoglobulin (see, Kabat et al., Sequences of Proteins of Immunological constant regions, e.g., at least about 85-90%. Such as about Interest, U.S. Department of Health and Human Services, 95% or more identical. Hence, all parts of a humanized immu 1991, which is hereby incorporated by reference). The Kabat noglobulin, except possibly the CDRs, are substantially iden database is now maintained online. The sequences of the tical to corresponding parts of natural human immunoglobu US 2013/02743 15 A1 Oct. 17, 2013
lin sequences. Humanized immunoglobulins can be ated proteins, such as any combination of those listed in Table constructed by means of genetic engineering (see for 1, such as at least 2, least 5, at least 7, at least 10, at least 20, example, U.S. Pat. No. 5,585,089). at least 30, at least 40, at least 50, at least 60, at least 70, at least 0060 Array: An arrangement of molecules, such as bio 80, at least 90, at least 100, at least 150, at least 200, at least logical macromolecules (such as peptides or nucleic acid 250, at least 300, at least 400, at least 500, at least 600, at least molecules) or biological samples (such as tissue sections), in 700, at least 800, at least 900, at least 1000, or at least 1100 addressable locations on or in a substrate. A “microarray' is sequences listed in Table 1 (for example, 2, 6, 8, 9, 10, 11, 12. an array that is miniaturized so as to require or be aided by 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, microscopic examination for evaluation or analysis. 70, 75, 80, 85,90, 95, 100,105,110, 120, 130, 140, 150, 175, 0061 The array of molecules (“features') makes it pos 225, 275,325, 350,375, 450, 550, 650, 750, 850, 950, 1050 sible to carry out a very large number of analyses on a sample or 1149 of those listed). at one time. In certain example arrays, one or more molecules 0064 Binding or stable binding: An association between (such as an oligonucleotide probe) will occur on the array a two Substances or molecules. Such as the hybridization of one plurality of times (such as twice), for instance to provide nucleic acid molecule to another (or itself), the association of internal controls. The number of addressable locations on the an antibody with a peptide, or the association of a protein with array can vary, for example from at least one, to at least 2, to another protein or nucleic acid molecule. An oligonucleotide at least 5, to at least 10, at least 20, at least 30, at least 50, at molecule binds or stably binds to a target nucleic acid mol least 75, at least 100, at least 150, at least 200, at least 300, at ecule if a sufficient amount of the oligonucleotide molecule least 500, least 550, at least 600, at least 800, at least 1000, at forms base pairs or is hybridized to its target nucleic acid least 10,000, or more. In particular examples, an array molecule, to permit detection of that binding. “Preferentially includes nucleic acid molecules, such as oligonucleotide binds' indicates that one molecule binds to another with high sequences that are at least 15 nucleotides in length, Such as affinity, and binds to heterologous molecules at a low affinity. about 15-40 nucleotides in length. In particular examples, an 0065 Binding can be detected by any procedure known to array includes oligonucleotide probes or primers which can one skilled in the art, Such as by physical or functional prop be used to detect sensitive to ovarian endothelial cell tumor erties of a target:oligonucleotide complex or a protein:anti associated molecule sequences, such as at least one of those body complex. For example, binding can be detected func listed in Table 1, such as at least 5, at least 7, at least 10, at least tionally by determining whether binding has an observable 20, at least 30, at least 40, at least 50, at least 60, at least 70, effect upon a biosynthetic process Such as expression of a at least 80, at least 90, at least 100, at least 150, at least 200, gene, DNA replication, transcription, translation, and the at least 250, at least 300, at least 400, at least 500, at least 600, like. at least 700, at least 800, at least 900, at least 1000, or at least 0.066 Physical methods of detecting the binding of 1100 sequences listed in Table 1 (for example, 2, 6, 8, 9, 10, complementary Strands of nucleic acid molecules, includebut 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,55, are not limited to, such methods as DNase I or chemical 60, 65, 70, 75, 80, 85,90, 95, 100,105, 110, 120, 130, 140, footprinting, gel shift and affinity cleavage assays, Northern 150, 175, 225, 275,325, 350, 375,450, 550, 650, 750, 850, blotting, dot blotting and light absorption detection proce 950, 1050 or 1149 of those listed). In an example, the array is dures. For example, one method involves observing a change a commercially available such as a U133 Plus 2.0 oligonucle in light absorption of a solution containing an oligonucleotide otide array from Affymetrix. (Affymetrix, Santa Clara, Calif.). (or an analog) and a target nucleic acid at 220 to 300 nm as the 0062. Within an array, each arrayed sample is addressable, temperature is slowly increased. If the oligonucleotide or in that its location can be reliably and consistently determined analog has bound to its target, there is a Sudden increase in within at least two dimensions of the array. The feature appli absorption at a characteristic temperature as the oligonucle cation location on an array can assume different shapes. For otide (or analog) and target disassociate from each other, or example, the array can be regular (such as arranged in uni melt. In another example, the method involves detecting a form rows and columns) or irregular. Thus, in ordered arrays signal. Such as a detectable label, present on one or both the location of each sample is assigned to the sample at the nucleic acid molecules (or antibody or protein as appropri time when it is applied to the array, and a key may be provided ate). in order to correlate each location with the appropriate target 0067. The binding between an oligomer and its target or feature position. Often, ordered arrays are arranged in a nucleic acid is frequently characterized by the temperature symmetrical grid pattern, but samples could be arranged in (T) at which 50% of the oligomer is melted from its target. other patterns (such as in radially distributed lines, spiral A higher (T) means a stronger or more stable complex lines, or ordered clusters). Addressable arrays usually are relative to a complex with a lower (T). computer readable, in that a computer can be programmed to 006.8 Biological activity: An expression describing the correlate a particular address on the array with information beneficial or adverse effects of an agent on living matter. about the sample at that position (such as hybridization or When the agent is a complex chemical mixture, this activity is binding data, including for instance signal intensity). In some exerted by the Substance's active ingredient or pharmacoph examples of computer readable formats, the individual fea ore, but can be modified by the other constituents. Activity is tures in the array are arranged regularly, for instance in a generally dosage-dependent and it is not uncommon to have Cartesian grid pattern, which can be correlated to address effects ranging from beneficial to adverse for one Substance information by a computer. when going from low to high doses. In one example, a specific 0063 Protein-based arrays include probe molecules that binding agent significantly reduces the biological activity of are or include proteins, or where the target molecules are or the one or more ovarian endothelial cell tumor-associated include proteins, and arrays including nucleic acids to which molecules that is upregulated in ovarian endothelial tumor proteins are bound, or vice versa. In some examples, an array cells (such as those listed in Tables 2 and 4) which reduces or contains antibodies to ovarian endothelial cell tumor-associ eliminates ovarian cancer, such as by reducing or inhibiting US 2013/02743 15 A1 Oct. 17, 2013
angiogenesis. In some examples, a specific binding agent least 85%, 90%, 95%, or 98% sequence identity to GenBank significantly increase the biological activity of one or more Accession No. AAH50524, NP 004376, or AAH96495. In ovarian endothelial cell tumor-associated molecules that is other examples, CSPG2 has a sequence that hybridizes to downregulated in ovarian endothelial tumor cells (such as AFFYMETRIX(R) Probe ID No. 204619 s at and 221731 those listed in Table 3). X a and retains CSPG2 activity (such as the capability to be 0069 Cancer: The “pathology' of cancer includes all phe expressed in ovarian tumor endothelial cells and/or modulate nomena that compromise the well-being of the subject. This tumor and/or vascular growth). includes, without limitation, abnormal or uncontrollable cell 0074 Complementarity and percentage complementarity: growth, metastasis, interference with the normal functioning Molecules with complementary nucleic acids form a stable of neighboring cells, release of cytokines or other secretory duplex or triplex when the strands bind, (hybridize), to each products at abnormal levels, suppression or aggravation of other by forming Watson-Crick, Hoogsteen or reverse Hoogs inflammatory or immunological response, neoplasia, prema teen base pairs. Stable binding occurs when an oligonucle lignancy, malignancy, invasion of Surrounding or distant tis otide molecule remains detectably bound to a target nucleic Sues or organs, such as lymph nodes, etc. "Metastatic disease' acid sequence (such as an ovarian endothelial cell tumor refers to cancer cells that have left the original tumor site and associated molecule) under the required conditions. migrate to other parts of the body for example via the blood 0075 Complementarity is the degree to which bases in stream or lymph system. one nucleic acid strand base pair with the bases in a second 0070 Chemotherapeutic agent or Chemotherapy: Any nucleic acid strand. Complementarity is conveniently chemical agent with therapeutic usefulness in the treatment of described by percentage, that is, the proportion of nucleotides diseases characterized by abnormal cell growth. Such dis that form base pairs between two strands or within a specific eases include tumors, neoplasms, and cancer as well as dis region or domain of two strands. For example, if 10 nucle eases characterized by hyperplastic growth Such as psoriasis. otides of a 15-nucleotide oligonucleotide form base pairs In one embodiment, a chemotherapeutic agent is an agent of with a targeted region of a DNA molecule, that oligonucle use in treating ovarian cancer, Such as papillary serous ova otide is said to have 66.67% complementarity to the region of rian cancer. In one example, a chemotherapeutic agent is a DNA targeted. radioactive compound. One of skill in the art can readily 0076. In the present disclosure, “sufficient complementa identify a chemotherapeutic agent of use (see for example, rity” means that a sufficient number of base pairs exist Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in between an oligonucleotide molecule and a target nucleic Harrison's Principles of Internal Medicine, 14th edition: acid sequence (such as a ovarian endothelial cell tumor-asso Perry et al., Chemotherapy,Ch. 17 in Abeloff, Clinical Oncol ciated molecule, for example any of the genes listed in Table ogy 2nd ed., 2000 Churchill Livingstone, Inc.; Baltzer and 1) to achieve detectable binding. When expressed or mea Berkery. (eds): Oncology Pocket Guide to Chemotherapy, Sured by percentage of base pairs formed, the percentage 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer Knobf, complementarity that fulfills this goal can range from as little and Durivage (eds): The Cancer Chemotherapy Handbook, as about 50% complementarity to full (100%) complemen 4th ed. St. Louis, Mosby-Year Book, 1993). Chemotherapeu tary. In general, Sufficient complementarity is at least about tic agents used for treating ovarian cancer include, but are not 50%, for example at least about 75% complementarity, at limited to, carboplatin, cisplatin, paclitaxel, docetaxel, doxo least about 90% complementarity, at least about 95% comple rubicin, epirubicin, topotecan, irinotecan, gemcitabine, iaZo mentarity, at least about 98% complementarity, or even at furine, gemcitabine, etoposide, Vinorelbine, tamoxifen, Val least about 100% complementarity. spodar, cyclophosphamide, methotrexate, fluorouracil, 0077. A thorough treatment of the qualitative and quanti mitoxantrone and vinorelbine. Combination chemotherapy is tative considerations involved in establishing binding condi the administration of more than one agent (Such as more than tions that allow one skilled in the art to design appropriate one chemotherapeutic agent) to treat cancer. oligonucleotides for use under the desired conditions is pro (0071 Chrondroitin sulfate proteoglycan 2 (CSPG2): An vided by Beltz et al. Methods Enzymol. 100:266-285, 1983, extracellular matrix component of the vitreous gel that has and by Sambrook et al. (ed.), Molecular Cloning: A Labora been reported to be an anti-cell adhesive. In particular tory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor Labora examples, expression of CSPG2 is increased in ovarian can tory Press, Cold Spring Harbor, N.Y., 1989. cer endothelial cells. The term CSPG2 includes any CSPG2 0078 Contacting: Placement in direct physical associa gene, cDNA, mRNA, or protein from any organism and that is tion, including both a solid and liquid form. Contacting can CSPG2 and is expressed and in some examples overex occur in vitro, for example, with isolated cells or in vivo by pressed in ovarian cancer endothelial cells. administering to a Subject. 0072 Nucleic acid and protein sequences for CSPG2 are 0079 Decrease: To reduce the quality, amount, or strength publicly available. For example, GenBank Accession Nos.: of Something. In one example, a therapy decreases a tumor NM 004385 and BCO96495 disclose CSPG2 nucleic acid (such as the size of a tumor, the growth of a tumor, number of sequences, and GenBank Accession Nos.: AAH50524, tumors, the metastasis of a tumor, or combinations thereof), NP 004376, and AAH96495 disclose CSPG2 protein or one or more symptoms associated with a tumor, for sequences, all of which are incorporated by reference as pro example as compared to the response in the absence of the vided by GenBank on Feb. 14, 2007. therapy (Such as a therapy administered to affect tumor size 0073. In one example, CSPG2 includes a full-length wild by inhibiting angiogenesis via administration of a binding type (or native) sequence, as well as CSPG2 allelic variants agent capable of binding to one or more of the ovarian endot that retain the ability to be expressed in ovarian tumor endot helial cell tumor-associated markers listed in Tables 1 helial cells and/or modulate ovarian tumor endothelial cells, through 5 that is involved in promoting angiogenesis, such as Such as increase vascular growth. In certain examples, by inhibiting an ovarian endothelial cell tumor-associated CSPG2 has at least 80% sequence identity, for example at marker that is upregulated in ovarian endothelial tumor cells US 2013/02743 15 A1 Oct. 17, 2013 or by increasing activity of an ovarian endothelial cell tumor examples, production of a gene product decreases by at least associated marker that is downregulated in ovarian endothe 2-fold, for example at least 3-fold or at least 4-fold, as com lial tumor cells). In a particular example, a therapy decreases pared to a control (Such an amount of gene expression in a the size of a tumor, the growth of a tumor, the number of normal endothelial cell). In one example, a control is a rela tumors, the metastasis of a tumor, or combinations thereof, tive amount of gene expression or protein expression in a Subsequent to the therapy, such as a decrease of at least 10%, biological sample taken from a subject who does not have at least 20%, at least 50%, or even at least 90%. Such ovarian cancer. decreases can be measured using the methods disclosed 0087 Endothelial cell: Cells that line the interior surface herein. of blood vessels, forming an interface between circulating 0080 Determining expression of a gene product: Detec blood in the lumen and the rest of the vessel wall. For tion of a level expression in either a qualitative or quantitative example, endothelial cells line the entire circulatory system. a. Further, both blood and lymphatic capillaries are composed 0081 Diagnosis: The process of identifying a disease by of a single layer of endothelial cells. its signs, symptoms and results of various tests. The conclu I0088 Epidermal Growth Factor-like domain multiple 6 sion reached through that process is also called “a diagnosis.” (EGFL6): A member of the epidermal growth factor (EGF) Forms of testing commonly performed include blood tests, repeat Superfamily of genes known to encode proteins that medical imaging, urinalysis, and biopsy. govern cellular proliferative responses. EGFL6 has been 0082 DNA (deoxyribonucleic acid): A long chain poly identified as a possible regulator of cell cycle and oncogen mer which includes the genetic material of most living organ CS1S. isms (some viruses have genes including ribonucleic acid, I0089. In particular examples, expression of EGFL6 is RNA). The repeating units in DNA polymers are four differ increased in ovarian cancer endothelial cells. The term ent nucleotides, each of which includes one of the four bases, EGFL6 includes any EGFL6 gene, cDNA, mRNA, or protein adenine, guanine, cytosine and thymine bound to a deoxyri from any organism and that is EGFL6 and is expressed and in bose Sugar to which a phosphate group is attached. Triplets of Some examples overexpressed in ovarian cancer endothelial nucleotides, referred to as codons, in DNA molecules code cells. for amino acid in a polypeptide. The term codon is also used 0090 Nucleic acid and protein sequences for EGFL6 are for the corresponding (and complementary) sequences of publicly available. For example, GenBank Accession Nos.: three nucleotides in the mRNA into which the DNA sequence NM O15507, NM 019397 and BCO38587 disclose EGFL6 is transcribed. nucleic acid sequences, and GenBank Accession Nos.: 0083. Differential expression: A difference, such as an AAQ88699, CAM23572, and AAF27812 disclose EGFL6 increase or decrease, in the conversion of the information protein sequences, all of which are incorporated by reference encoded in a gene (Such as an ovarian endothelial cell tumor as provided by GenBank on Feb. 14, 2007. associated molecule) into messenger RNA, the conversion of 0091. In one example, EGFL6 includes a full-length wild mRNA to a protein, or both. In some examples, the difference type (or native) sequence, as well as EGFL6 allelic variants is relative to a control or reference value. Such as an amount of that retain the ability to be expressed in ovarian tumor endot gene expression that is expected in a Subject who does not helial cells and/or modulate ovarian tumor endothelial cells, have ovarian cancer or in a normal (non-cancerous) endothe Such as increase vascular growth. In certain examples, lial cell Sample. Detecting differential expression can include EGFL6 has at least 80% sequence identity, for example at measuring a change in gene expression. least 85%, 90%, 95%, or 98% sequence identity to GenBank 0084 Downregulated or inactivation: When used in refer Accession No. AAQ88699, CAM23572, or AAF27812. In ence to the expression of a nucleic acid molecule. Such as a other examples, EGFL6 has a sequence that hybridizes to gene, refers to any process which results in a decrease in Affymetrix Probe ID No. 219454 at and retains EGFL6 production of a gene product. A gene product can be RNA activity (such as the capability to be expressed in ovarian (such as mRNA, rRNA, tRNA, and structural RNA) or pro tumor endothelial cells and/or modulate tumor and/or vascu tein. Therefore, gene downregulation or deactivation includes lar growth). processes that decrease transcription of a gene or translation 0092 Expression: The process by which the coded infor of mRNA. Examples of genes whose expression is downregu mation of a gene is converted into an operational, non-opera lated in ovarian tumor endothelial cells can be found in Table tional, or structural part of a cell. Such as the synthesis of a 1 (indicated by a negative fold change. Such as TLOC1 and protein. Gene expression can be influenced by external sig HS6ST2) and Table 3 (such as PLN, SELE, GREB1, OGN nals. For instance, exposure of a cell to a hormone may and LCXD3). stimulate expression of a hormone-induced gene. Different 0085 Examples of processes that decrease transcription types of cells can respond differently to an identical signal. include those that facilitate degradation of a transcription Expression of a gene also can be regulated anywhere in the initiation complex, those that decrease transcription initiation pathway from DNA to RNA to protein. Regulation can rate, those that decrease transcription elongation rate, those include controls on transcription, translation, RNA transport that decrease processivity of transcription and those that and processing, degradation of intermediary molecules Such increase transcriptional repression. Gene downregulation can as mRNA, or through activation, inactivation, compartmen include reduction of expression above an existing level. talization or degradation of specific protein molecules after Examples of processes that decrease translation include those they are produced. that decrease translational initiation, those that decrease 0093. The expression of a nucleic acid molecule can be translational elongation and those that decrease mRNA sta altered relative to a normal (wildtype) nucleic acid molecule. bility. Alterations in gene expression, such as differential expres I0086 Gene downregulation includes any detectable Sion, include but are not limited to: (1) overexpression; (2) decrease in the production of a gene product. In certain underexpression; or (3) Suppression of expression. Alterna US 2013/02743 15 A1 Oct. 17, 2013
tions in the expression of a nucleic acid molecule can be referred to as a fingerprint) can be linked to a tissue or cell associated with, and in fact cause, a change in expression of type (Such as ovarian cancer cell), to a particular stage of the corresponding protein. Specific examples of ovarian normal tissue growth or disease progression (such as endothelial cell tumor-associated molecules that are up-regu advanced ovarian cancer), or to any other distinct or identifi lated in ovarian tumor endothelial cells are provided in Tables able condition that influences gene expression in a predict 2 and 4. Specific examples of ovarian endothelial cell tumor able way. Gene expression profiles can include relative as associated molecules that are down-regulated in ovarian well as absolute expression levels of specific genes, and can tumor endothelial cells are listed in Table 3. For example, be viewed in the context of a test sample compared to a EZH2, EGFL6, TNFAIP6, TWIST1, STC1, HOP, CSPG2, baseline or control sample profile (such as a sample from a and PLXDC1 are upregulated or increased in expression in Subject who does not have ovarian cancer or normal endot ovarian tumor endothelial cells, while TLOC1 and HS6ST2 helial cells). In one example, a gene expression profile in a are downregulated or decreased in expression in Such cells. Subject is read on an array (Such as a nucleic acid or protein 0094 Protein expression can also be altered in some man array). For example, a gene expression profile is performed ner to be different from the expression of the protein in a using a commercially available array Such as a Human normal (wild type) situation. This includes but is not neces Genome U133 2.0 Plus Microarray from AFFYMETRIX(R) sarily limited to: (1) a mutation in the protein such that one or (AFFYMETRIX(R), Santa Clara, Calif.). more of the amino acid residues is different; (2) a short 0.098 Homeodomain-only protein, transcript variant 2 deletion or addition of one or a few (Such as no more than (HOP): A transcriptional repressor that modulates serum 10-20) amino acid residues to the sequence of the protein; (3) response factor-dependent cardiac-specific gene expression a longer deletion or addition of amino acid residues (such as and cardiac development. In particular examples, expression at least 20 residues). Such that an entire protein domain or of HOP is increased in ovarian cancer endothelial cells. The Sub-domain is removed or added; (4) expression of an term HOP includes any HOP gene, cDNA, mRNA, or protein increased amount of the protein compared to a control or from any organism and that is HOP and is expressed and in standard amount; (5) expression of a decreased amount of the Some examples overexpressed in ovarian cancer endothelial protein compared to a control or standard amount; (6) alter cells. ation of the Subcellular localization or targeting of the protein; (0099. Nucleic acid and protein sequences for HOP are (7) alteration of the temporally regulated expression of the publicly available. For example, GenBank Accession Nos.: protein (such that the protein is expressed when it normally NM 1392 11, XM 001083738, and XM 001137349 dis would not be, or alternatively is not expressed when it nor close HOP nucleic acid sequences, and GenBank Accession mally would be); (8) alteration instability of a protein through Nos.: AAH14225, NP 631958, and NP 631957 disclose increased longevity in the time that the protein remains local HOP protein sequences, all of which are incorporated by ized in a cell; and (9) alteration of the localized (such as organ reference as provided by GenBank on Feb. 14, 2007. or tissue specific or Subcellular localization) expression of the 0100. In one example, HOP includes a full-length wild protein (such that the protein is not expressed where it would type (or native) sequence, as well as HOPallelic variants that normally be expressed or is expressed where it normally retain the ability to be expressed in ovarian tumor endothelial would not be expressed), each compared to a control or stan cells and/or modulate ovarian tumor endothelial cells, such as dard. increase vascular growth. In certain examples, HOP has at 0095 Controls or standards for comparison to a sample, least 80% sequence identity, for example at least 85%, 90%, for the determination of differential expression, include 95%, or 98% sequence identity to GenBank Accession No.: samples believed to be normal (in that they are not altered for AAH14225, NP 631958, or NP 631957. In other the desired characteristic, for example a sample from a Sub examples, HOP has a sequence that hybridizes to AFFYME ject who does not have cancer, Such as ovarian cancer) as well TRIX(R) Probe ID No. 211597 s at and retains HOP activity as laboratory values, even though possibly arbitrarily set, (such as the capability to be expressed in ovarian tumor endot keeping in mind that such values can vary from laboratory to helial cells and/or modulate tumor and/or vascular growth). laboratory. 0101 Hybridization: To form base pairs between comple 0096 Laboratory standards and values may be set based mentary regions of two strands of DNA, RNA, or between on a known or determined population value and can be Sup DNA and RNA, thereby forming a duplex molecule. Hybrid plied in the format of a graph or table that permits comparison ization conditions resulting in particular degrees of Strin of measured, experimentally determined values. gency will vary depending upon the nature of the hybridiza 0097 Gene expression profile (or fingerprint): Differen tion method and the composition and length of the tial or altered gene expression can be detected by changes in hybridizing nucleic acid sequences. Generally, the tempera the detectable amount of gene expression (such as cDNA or ture of hybridization and the ionic strength (such as the Na" mRNA) or by changes in the detectable amount of proteins concentration) of the hybridization buffer will determine the expressed by those genes. A distinct or identifiable pattern of stringency of hybridization. Calculations regarding hybrid gene expression, for instance a pattern of high and low ization conditions for attaining particular degrees of Strin expression of a defined set of genes or gene-indicative nucleic gency are discussed in Sambrook et al., (1989) Molecular acids such as ESTs; in Some examples, as few as one or two Cloning, second edition, Cold Spring Harbor Laboratory, genes provides a profile, but more genes can be used in a Plainview, N.Y. (chapters 9 and 11). The following is an profile, for example at least 3, at least 4, at least 5, at least 6, exemplary set of hybridization conditions and is not limiting: at least 10, at least 20, at least 25, at least 30, at least 50, at least 0102 Very High Stringency (Detects Sequences that 80, at least 100, at least 190, at least 200, at least 300, at least Share at Least 90% Identity) 400, at least 500, at least 550, at least 600, at least 700, at least (0103 Hybridization: 5xSSC at 65° C. for 16 hours 800, at least 900, at least 1000, at least 1100 or more of those 0104 Washtwice: 2xSSC at room temperature (RT) for 15 listed in any of Tables 1-5. A gene expression profile (also minutes each US 2013/02743 15 A1 Oct. 17, 2013
0105 Wash twice: 0.5xSSC at 65° C. for 20 minutes each gated to a binding agent that specifically binds to one or more 0106 High Stringency (Detects Sequences that Share at of the ovarian endothelial cell tumor-associated molecules Least 80% Identity) disclosed in Tables 1 through 5 to allow for the detection/ 0107 Hybridization: 5x-6xSSC at 65° C.-70° C. for 16-20 screening for angiogenesis and/or the presence of a tumor in hours a Subject. 01.08 Wash twice: 2XSSC at RT for 5-20 minutes each 0116 Malignant: Cells that have the properties of anapla 0109 Wash twice: 1XSSC at 55° C.-70° C. for 30 minutes sia invasion and metastasis. each 0117 Mammal: This term includes both human and non 0110 Low Stringency (Detects Sequences that Share human mammals. Examples of mammals include, but are not Greater than 50% Identity) limited to: humans and Veterinary and laboratory animals, 0111 Hybridization: 6xSSC at RT to 55° C. for 16-20 Such as pigs, cows, goats, cats, dogs, rabbits and mice. hours 0118 Neoplasm: Abnormal growth of cells. 0112 Wash at least twice: 2x-3XSSC at RT to 55° C. for 0119 Normal Cell: Non-tumor cell, non-malignant, unin 20-30 minutes each. fected cell. 0113. Inhibitor: Any chemical compound, nucleic acid I0120 Nucleic acid array: An arrangement of nucleic acids molecule, peptide or polypeptide Such as an antibody or (such as DNA or RNA) in assigned locations on a matrix. Such RNAi that can reduce activity of a gene product or interfere as that found in cDNA arrays, or oligonucleotide arrays. Such with expression of a gene, respectively. In some examples, an as those listed in Tables 1-5. inhibitor can reduce or inhibit the activity of a protein that is encoded by a gene either directly or indirectly. Direct inhibi 0121 Nucleic acid molecules representing genes: Any tion can be accomplished, for example, by binding to a pro nucleic acid, for example DNA (intron or exon or both), tein and thereby preventing the protein from binding an cDNA, or RNA (such as mRNA), of any length suitable for intended target, Such as a receptor. Indirect inhibition can be use as a probe or other indicator molecule, and that is infor accomplished, for example, by binding to a protein's mative about the corresponding gene. intended target, such as a receptor orbinding partner, thereby I0122) Nucleic acid molecules: A deoxyribonucleotide or blocking or reducing activity of the protein. In some ribonucleotide polymer including, without limitation, cDNA, examples, an inhibitor of the disclosure can inhibit a gene by mRNA, genomic DNA, and synthetic (such as chemically reducing or inhibiting expression of the gene, inter alia by synthesized) DNA. The nucleic acid molecule can be double interfering with gene expression (transcription, processing, Stranded or single-stranded. Where single-stranded, the translation, post-translational modification), for example, by nucleic acid molecule can be the sense Strand or the antisense interfering with the genes mRNA and blocking translation of Strand. In addition, nucleic acid molecule can be circular or the gene product or by post-translational modification of a linear. gene product, or by causing changes in intracellular localiza (0123. The disclosure includes isolated nucleic acid mol tion. ecules that include specified lengths of an ovarian endothelial 0114 Isolated: An "isolated' biological component (such cell tumor-associated molecule nucleotide sequence, for as a nucleic acid molecule, protein, or cell) has been Substan sequences for genes listed in Tables 1 through 4. Such mol tially separated or purified away from other biological com ecules can include at least 10, at least 15, at least 20, at least ponents in the cell of the organism, or the organism itself, in 25, at least 30, at least 35, at least 40, at least 45 or at least 50 which the component naturally occurs, such as other chromo consecutive nucleotides of these sequences or more, and can somal and extra-chromosomal DNA and RNA, proteins and be obtained from any region of a ovarian endothelial cell cells. Nucleic acid molecules and proteins that have been tumor-associated molecule. "isolated include nucleic acid molecules and proteins puri 0.124 Oligonucleotide: A plurality of joined nucleotides fied by standard purification methods. The term also joined by native phosphodiester bonds, between about 6 and embraces nucleic acid molecules and proteins prepared by about 300 nucleotides in length. An oligonucleotide analog recombinant expression in a host cell as well as chemically refers to moieties that function similarly to oligonucleotides synthesized nucleic acid molecules and proteins. For but have non-naturally occurring portions. For example, oli example, an isolated serous papillary ovarian cancer cell is gonucleotide analogs can contain non-naturally occurring one that is substantially separated from other ovarian cell portions. Such as altered Sugar moieties or inter-Sugar link Subtypes, such as endometrioid, clear cell or mucinous Sub ages, such as a phosphorothioate oligodeoxynucleotide. types. 0.125 Particular oligonucleotides and oligonucleotide 0115 Label: An agent capable of detection, for example analogs can include linear sequences up to about 200 nucle by ELISA, spectrophotometry, flow cytometry, or micros otides in length, for example a sequence (such as DNA or copy. For example, a label can be attached to a nucleic acid RNA) that is at least 6 nucleotides, for example at least 8, at molecule or protein, thereby permitting detection of the least 10, at least 15, at least 20, at least 21, at least 25, at least nucleic acid molecule or protein. Examples of labels include, 30, at least 35, at least 40, at least 45, at least 50, at least 100 but are not limited to, radioactive isotopes, enzyme Sub or even at least 200 nucleotides long, or from about 6 to about strates, co-factors, ligands, chemiluminescent agents, fluoro 50 nucleotides, for example about 10-25 nucleotides, such as phores, haptens, enzymes, and combinations thereof. Meth 12, 15 or 20 nucleotides. ods for labeling and guidance in the choice of labels 0.126 Oligonucleotide probe: A short sequence of nucle appropriate for various purposes are discussed for example in otides, such as at least 8, at least 10, at least 15, at least 20, at Sambrook et al. (Molecular Cloning: A Laboratory Manual, least 21, at least 25, or at least 30 nucleotides in length, used Cold Spring Harbor, N.Y., 1989) and Ausubel et al. (In Cur to detect the presence of a complementary sequence by rent Protocols in Molecular Biology, John Wiley & Sons, molecular hybridization. In particular examples, oligonucle New York, 1998). In a particular example, a label is conju otide probes include a label that permits detection of oligo US 2013/02743 15 A1 Oct. 17, 2013
nucleotide probe: target sequence hybridization complexes, ences, by E. W. Martin, Mack Publishing Co., Easton, Pa., Such as with an ovarian endothelial cell tumor-associated 15th Edition (1975), describes compositions and formula molecule listed in Tables 1-5. tions suitable for pharmaceutical delivery of one or more 0127 Ovarian cancer: A malignant ovarian neoplasm (an therapeutic agents, such as one or more compositions that abnormal growth located on the ovaries). Cancer of the ova include a binding agent that specifically binds to at least one ries includes ovarian carcinoma, papillary serous cystadeno of the disclosed ovarian endothelial cell tumor-associated carcinoma, mucinous cystadenocarcinoma, endometrioid molecules. tumors, celioblastoma, clear cell carcinoma, unclassified car I0134. In general, the nature of the carrier will depend on cinoma, granulosa-thecal cell tumors, Sertoli-Leydig cell the particular mode of administration being employed. For tumors, dysgerminoma, and malignant teratoma. The most instance, parenteral formulations can include injectable fluids common type of ovarian cancer is papillary serous carci that include pharmaceutically and physiologically acceptable Oa. fluids such as water, physiological saline, balanced salt solu 0128 Surgery is an exemplary treatment for ovarian can tions, aqueous dextrose, glycerol or the like as a vehicle. In cer and can be necessary for diagnosis. The type of Surgery addition to biologically-neutral carriers, pharmaceutical depends upon how widespread the cancer is when diagnosed compositions to be administered can contain minor amounts (the cancer stage), as well as the type and grade of cancer. The of non-toxic auxiliary Substances, such as wetting or emulsi Surgeon may remove one (unilateral oophorectomy) or both fying agents, preservatives, and pH buffering agents and the ovaries (bilateral oophorectomy), the fallopian tubes (salp like, for example sodium acetate or Sorbitan monolaurate, ingectomy), and the uterus (hysterectomy). For Some very Sodium lactate, potassium chloride, calcium chloride, and early tumors (stage 1, low grade or low-risk disease), only the triethanolamine oleate. involved ovary and fallopian tube will be removed (called a 0.135 Plexin domain containing 1 (PLXDC1): A large “unilateral salpingo-oophorectomy,” USO), especially in transmembrane receptor. In vitro, plexin-C1 has been shown young females who wish to preserve their fertility. In to bind the GPI-anchored semaphorin Sema7A and the advanced disease as much tumor as possible is removed (de soluble viral semaphorins SemaVA (A39R) and SemaVB bulking Surgery). In cases where this type of Surgery is suc (AHV). Plexin C1 engagement by SemaVA inhibits integrin cessful, the prognosis is improved compared to Subjects mediated dendritic cell adhesion and chemotaxis in vitro, where large tumor masses (more than 1 cm in diameter) are Suggesting a role for plexin C1 in dendritic cell migration. left behind. 0.136. In an example, expression of PLXDL1 is increased 0129. Chemotherapy is often used after surgery to treat in ovarian tumor endothelial cells. The term PLXDC1 any residual disease. At present systemic chemotherapy often includes any plexin C1 gene, cDNA, mRNA, or protein from includes a platinum derivative with a taxane as a method of any organism and that is a PLXDC1 and is expressed and in treating advanced ovarian cancer. Chemotherapy is also used Some examples overexpressed in ovarian tumor endothelial to treat Subjects who have a recurrence. cells. 0130 Ovarian endothelial cell tumor-associated (or 0.137 Exemplary nucleic acid and protein sequences for related) molecule: A molecule whose expression is altered in PLXDC1 are publicly available. For example, GenBank ovarian tumor endothelial cells. Such molecules include, for Accession Nos.: NM 018797, XM 622776, AB208934, instance, nucleic acid sequences (such as DNA, cDNA, or and NM 005761 disclose PLXDC1 nucleic acid sequences mRNAs) and proteins. Specific genes include those listed in and GenBank Accession Nos.: NP 061267, XP 622776, Tables 1 through 5. Thus, the presence of the respective BAD921.71, and NP 005752 disclose PLXDC1 protein ovarian endothelial cell tumor-associated molecules can be sequences, all of which are incorporated by reference as pro used to diagnose, or determine the prognosis of, an ovarian vided by GenBank on Feb. 14, 2007. tumor in a Subject. 0.138. In one example, a PLXDC1 sequence includes a 0131. In an example, an ovarian endothelial cell tumor full-length wild-type (or native) sequence, as well as associated molecule is any molecule listed in Tables 1 PLXDC1 allelic variants, fragments, homologs or fusion through 5. Specific examples of ovarian endothelial cell sequences that retain the ability to be expressed in ovarian tumor-associated molecules that are up-regulated in ovarian tumor endothelial cells and/or modulate ovarian tumor endot tumor endothelial cells are provided in Tables 2 and 4. Spe helial cells, such as increase vascular growth. In certain cific examples of ovarian endothelial cell tumor-associated examples, PLXDC1 has at least 80% sequence identity, for molecules that are down-regulated in ovarian tumor endothe example at least 85%, 90%, 95%, or 98% sequence identity to lial cells are listed in Table 3. As illustrated in Table 4, a GenBank Accession No.: NP 061267, XP 622776, number of the identified ovarian cell tumor-associated mol BAD921.71, or NP 005752. In other examples, a PLXDC1 ecules are related to cell proliferation, tube formation and cell has a sequence that hybridizes to AFFYMETRIX(R) Probe ID motility. No. 214081 at and retains PLXDC1 activity (such as the 0132. Ovarian endothelial cell tumor-associated mol capability to be expressed in ovarian tumor endothelial cells ecules can be involved in or influenced by cancer in different and/or modulate tumor and/or vascular growth). ways, including causative (in that a change in a ovarian endot I0139 Polymerase Chain Reaction (PCR): An in vitro helial cell tumor-associated molecule leads to development of amplification technique that increases the number of copies or progression of ovarian cancer) or resultive (in that devel of a nucleic acid molecule (for example, a nucleic acid mol opment of or progression of ovarian cancer causes or results ecule in a sample or specimen). In an example, a biological in a change in the ovarian endothelial cell tumor-associated sample collected from a subject (e.g., with ovarian cancer) is molecule). contacted with a pair of oligonucleotide primers, under con 0.133 Pharmaceutically Acceptable Carriers: The phar ditions that allow for the hybridization of the primers to maceutically acceptable carriers (vehicles) useful in this dis nucleic acid template in the sample. The primers are extended closure are conventional. Remington's Pharmaceutical Sci under Suitable conditions, dissociated from the template, and US 2013/02743 15 A1 Oct. 17, 2013
then re-annealed, extended, and dissociated to amplify the 0145 Sample (or biological sample): A biological speci number of copies of the nucleic acid. The product of a PCR men containing genomic DNA, RNA (including mRNA), can be characterized by methods known in the art such as protein, or combinations thereof, obtained from a Subject. electrophoresis, restriction endonuclease cleavage patterns, Examples include, but are not limited to, peripheral blood, oligonucleotide hybridization or ligation, and/or nucleic acid urine, saliva, tissue biopsy, Surgical specimen, amniocentesis sequencing, using standard techniques. samples and autopsy material. In one example, a sample 0140 Primers: Short nucleic acid molecules, for instance includes an ovarian cancertissue biopsy. DNA oligonucleotides 10-100 nucleotides in length, such as 0146 Sensitivity: A measurement of activity, such as bio about 15, 20, 25, 30 or 50 nucleotides or more in length. logical activity, of a molecule or a collection of molecules in Primers can be annealed to a complementary target DNA a given condition. In an example, sensitivity refers to the Strand (such as a gene listed in Table 1) by nucleic acid activity of an agent, such as a binding agent that preferentially hybridization to form a hybrid between the primer and the binds to one or more ovarian endothelial cell tumor-associ target DNA strand. Primer pairs can be used for amplification ated molecules, to alter the growth, development or progres of a nucleic acid sequence, such as by PCR or other nucleic sion of a disease, such as ovarian cancer. In certain examples, acid amplification methods known in the art. sensitivity or responsiveness can be assessed using any end 0141 Methods for preparing and using nucleic acid prim point indicating a benefit to the Subject, including, without ers are described, for example, in Sambrooketal. (In Molecu limitation, (1) inhibition, to some extent, of tumor growth, lar Cloning: A Laboratory Manual. CSHL, New York, 1989), including slowing down and complete growth arrest; (2) Ausubel et al. (ed.) (In Current Protocols in Molecular Biol reduction in the number of tumor cells; (3) reduction in tumor ogy, John Wiley & Sons, New York, 1998), and Innis et al. size; (4) inhibition (such as reduction, slowing down or com (PCR Protocols, A Guide to Methods and Applications, Aca plete stopping) of tumor cell infiltration into adjacent periph demic Press, Inc., San Diego, Calif., 1990). PCR primer pairs eral organs and/or tissues; (5) inhibition (such as reduction, can be derived from a known sequence, for example, by using slowing down or complete stopping) of metastasis; (6) computer programs intended for that purpose such as Primer enhancement of anti-tumor immune response, which may, (Version 0.5, (C) 1991, Whitehead Institute for Biomedical but does not have to, result in the regression or rejection of the Research, Cambridge, Mass.). One of ordinary skill in the art tumor, (7) relief, to some extent, of one or more symptoms will appreciate that the specificity of a particular primer associated with the tumor; (8) increase in the length of Sur increases with its length. Thus, for example, a primer includ vival following treatment; (9) decreased mortality at a given ing 30 consecutive nucleotides of an ovarian endothelial cell point of time following treatment; and/or (10) reducing or tumor-associated molecule will anneal to a target sequence, inhibiting angiogenesis. Such as another homolog of the designated endothelial cell 0147 Sequence identity/similarity: The identity/similar tumor-associated protein, with a higher specificity than a ity between two or more nucleic acid sequences, or two or corresponding primer of only 15 nucleotides. Thus, in order more amino acid sequences, is expressed in terms of the to obtain greater specificity, primers can be selected that identity or similarity between the sequences. Sequence iden include at least 20, at least 25, at least 30, at least 35, at least tity can be measured in terms of percentage identity; the 40, at least 45, at least 50 or more consecutive nucleotides of higher the percentage, the more identical the sequences are. an ovarian endothelial cell tumor-associated nucleotide Sequence similarity can be measured in terms of percentage Sequence. similarity (which takes into account conservative amino acid Substitutions); the higher the percentage, the more similar the 0142 Prognosis: A prediction of the course of a disease, sequences are. Homologs or orthologs of nucleic acid or Such as ovarian cancer. The prediction can include determin amino acid sequences possess a relatively high degree of ing the likelihood of a subject to develop aggressive, recurrent sequence identity/similarity when aligned using standard disease, to Survive a particular amount of time (e.g., deter methods. This homology is more significant when the mine the likelihood that a subject will survive 1, 2, 3 or 5 orthologous proteins or cDNAs are derived from species years), to respond to a particular therapy (e.g., chemo which are more closely related (such as human and mouse therapy), or combinations thereof. sequences), compared to species more distantly related (Such 0143 Purified: The term “purified' does not require abso as human and C. elegans sequences). lute purity; rather, it is intended as a relative term. Thus, for 0148 Methods of alignment of sequences for comparison example, a purified protein preparation is one in which the are well known in the art. Various programs and alignment protein referred to is more pure than the protein in its natural algorithms are described in: Smith & Waterman, Adv. Appl. environment within a cell. For example, a preparation of a Math. 2:482, 1981; Needleman & Wunsch, J. Mol. Biol. protein is purified such that the protein represents at least 50% 48:443, 1970: Pearson & Lipman, Proc. Natl. Acad. Sci. USA of the total protein content of the preparation. Similarly, a 85:2444, 1988: Higgins & Sharp, Gene, 73:237-44, 1988: purified oligonucleotide preparation is one in which the oli Higgins & Sharp, CABIOS 5:151-3, 1989; Corpet et al., Nuc. gonucleotide is more pure than in an environment including a Acids Res. 16:10881-90, 1988; Huang et al. Computer Appls. complex mixture of oligonucleotides. in the Biosciences 8, 155-65, 1992; and Pearson et al., Meth. 0144. Recombinant: A recombinant nucleic acid molecule Mol. Bio. 24:307-31, 1994. Altschulet al., J. Mol. Biol. 215: is one that has a sequence that is not naturally occurring or has 403-10, 1990, presents a detailed consideration of sequence a sequence that is made by an artificial combination of two alignment methods and homology calculations. otherwise separated segments of sequence. This artificial 0149. The NCBI Basic Local Alignment Search Tool combination can be accomplished for example, by chemical (BLAST) (Altschulet al., J. Mol. Biol. 215:403-10, 1990) is synthesis or by the artificial manipulation of isolated seg available from several sources, including the National Center ments of nucleic acid molecules, such as by genetic engineer for Biological Information (NCBI, National Library of Medi ing techniques. cine, Building 38A, Room 8N805, Bethesda, Md. 20894) and US 2013/02743 15 A1 Oct. 17, 2013
on the Internet, for use in connection with the sequence analy 0154) One indication that two nucleic acid molecules are sis programs blastp, blastin, blastX, thlastin and thlastX. Addi closely related is that the two molecules hybridize to each tional information can be found at the NCBI web site. other under stringent conditions, as described above. Nucleic 0150 BLASTN is used to compare nucleic acid acid sequences that do not show a high degree of identity may sequences, while BLASTP is used to compare amino acid nevertheless encode identical or similar (conserved) amino sequences. If the two compared sequences share homology, acid sequences, due to the degeneracy of the genetic code. then the designated output file will present those regions of Changes in a nucleic acid sequence can be made using this homology as aligned sequences. If the two compared degeneracy to produce multiple nucleic acid molecules that sequences do not share homology, then the designated output all encode Substantially the same protein. Such homologous file will not present aligned sequences. nucleic acid sequences can, for example, possess at least about 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence 0151. Once aligned, the number of matches is determined identity with the genes listed in Table 1 as determined by this by counting the number of positions where an identical nucle method. An alternative (and not necessarily cumulative) indi otide or amino acid residue is presented in both sequences. cation that two nucleic acid sequences are substantially iden The percent sequence identity is determined by dividing the tical is that the polypeptide which the first nucleic acid number of matches either by the length of the sequence set encodes is immunologically cross reactive with the polypep forth in the identified sequence, or by an articulated length tide encoded by the second nucleic acid. (such as 100 consecutive nucleotides or amino acid residues 0155 One of skill in the art will appreciate that the par from a sequence set forth in an identified sequence), followed ticular sequence identity ranges are provided for guidance by multiplying the resulting value by 100. For example, a only; it is possible that strongly significant homologs could be nucleic acid sequence that has 1166 matches when aligned obtained that fall outside the ranges provided. with a test sequence having 1154 nucleotides is 75.0 percent identical to the test sequence (1166-1554*100–75.0). The 0156 Short interfering RNA (siRNA): A double stranded percent sequence identity value is rounded to the nearest nucleic acid molecule capable of RNA interference or tenth. For example, 75.11, 75.12, 75.13, and 75.14 are “RNAi (See, for example, Bass Nature 411: 428-429, 2001; rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and Elbashir et al., Nature 411: 494-498, 2001; and Kreutzer et 75.19 are rounded up to 75.2. The length value will always be al., International PCT Publication No. WO 00/44895; Zer an integer. In another example, a target sequence containing a nicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 20-nucleotide region that aligns with 20 consecutive nucle 99/32619; Plaetincket al., International PCT Publication No. otides from an identified sequence as follows contains a WO 00/01846; Mello and Fire, International PCT Publication region that shares 75 percent sequence identity to that iden No. WO 01/29058: Deschamps-Depaillette, International tified sequence (that is, 15-20*100–75). PCT Publication No. WO99/07409; and Li et al., Interna 0152 For comparisons of amino acid sequences of greater tional PCT Publication No. WO 00/44914.) As used herein, than about 30 amino acids, the Blast 2 sequences function is siRNA molecules need not be limited to those molecules employed using the default BLOSUM62 matrix set to default containing only RNA, but further encompasses chemically parameters, (gap existence cost of 11, and a per residue gap modified nucleotides and non-nucleotides having RNAi cost of 1). Homologs are typically characterized by posses capacity or activity. In an example, an siRNA molecule is one sion of at least 70% sequence identity counted over the full that reduces or inhibits the biological activity or expression of length alignment with an amino acid sequence using the one or more ovarian endothelial cell tumor-associated mol NCBI Basic Blast 2.0, gapped blastp with databases such as ecules disclosed in Tables 1, 2, 4 or 5 that are upregulated in the nr or Swissprot database. Queries searched with the blastin ovarian tumor endothelial cells, such as EGFL6, TNFAIP6, program are filtered with DUST (Hancock and Armstrong, TWIST1, STC1, HOP, CSPG2, PLXDC1, EZH2, the Notch 1994, Comput. Appl. Biosci. 10:67-70). Other programs use ligand Jagged 1 or PTK2. SEG. In addition, a manual alignment can be performed. 0157 Specific Binding Agent: An agent that binds sub Proteins with even greater similarity will show increasing stantially or preferentially only to a defined target (for percentage identities when assessed by this method, such as at example, those listed in Table 1). Such as a protein, enzyme, least about 75%, 80%, 85%, 90%, 95%, 98%, or 99% polysaccharide, oligonucleotide, DNA, RNA, recombinant sequence identity with the proteins listed in Table 1. vector or a small molecule. In an example, a “specific binding 0153. When aligning short peptides (fewer than around 30 agent' is capable of binding to at least one of the disclosed amino acids), the alignment is be performed using the Blast 2 ovarian endothelial cell tumor-associated molecules. Thus, a sequences function, employing the PAM30 matrix set to RNA-specific binding agent binds substantially only to the default parameters (open gap 9. extension gap 1 penalties). defined RNA, or to a specific region within the RNA. For Proteins with even greater similarity to the reference example, a “specific binding agent' includes a siRNA that sequence will show increasing percentage identities when binds substantially to a specified RNA. assessed by this method, such as at least about 60%, 70%, 0158. A protein-specific binding agent binds substantially 75%, 80%, 85%, 90%. 95%, 98%, 99% sequence identity only the defined protein, or to a specific region within the with the proteins listed in Table 1. When less than the entire protein. For example, a “specific binding agent' includes sequence is being compared for sequence identity, homologs antibodies and other agents that bind Substantially to a speci will typically possess at least 75% sequence identity over fied polypeptide. The antibodies can be monoclonal or poly short windows of 10-20 amino acids, and can possess clonal antibodies that are specific for the polypeptide, as well sequence identities of at least 85%, 90%. 95% or 98% as immunologically effective portions (“fragments') thereof. depending on their identity to the reference sequence. Meth The determination that a particular agent binds Substantially ods for determining sequence identity over Such short win only to a specific polypeptide may readily be made by using dows are described at the NCBI web site. or adapting routine procedures. One Suitable in vitro assay US 2013/02743 15 A1 Oct. 17, 2013
makes use of the Western blotting procedure (described in amount, for example by at least 20%, at least 50%, at least many standard texts, including Harlow and Lane. Using Anti 60%, at least 70%, at least 80%, at least 90%, at least 95%, at bodies: A Laboratory Manual, CSHL, New York, 1999). least 98%, or even at least 100% (elimination of detectable 0159 Stanniocalcin 1 (STC1): A hormone that plays a role cancer cells), as compared to the number of cancer cells in the in calcium regulation, phosphate homeostasis and cell absence of the composition. metabolism. In particular examples, expression of STC1 is 0167. In other examples, it is an amount of the specific increased in ovarian tumor endothelial cells. The term STC1 binding agent for one or more of the disclosed ovarian endot includes any STC1 gene, cDNA, mRNA, or protein from any helial cell tumor-associated molecules capable of reducing organism and that is STC1 and is expressed or overexpressed angiogenesis by least 20%, at least 50%, at least 60%, at least in Some examples in ovarian tumor endothelial cells. 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 0160 Nucleic acid and protein sequences for STC1 are even at least 100% (elimination of detectable angiogenesis) publicly available. For example, GenBank Accession Nos.: by the specific binding agent, or both, effective to decrease the NM 009285, NM 00003155, and NM 031123 disclose metastasis of a tumor. STC1 nucleic acid sequences, and GenBank Accession Nos.: 0168 A therapeutically effective amount of a specific AAH21425, NP 112385, and NP 033311 disclose STC1 binding agent for at least one of the disclosed ovarian endot protein sequences, all of which are incorporated by reference helial cell tumor-associated molecules, or cancer cells lysed as provided by GenBank on Feb. 14, 2007. by a therapeutic molecule conjugated to the agent, can be 0161 In one example, STC1 includes a full-length wild administered in a single dose, or in several doses, for example type (or native) sequence, as well as STC1 allelic variants that daily, during a course of treatment. However, the therapeuti retain the ability to be expressed in ovarian tumor endothelial cally effective amount can depend on the Subject being cells and/or modulate ovarian tumor endothelial cells, such as treated, the severity and type of the condition being treated, increase vascular growth. In certain examples, STC1 has at and the manner of administration. For example, a therapeu least 80% sequence identity, for example at least 85%, 90%, tically effective amount of Such agent can vary from about 1 95%, or 98% sequence identity to GenBank Accession No.: ug-10 mg per 70 kg body weight if administered intrave AAH21425, NP 112385, or NP 033311. In other nously and about 10 ug-100 mg per 70 kg body weight if examples, STC1 has a sequence that hybridizes to AFFYME administered intratumorally. TRIX(R) Probe ID No. 230746 s at, 204595 s at, and 0169 Tissue: A plurality of functionally related cells. A 204597 X at and retains STC1 activity (such as the capability tissue can be a suspension, a semi-solid, or Solid. Tissue to be expressed in ovarian tumor endothelial cells and/or includes cells collected from a subject such as the ovaries. modulate tumor and/or vascular growth). 0170 Treating a disease: “Treatment” refers to a therapeu 0162. Subject: Living multi-cellular vertebrate organisms, tic intervention that ameliorates a sign or symptom of a dis a category that includes human and non-human mammals. ease or pathological condition, Such as a sign or symptom of 0163 Target sequence: A sequence of nucleotides located ovarian cancer. Treatment can also induce remission or cure in a particular region in the human genome that corresponds of a condition, such as ovarian cancer. In particular examples, to a desired sequence, such as an ovarian endothelial cell treatment includes preventing a disease, for example by tumor-associated sequence. The target can be for instance a inhibiting the full development of a disease. Prevention of a coding sequence; it can also be the non-coding strand that disease does not require a total absence of disease. For corresponds to a coding sequence. Examples of target example, a decrease of at least 50% can be sufficient. sequences include those sequences associated with ovarian 0171 Tumor: All neoplastic cell growth and proliferation, tumor endothelial cells, such as any of those listed in Tables 1 whether malignant or benign, and all pre-cancerous and can through 5. cerous cells and tissues. In an example, a tumor is an ovarian 0164. Therapeutically Effective Amount: An amount of a tumor. composition that alone, or together with an additional thera 0172 Tumor-necrosis factor, alpha-induced protein 6 peutic agent(s) (for example a chemotherapeutic agent), (TNFAIP6): A protein capable of regulating the expression of induces the desired response (e.g., treatment of a tumor). The various molecules involved in the control of inflammation. In preparations disclosed herein are administered in therapeuti particular examples, expression of TNFAIP6 is increased in cally effective amounts. ovarian cancer endothelial cells. The term TNFAIP6 includes 0.165. In one example, a desired response is to decrease any TNFAIP6 gene, cDNA, mRNA, or protein from any ovarian tumor size or metastasis in a Subject to whom the organism and that is TNFAIP6 and is expressed or overex therapy is administered. Tumor metastasis does not need to be pressed in Some examples in ovarian cancer endothelial cells. completely eliminated for the composition to be effective. For (0173 Nucleic acid and protein sequences for TNFAIP6 example, a composition can decrease metastasis by a desired are publicly available. For example, GenBank Accession amount, for example by at least 20%, at least 50%, at least Nos.: NM 007115, BC021155 and NM 009398 disclose 60%, at least 70%, at least 80%, at least 90%, at least 95%, at TNFAIP6 nucleic acid sequences, and GenBank Accession least 98%, or even at least 100% (elimination of the tumor), as Nos.: AAH21155, NP 009046 and NP 033424 disclose compared to metastasis in the absence of the composition. TNFAIP6 protein sequences, all of which are incorporated by 0166 In particular examples, it is an amount of the thera reference as provided by GenBank on Feb. 14, 2007. peutic agent conjugated to the specific binding agent effective 0.174. In one example, TNFAIP6 includes a full-length to decrease a number of ovarian cancer cells, such as in a wild-type (or native) sequence, as well as TNFAIP6 allelic Subject to whom it is administered, for example a subject variants that retain the ability to be expressed in ovarian tumor having one or more ovarian carcinomas. The cancer cells do endothelial cells and/or modulate ovarian tumor endothelial not need to be completely eliminated for the composition to cells. Such as Suppression of vascular growth. In certain be effective. For example, a composition can decrease the examples, TNFAIP6 has at least 80% sequence identity, for number of cancer cells or growth of such cells by a desired example, at least 85%, 90%. 95%, or 98% sequence identity US 2013/02743 15 A1 Oct. 17, 2013
to GenBank Accession No.: AAH21155, NP 009046 or and PLXDC1 are upregulated or increased in expression in NP 033424. In other examples, TNFAIP6 has a sequence ovarian tumor endothelial cells. that hybridizes to AFFYMETRIX(R) Probe ID No. 206026 0181 Examples of processes that increase transcription s at and retains TNFAIP6 activity (such as the capability to be include those that facilitate formation of a transcription ini expressed in ovarian tumor endothelial cells and/or modulate tiation complex, those that increase transcription initiation tumor and/or vascular growth). rate, those that increase transcription elongation rate, those (0175 Twist homologue 1 (TWIST1): Overexpression of that increase processivity of transcription and those that TWIST1 has been reported to play a role in destabilizing the relieve transcriptional repression (for example by blocking genome, thus promoting chromosomal instability. For the binding of a transcriptional repressor). Gene upregulation example, TWIST1 is capable of inhibiting chrondrogenesis. can include inhibition of repression as well as stimulation of TWIST1 protein has also been noted to be involved in the expression above an existing level. Examples of processes regulation of tumor necrosis factor alpha production by anti that increase translation include those that increase transla inflammatory factors and pathways. In particular examples, tional initiation, those that increase translational elongation expression of TWIST1 is increased in ovarian cancer endot and those that increase mRNA stability. helial cells. The term TWIST1 includes any TWIST1 gene, 0182 Gene upregulation includes any detectable increase cDNA, mRNA, or protein from any organism and that is in the production of a gene product. In certain examples, TWIST1 and is expressed or overexpressed in some examples production of a gene product increases by at least 2-fold, for in ovarian cancer endothelial cells. example at least 3-fold or at least 4-fold, as compared to a 0176) Nucleic acid and protein sequences for TWIST1 are control (such an amount of gene expression in a normal publicly available. For example, GenBank Accession Nos.: endothelial cell). In one example, a control is a relative NM 000474, NM 053530 and XM 001076553 and dis amount of gene expression in a biological sample, such as in close TWIST1 nucleic acid sequences, and GenBank Acces an ovarian tissue biopsy obtained from a Subject that does not Sion Nos.: NP OOO465 and ABM87769 disclose TWIST1 have ovarian cancer. protein sequences, all of which are incorporated by reference 0183 Vasohibin 1 (VASH1): a protein that is expressed in as provided by GenBank on Feb. 14, 2007. a variety of tissues and inhibits functions relevant to neovas (0177. In one example, TWIST1 includes a full-length cularization (migration, proliferation, and network formation wild-type (or native) sequence, as well as TWIST1 allelic by endothelial cells). Vasohibin also inhibits angiogenesis in variants that retain the ability to be expressed in ovariantumor vivo. The unglycosylated protein (42 kDa) does not contain a endothelial cells and/or modulate ovarian tumor endothelial classical Secretory Secretion sequence and appears in the cells. Such as increase vascular growth. In certain examples, medium as a protein of 30 kDa, Suggesting proteolytic pro TWIST1 has at least 80% sequence identity, for example at cessing during secretion. In particular examples, VASH1 is least 85%, 90%, 95%, or 98% sequence identity to GenBank regulated by EZH2 wherein EZH2 binds to the VASH1 pro Accession No.: NP 000465 or ABM87769. In other moter and decreases or inhibits VASH1 anti-angiogenesis examples, TWIST1 has a sequence that hybridizes to activity. The term VASH1 includes any VASH1 gene, cDNA, AFFYMETRIX(R) Probe ID No. 206026 s at and retains mRNA, or protein from any organism and that is VASH1 and TWIST1 activity (such as the capability to be expressed in is expressed or overexpressed in some examples in ovarian ovariantumor endothelial cells and/or modulate tumor and/or cancer endothelial cells. vascular growth). 0.184 Nucleic acid and protein sequences for VASH1 are 0.178 Under conditions sufficient for: A phrase that is used publicly available. For example, GenBank Accession Nos.: to describe any environment that permits the desired activity. NP 055724 (human); NP 796328 (mouse); and In one example, includes administering a test agent to an NP 659 128 disclose VASH1 amino acid sequences which ovarian cancer cell or a subject sufficient to allow the desired are incorporated by reference as provided by GenBank on activity. In particular examples, the desired activity is altering Aug. 14, 2009. Further, GenBank Accession Nos.: the activity (Such as the expression) of an ovarian endothelial NM 014909 (human) and NM 177354 (mouse) disclose cell tumor-associated molecule. nucleic acid sequences which are incorporated by reference 0179 Unit dose: A physically discrete unit containing a as provided by GenBank on Aug. 14, 2009. predetermined quantity of an active material calculated to 0185. In one example, vasohibin includes a full-length individually or collectively produce a desired effect, such as a wild-type (or native) sequence, as well as VASH1 allelic therapeutic effect. A single unit dose or a plurality of unit variants that retain the ability to be expressed in ovarian tumor doses can be used to provide the desired effect, such as treat endothelial cells and/or modulate ovarian tumor endothelial ment of a tumor, for example a metastatic tumor. In one cells, such as increase vascular growth. In certain examples, example, a unit dose includes a desired amount of an agent VASH1 has at least 80% sequence identity, for example at that decreases or inhibits angiogenesis. least 85%, 90%, 95%, or 98% sequence identity to GenBank 0180 Upregulated or activation: When used in reference Accession No.: NP 055724; NP 796328 or NP 659 128 to the expression of a nucleic acid molecule. Such as a gene, and retains VASH1 activity (such as the capability to be refers to any process which results in an increase in produc expressed in ovarian tumor endothelial cells and/or modulate tion of a gene product. A gene product can be RNA (such as tumor and/or vascular growth). mRNA, rRNA, tRNA, and structural RNA) or protein. There 0186 Zeste homologue 2 (EZH2): A member of the poly fore, gene upregulation or activation includes processes that comb group of genes that has been reported to be involved in increase transcription of a gene or translation of mRNA. cell cycle regulation. EZH2, a component of the polycomb Specific examples of ovarian endothelial cell tumor-associ repressive complex 2 (PRC2), has intrinsic histone methyl ated molecules that are up-regulated in ovarian tumor endot transferase (HMTase) activity and has been implicated in the helial cells are provided in Tables 2 and 4. For example, progression and metastasis of several cancers. EZH2 is also a EZH2, EGFL6, TNFAIP6, TWIST1, STC1, HOP, CSPG2, transcriptional repressor that has multiple targets, including US 2013/02743 15 A1 Oct. 17, 2013 anti-angiogenic, pro-apoptotic, and tumor Suppressor genes. composition to a Subject. The composition can include a In particular examples, expression of EZH2 is increased in binding agent that is specific for one of the ovarian endothe ovarian cancer endothelial cells. In one example, expression lial cell tumor-associated molecules listed in any of Tables 1, of EZH2 is an indicator of poor prognosis. The term EZH2 2, 4 or 5 that are upregulated in ovarian tumor cells. Admin includes any EZH2 gene, cDNA, mRNA, or protein from any istration of Such compounds decreases the expression or organism and that is EZH2 and is expressed or overexpressed activity of the molecule that is undesirably upregulated in in Some examples in ovarian cancer endothelial cells. ovarian cancer cells. The molecules in Tables 1, 2, 4, or 5 0187. Nucleic acid and protein sequences for EZH2 are include, for instance, nucleic acid sequences (such as DNA, publicly available. For example, GenBank Accession Nos.: cDNA, or mRNAs) and proteins. Specific genes include those NM 004456 and AY519465.1 disclose EZH2 nucleic acid listed in Tables 1, 2, 4 or 5 as well as fragments of the sequences, and GenBank Accession No. AASO9975 dis full-length genes, cDNAs, or mRNAs (and proteins encoded closes a EZH2 protein sequence, which are incorporated by thereby) whose expression is upregulated in response to an reference as provided by GenBank on Feb. 14, 2007. ovarian tumor, Such as ovarian cancer. 0188 In one example, EZH2 includes a full-length wild 0.192 In particular examples, the specific binding agent is type (or native) sequence, as well as EZH2 allelic variants, an inhibitor such as a siRNA or an antibody to one of the fragments, homologs or fusion sequences that retain the abil disclosed ovarian endothelial cell tumor-associated mol ity to be expressed in ovarian tumor endothelial cells and/or ecules that is upregulated in ovarian tumor cells. For example, modulate ovarian tumor endothelial cells, such as increase the specific binding agent can be a siRNA that interferes with vascular growth. In certain examples, EZH2 has at least 80% mRNA expression of one of the disclosed ovarian endothelial sequence identity, for example at least 85%, 90%. 95%, or cell tumor-associated molecules that are involved in angio 98% sequence identity to AASO9975. In other examples, genesis, such as a molecule involved in regulating cell motill EZH2 has a sequence that hybridizes to AFFYMETRIX(R) ity, cell proliferation or tube formation, thereby inhibiting cell Probe ID No. 203358 s at and retains EZH2 activity (such as motility, cell proliferation or tube formation. For example, the the capability to be expressed in ovarian tumor endothelial specific binding agent can be a siRNA that inhibits the expres cells and/or modulate tumor and/or vascular growth). sion of PTK2, EZH2 or Jagged 1. In other particular 0189 Additional terms commonly used in molecular examples, ovarian tumor growth is reduced or inhibited by genetics can be found in Benjamin Lewin, Genes V published administering a specific binding agent to inhibit or reduce the by Oxford University Press, 1994 (ISBN 0-19-854287-9); expression or production of EZH2, EGFL6, TNFAIP6, Kendrew et al. (eds.), The Encyclopedia of Molecular Biol TWIST1, STC1, HOP, CSPG2, and PLXDC1. In additional ogy, published by Blackwell Science Ltd., 1994 (ISBN examples, a composition includes at least two specific bind 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biol ing agents such as two specific siRNAS that each bind to their ogy and Biotechnology: a Comprehensive Desk Reference, respective ovarian endothelial cell tumor-associated nucle published by VCH Publishers, Inc., 1995 (ISBN 1-56081 otide sequences and inhibitovarian tumor growth in a Subject. 569-8). In Some examples, the composition includes at least 2, 3, 4, 5, 5, 8 or 10 different siRNA molecules. For example, the com Methods of Treatment position can include PTK2, EZH2 and Jagged 1 siRNAs. 0190. It is shown herein that ovarian cancer is associated with differential expression of ovarian endothelial cell tumor Treating Ovarian Cancer by Altering Activity of an Ovarian associated molecules. For example, the disclosed gene Endothelial Cell Tumor-Associated Molecule expression profile has identified ovarian endothelial cell 0193 Methods are provided to inhibit ovarian endothelial tumor-associated molecules. Based on these observations, cell tumor-associated molecule activity or expression to treat methods of treatment to reduce or eliminate ovarian cancer an ovarian tumor. Treatment of tumors by reducing the num are disclosed. For example, the method can include inhibiting ber of ovarian endothelial cell tumor-associated molecules the expression or biological activity of at least one of the can include delaying the development of the tumor in a Sub ovarian endothelial cell tumor associated molecules from ject (such as preventing metastasis of a tumor). Treatment of Tables 1, 2, 4, and/or 5 that are upregulated in ovarian tumor a tumor also includes reducing signs or symptoms associated cells or increasing the expression or biological activity of at with the presence of Such a tumor (for example by reducing least one of the ovarian endothelial cell tumor associated the size, growth or Volume of the tumor or a metastasis molecules from Tables 1,3 and/or 5 that are downregulated in thereof). Such reduced growth can in Some examples ovarian tumor cells or combinations thereof. As used herein, decrease or slow metastasis of the tumor, or reduce the size or “inhibit does not require 100% inhibition of expression or volume of the tumor by at least 10%, at least 20%, at least activity. For example, a Substantial reduction may be 50%, or at least 75%, such as by inhibiting angiogenesis by at adequate, such as reduction in expression oractivity of at least least 10%, at least 20%, at least 50%, or at least 75%. For 20%, at least 30%, at least 50%, at least 75%, or at least 95% example, ovarian endothelial cell tumor-associated mol may be sufficient to obtain desired therapeutic results. In ecules involved in angiogenesis, Such as molecules involved Some examples, an “increase' in expression or activity is an in promoting cell proliferation, cell motility or tube formation increase of at least 20%, at least 30%, at least 50%, at least can be inhibited to treat an ovarian tumor, such as those 75%, or at least 95%. In some embodiments, the subject is a provided in any of Tables 1, 2, 4 or 5 that are upregulated in human, but the subject can alternatively be a veterinary or ovarian endothelial tumor cells. In other examples, ovarian laboratory Subject. In some embodiments, the ovarian cancer tumor growth is reduced or inhibited by inhibiting the expres is papillary serous ovarian cancer. sion or biological activity ovarian endothelial cell tumor 0191 Methods are disclosed herein for treating an ovarian associated molecules provided in any of Tables 1, 2, 4 or 5that tumor, Such as ovarian cancer. In one example, the method are upregulated in ovarian tumor endothelial cells. In further includes administeringatherapeutically effective amount of a examples, inhibition of ovarian endothelial cell tumor-asso US 2013/02743 15 A1 Oct. 17, 2013
ciated molecules includes reducing the invasive activity of the encoded by the genes listed in Tables 1, 2, 4 and/or 5 that are tumor in the Subject. In some examples, treatment using the upregulated in ovarian tumor endothelial cells, may substan methods disclosed herein prolongs the time of survival of the tially reduce the biological function of the protein (for Subject. example, the ability of the protein to promote angiogenesis). Such agents can be administered in therapeutically effective Specific Binding Agents amounts to subjects in need thereof. Such as a Subject having 0194 Specific binding agents are agents that selectively ovarian cancer, Such as papillary serous ovarian cancer. bind with higher affinity to a molecule of interest, than to other molecules. For example, a specific binding agent can be Pre-Screening Subjects one that binds with high affinity to one of the genes or gene 0199. In some examples, subjects are initially screened to products of the ovarian endothelial cell tumor-associated determine if they have ovarian cancer. In an example, Subjects molecules listed in any of Tables 1, 2, 4 or 5 that are upregu are initially screened for ovarian cancer by using one of the lated in ovarian tumor endothelial cells, but does not substan disclosed gene expression profiles (as discussed in detail tially bind to another gene or gene product. In a specific below). In some examples, if one or more of the disclosed example, a specific binding agent binds to one gene listed in endothelial cell tumor-associated molecules upregulated in Tables 1, 2, 4 or 5 that is upregulated in ovarian tumor endot ovarian endothelial cells (such as those listed in Tables 2 and helial cells thereby reducing or inhibiting expression of the 4) is detected, a specific binding agent capable of reducing or gene, but does not bind to the other genes (or gene product) inhibiting ovarian cancer is adminstered. listed in such Tables under similar conditions. For example, the agent can interfere with gene expression (transcription, Pre-Screening Specific Binding Agents processing, translation, post-translational modification), 0200. In some examples, specific binding agents are ini such as, by interfering with the genes mRNA and blocking tially screened for treating ovarian cancer by use of the dis translation of the gene product or by post-translational modi closed gene expression profile (see below). For example, the fication of a gene product, or by causing changes in intracel disclosed gene expression profile can be used to identify lular localization. In another specific example, a specific specific binding agents capable of reducing or inhibiting ova binding agent binds to a protein encoded by of one of the rian cancer. In an example, the disclosed gene expression genes listed in Table 1, 2, 4 or 5 that is upregulated in ovarian profile is used to identify compositions that can be employed tumor endothelial cells with a binding affinity in the range of to reduce or inhibit angiogenesis in ovarian tumors. 0.1 to 20 nM and reduces or inhibits the activity of such protein. Exemplary Tumors 0.195 Examples of specific binding agents include, but are not limited to, siRNAS, antibodies, ligands, recombinant pro 0201 A tumor is an abnormal growth of tissue that results teins, peptide mimetics, and soluble receptor fragments. One from excessive cell division. A particular example of a tumor example of a specific binding agent is a siRNA. Methods of is cancer. For example, the current application provides meth making siRNA that can be used clinically are known in the art. ods for the treatment (such as the prevention or reduction of Particular siRNAs and methods that can be used to produce metastasis) of tumors (such as cancers) by altering the expres and administer them are described in detail below. In some sion/production of one or more disclosed ovarian endothelial examples, the siRNA is incorporated into a chitosan (CH) cell tumor-associated molecules. In some examples, the nanoparticle. Such as chitosan obtained from shellfish or tumor is treated in Vivo, for example in a mammalian Subject, fungi. Such as a human Subject. Exemplary tumors that can be 0196. Another specific example of a specific binding agent treated using the disclosed methods include, but are not lim is an antibody, Such as a monoclonal or polyclonal antibody. ited to ovarian cancer, including metastases of such tumors to Methods of making antibodies that can be used clinically are other organs. Generally, the tumor is an ovarian cancer. Such known in the art. Particular antibodies and methods that can as papillary serous ovarian cancer. be used to produce them are described in detail below. 0197) Inafurther example, small molecular weight inhibi Administration tors or antagonists of the receptor protein can be used to 0202 Methods of administrating the disclosed composi regulate activity Such as the expression or production of ova tions are routine, and can be determined by a skilled clinician. rian endothelial cell tumor-associated molecules. In a particu For example, the disclosed therapies (such as those that lar example, Small molecular weight inhibitors orantagonists include a binding agent specific for one of the disclosed of the proteins encoded by the genes listed in Tables 2 and/or ovarian endothelial cell tumor-associated molecules listed in 4 are employed. Tables 1, 2, 4 or 5 whose expression is increased in ovarian 0198 Specific binding agents can be therapeutic, for endothelial tumor-associated cells) can be administered via example by reducing or inhibiting the biological activity of a injection, intratumorally, orally, topically, transdermally, nucleic acid or protein. Complete inhibition is not required. parenterally, or via inhalation or spray. In a particular For example, a reduction by at least 10%, at least 20%, at least example, a composition is administered intravenously to a 30%, at least 50%, at least 70%, or even at least 90% can be mammalian Subject, Such as a human. Sufficient. For example, a specific binding agent that binds 0203 The therapeutically effective amount of the agents with high affinity to a gene listed in Tables 1, 2, 4 and/or 5 that administered can vary depending upon the desired effects and are upregulated in ovarian tumor endothelial cells, may Sub the subject to be treated. In one example, the method includes stantially reduce the biological function of the gene or gene daily administration of at least 1 lug of the composition to the product (for example, the ability of the gene or gene product Subject (such as a human Subject). For example, a human can to facilitate angiogenesis). In other examples, a specific bind be administered at least 1 Lug or at least 1 mg of the compo ing agent that binds with high affinity to one of the proteins sition daily, such as 10 ug to 100 ug daily, 100 g to 1000 g US 2013/02743 15 A1 Oct. 17, 2013
daily, for example 10 ug daily, 100 ug daily, or 1000 ug daily. bolization), as well as anti-tumor pharmaceutical treatments In one example, the Subject is administered at least 1 Lug (Such which can include radiotherapeutic agents, anti-neoplastic as 1-100 ug) intravenously of the composition including a chemotherapeutic agents, antibiotics, alkylating agents and binding agent that specifically binds to one of the disclosed antioxidants, kinase inhibitors, and other agents. Particular ovarian endothelial cell tumor-associated molecules provided examples of additional therapeutic agents that can be used herein. In one example, the Subject is administered at least 1 include microtubule binding agents, DNA intercalators or mg intramuscularly (for example in an extremity) of Such cross-linkers, DNA synthesis inhibitors, DNA and/or RNA composition. In a specific example, the dose is 50 to 350 transcription inhibitors, antibodies, enzymes, enzyme inhibi ug/kg twice weekly, Such as 150 ug/kg twice weekly (for tors, and gene regulators. These agents (which are adminis example via iv injection). The dosage can be administered in tered at a therapeutically effective amount) and treatments divided doses (such as 2, 3, or 4 divided doses per day), or in can be used alone or in combination. Methods and therapeutic a single dosage daily. dosages of Such agents are known to those skilled in the art, 0204. In particular examples, the subject is administered and can be determined by a skilled clinician. the therapeutic composition that includes a binding agent 0209 “Microtubule binding agent” refers to an agent that specific for one of the disclosed ovarian endothelial cell interacts with tubulin to stabilize or destabilize microtubule tumor-associated molecules on a multiple daily dosing sched formation thereby inhibiting cell division. Examples of ule, such as at least two consecutive days, 10 consecutive microtubule binding agents that can be used in conjunction days, and so forth, for example for a period of weeks, months, with the disclosed therapy include, without limitation, pacli or years. In one example, the Subject is administered the taxel, docetaxel, vinblastine, Vindesine, Vinorelbine (navel therapeutic composition that a binding agent specific for one bine), the epothilones, colchicine, dolastatin 15, nocodazole, of the disclosed ovarian endothelial cell tumor-associated podophyllotoxin and rhizoxin. Analogs and derivatives of molecules daily for a period of at least30days, such as at least Such compounds also can be used and are known to those of 2 months, at least 4 months, at least 6 months, at least 12 ordinary skill in the art. For example, suitable epothilones and months, at least 24 months, or at least 36 months. epothilone analogs are described in International Publication 0205 The therapeutic compositions, such as those that No. WO 2004/018478. Taxoids, such as paclitaxel and doc include a binding agent specific for one of the ovarian endot etaxel, as well as the analogs of paclitaxel taught by U.S. Pat. helial cell tumor-associated molecules, can further include Nos. 6,610,860: 5,530,020; and 5,912,264 can be used. one or more biologically active or inactive compounds (or 0210. The following classes of compounds are of use in both). Such as anti-neoplastic agents and conventional non the methods disclosed herein: Suitable DNA and/or RNA toxic pharmaceutically acceptable carriers, respectively. transcription regulators, including, without limitation, acti 0206. In a particular example, a therapeutic composition nomycin D, daunorubicin, doxorubicin and derivatives and that includes a therapeutically effective amount of a binding analogs thereofalso are suitable for use in combination with agent specific for one of the disclosed ovarian endothelial cell the disclosed therapies. DNA intercalators and cross-linking tumor-associated molecules further includes one or more bio agents that can be administered to a subject include, without logically inactive compounds. Examples of such biologically limitation, cisplatin, carboplatin, oxaliplatin, mitomycins, inactive compounds include, but are not limited to: carriers, Such as mitomycin C, bleomycin, chlorambucil, cyclophos thickeners, diluents, buffers, preservatives, and carriers. The phamide and derivatives and analogs thereof. DNA synthesis pharmaceutically acceptable carriers useful for these formu inhibitors suitable for use as therapeutic agents include, with lations are conventional (see Remington’s Pharmaceutical out limitation, methotrexate, 5-fluoro-5'-deoxyuridine, Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 5-fluorouracil and analogs thereof. Examples of suitable 19th Edition (1995)). For solid compositions (for example, enzyme inhibitors include, without limitation, camptothecin, powder, pill, tablet, or capsule forms), conventional non-toxic etoposide, formestane, trichoStatin and derivatives and ana Solid carriers can include, for example, pharmaceutical logs thereof. Suitable compounds that affect gene regulation grades of mannitol, lactose, starch, or magnesium Stearate. In include agents that result in increased or decreased expres addition to biologically-neutral carriers, pharmaceutical sion of one or more genes, such as raloxifene, 5-azacytidine, compositions to be administered can include minor amounts 5-aza-2'-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, of non-toxic auxiliary Substances, such as wetting or emulsi mifepristone and derivatives and analogs thereof. Kinase fying agents, preservatives, and pH buffering agents and the inhibitors include Gleevac, Iressa, and Tarceva that prevent like, for example sodium acetate or Sorbitan monolaurate. phosphorylation and activation of growth factors. 0211. Other therapeutic agents, for example anti-tumor Additional Treatments agents, that may or may not fall under one or more of the 0207. In particular examples, prior to, during, or following classifications above, also are Suitable for administration in administration of a therapeutic amount of an agent that combination with the disclosed therapies. By way of reduces or inhibits ovarian cancer due to the interaction of a example, such agents include adriamycin, apigenin, rapamy binding agent with one of the disclosed ovarian endothelial cin, Zebularine, cimetidine, and derivatives and analogs cell tumor-associated molecules, the Subject can receive one thereof. or more other therapies. In one example, the Subject receives 0212. In one example, the therapeutic composition (Such one or more treatments to remove or reduce the tumor prior to as one including a binding agent specific for one or more of administration of a therapeutic amount of a composition the disclosed ovarian endothelial cell tumor-associated mol including a binding agent specific for one of the disclosed ecules) is injected into the Subject in the presence of an ovarian endothelial cell tumor-associated molecules. adjuvant. An adjuvant is an agent that when used in combi 0208 Examples of such therapies include, but are not lim nation with an immunogenic agent augments or otherwise ited to, surgical treatment for removal or reduction of the alters or modifies a resultant immune response. In some tumor (Such as Surgical resection, cryotherapy, or chemoem examples, an adjuvant increases the titer of antibodies US 2013/02743 15 A1 Oct. 17, 2013 20 induced in a Subject by the immunogenic agent. In one QIAGENR Inc (Germantown, Md.), INVITROGENR) example, the one or more peptides are administered to the (Carlsbad, Calif.), AMBION (Austin, Tex.), DHARMA Subject as an emulsion with an adjuvant and sterile water for CONR) (Lafayette, Colo.) or OPENBIOSYSTEMS(R) (Hunts injection (for example an intravenous or intramuscular injec ville, Ala.). tion). Incomplete Freund's Adjuvant (Seppic, Inc.) can be 0217. In certain examples, expression vectors are used as the Freund's Incomplete Adjuvant (IFA) (Fairfield, employed to express the at least one siRNA molecule. For N.J.). In some examples, IFA is provided in 3 ml of a mineral example, an expression vector can include a nucleic acid oil solution based on mannide oleate (Montanide ISA-51). At sequence encoding at least one siRNA molecule correspond the time of injection, the peptide(s) is mixed with the Mon ing to at least one of the disclosed ovarian endothelial cell tanide ISA.51 and then administered to the subject. Other tumor-associated molecules listed in Tables 1, 2, 4 and/or adjuvants can be used, for example, Freund's complete adju Table 5 that are upregulated in ovarian endothelial cell tumor vant, B30-MDP. LA-15-PH, montanide, saponin, aluminum cells. For example, siRNA specific for EZH2 can be gener hydroxide, alum, lipids, keyhole lympet protein, hemocya ated using publicly available EZH2 nucleic acid sequences, nin, a mycobacterial antigen, and combinations thereof. Such as those described above. In a particular example, the 0213. In some examples, the subject receiving the thera vector contains a sequence(s) encoding both strands of a peutic peptide composition (such as one including a binding siRNA molecule comprising a duplex. In another example, agent specific for one of the disclosed ovarian endothelial cell the vector also contains sequence(s) encoding a single nucleic tumor-associated molecules) is also administered interleu acid molecule that is self-complementary and thus forms a kin-2 (IL-2), for example via intravenous administration. In siRNA molecule. Non-limiting examples of such expression particular examples, IL-2 (Chiron Corp., Emeryville, Calif.) vectors are described in Paul et al., Nature Biotechnology is administered at a dose of at least 500,000 IU/kg as an 19:505, 2002: Miyagishi and Taira, Nature Biotechnology intravenous bolus over a 15 minute period every eight hours 19:497, 2002: Lee et al., Nature Biotechnology 19:500, 2002: beginning on the day after administration of the peptides and and Novina et al., Nature Medicine, online publication Jun. 3, continuing for up to 5 days. Doses can be skipped depending 2003. on Subject tolerance. 0218. In other examples, siRNA molecules include a 0214. In some examples, the disclosed compositions can delivery vehicle, including interalia liposomes, for adminis be co-administered with a fully human antibody to cytotoxic tration to a subject, carriers and diluents and their salts, and T-lymphocyte antigen-4 (anti-CTLA-4). In some example can be present in pharmaceutical compositions. Nucleic acid Subjects receive at least 1 mg/kg anti-CTLA-4 (such as 3 molecules can be administered to cells by a variety of meth mg/kg every 3 weeks or 3 mg/kg as the initial dose with ods known to those of skill in the art, including, but not Subsequent doses reduced to 1 mg/kg every 3 weeks). restricted to, encapsulation in liposomes, by iontophoresis, or 0215. In one example, at least a portion of the ovarian by incorporation into other delivery vehicles, such as hydro tumor (Such as a metastatic tumor) is Surgically removed (for gels, cyclodextrins, biodegradable nanocapsules, and bioad example via cryotherapy), irradiated, chemically treated (for hesive microspheres, or by proteinaceous vectors (see, for example via chemoembolization) or combinations thereof, example, O'Hare and Normand, International PCT Publica prior to administration of the disclosed therapies (such as tion No. WO 00/53722). In one specific example, siRNAs are administration of a binding agent specific for one of the administered at according to the teachings of Soutschek et al. disclosed ovarian endothelial cell tumor-associated mol (Nature Vol. 432: 173-178, 2004) or Karpillow et al. (Pharma ecules). For example, a Subject having a metastatic tumor can Genomics 32-40, 2004) both of which are hereinincorporated have all or part of the tumor Surgically excised prior to admin by reference in their entireties. istration of the disclosed therapies (such as one including a 0219. In some examples, siRNAs are incorporated into binding agent specific for one of the disclosed ovarian endot neutral liposomes, such as DOPC or chitosan, and injected helial cell tumor-associated molecules). In an example, one or intraperitoneal or intravenously. For example, a siRNA can be more chemotherapeutic agents is administered following administered at at least 1 ug/kg twice weekly, such as at least treatment with a binding agent specific for one of the dis 50 ug/kg twice weekly, at least 100 ug/kg twice weekly, at closed ovarian endothelial cell tumor-associated molecules. least 125ug/kg twice weekly, at least 150 ug/kg twice weekly, In another particular example, the Subject has a metastatic at least 200 g/kg twice weekly for at least 1 week, Such as at tumor and is administered radiation therapy, chemoemboliza least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 tion therapy, or both concurrently with the administration of weeks, at least 12 weeks, or at least 24 weeks. In one example, the disclosed therapies (such as one including a binding agent about at least 1-500 ug/kg, Such 10-250 ug/kg, is adminstered specific for one of the disclosed ovarian endothelial cell at least twice weekly for at least 1 week, such as at least 2 tumor-associated molecules). weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 12 weeks, or at least 24 weeks. In a certain example, Generation and Administration of siRNA approximately 150 ug/kg is administered twice weekly, for 2 0216. In an example, certain inhibitors provided by this to 3 weeks. In other examples, approximately 1 ug/kg daily disclosure are species of siRNAs. One of ordinary skill in the for 3 weeks or 50 ug/kg every other day for 3 weeks is art can readily generate siRNAs which specifically bind to administered. one of the disclosed ovarian endothelial cell tumor-associated 0220 Alternatively, the nucleic acid/vehicle combination molecules that are upregulated in ovarian endothelial cell can be locally delivered by direct injection or by use of an tumor cells. In an example, commercially available kits, such infusion pump. Direct injection of the nucleic acid molecules as siRNA molecule synthesizing kits from PROMEGAR) of the disclosure, whether subcutaneous, intramuscular, or (Madison, Wis.) or AMBIONR) (Austin,Tex.) may be used to intradermal, can take place using standard needle and Syringe synthesize siRNA molecules. In another example, siRNAs methodologies, or by needle-free technologies such as those are obtained from commercial sources, such as from described by Barry et al., International PCT Publication No. US 2013/02743 15 A1 Oct. 17, 2013
WO99/31262. Other delivery routes, but are not limited to, cell tumor-associated molecules. For example, the antibody oral delivery (such as in tablet or pill form), intrathecal or can bind the specific ovarian endothelial cell tumor-associ intraperitoneal delivery. For example, intraperitoneal deliv ated molecules with an affinity constant of at least 10 M', ery can take place by injecting the treatment into the perito such as at least 107M", at least 10 M', at least 5x10 M', neal cavity of the subject in order to directly deliver the or at least 10 M'. molecules to the tumor site. More detailed descriptions of 0226. The specificantibodies can include a V, polypeptide nucleic acid delivery and administration are provided in Sul having amino acid sequences of the complementarity deter livan et al., PCT WO 94/02595, Draper et al., PCT WO93/ mining regions (CDRs) that are at least about 90% identical, 23569, Beigelman et al., PCT WO99/05094, and Klimuket such as at least about 95%, at least about 98%, or at least about al., PCT WO99/04819, all of which are incorporated by ref 99% identical to the amino acid sequences of the specific erence herein. ovarian endothelial cell tumor-associated molecules and a V 0221 Alternatively, certain siRNA molecules can be polypeptide having amino acid sequences of the CDRS that expressed within cells from eukaryotic promoters. Those are at least about 90% identical, such as at least about 95%, at skilled in the art will recognize that any nucleic acid can be least about 98%, or at least about 99% identical to the amino expressed in eukaryotic cells using the appropriate DNA/ acid sequences of the specific ovarian endothelial cell tumor RNA vector. The activity of such nucleic acids can be aug associated molecules. mented by their release from the primary transcript by an 0227. In one example, the sequence of the specificity enzymatic nucleic acid (Draperet al., PCTWO 93/23569, and determining regions of each CDR is determined. Residues Sullivan et al., PCTWO 94/02595). that are outside the SDR (non-ligand contacting sites) are 0222. In other examples, siRNA molecules can be substituted. For example, in any of the CDR sequences, at expressed from transcription units (see for example, Couture most one, two or three amino acids can be substituted. The et al., 1996, TIG 12:510) inserted into DNA or RNA vectors. production of chimeric antibodies, which include a frame The recombinant vectors can be DNA plasmids or viral vec work region from one antibody and the CDRs from a different tors. siRNA expressing viral vectors can be constructed based antibody, is well known in the art. For example, humanized on, for example, but not limited to, adeno-associated virus, antibodies can be routinely produced. The antibody or anti retrovirus, adenovirus, lentivirus or alphavirus. In another body fragment can be a humanized immunoglobulin having example, pol III based constructs are used to express nucleic CDRs from a donor monoclonal antibody that binds one of acid molecules (see for example, Thompson, U.S. Pat. Nos. the disclosed ovarian endothelial cell tumor-associated mol 5,902,880 and 6,146,886). ecules and immunoglobulin and heavy and light chain vari 0223) The recombinant vectors capable of expressing the able region frameworks from human acceptor immunoglobu siRNA molecules can be delivered as described above, and lin heavy and light chain frameworks. Generally, the persist in target cells. Alternatively, viral vectors can be used humanized immunoglobulin specifically binds to one of the that provide for transient expression of nucleic acid mol disclosed ovarian endothelial cell tumor-associated mol ecules. Such vectors can be repeatedly administered as nec ecules with an affinity constant of at least 107M", such as at essary. Once expressed, the siRNA molecule interacts with least 10 M' at least 5x10'M' or at least 10 M'. the target mRNA and generates an RNAi response. Delivery 0228. In another example, human monoclonal antibodies of siRNA molecule expressing vectors can be systemic, Such to the disclosed ovarian endothelial cell tumor-associated as by intravenous or intramuscular administration, by admin molecules in Tables 1, 2, 4 and 5 that are upregulated in istration to target cells ex-planted from a subject followed by ovarian endothelial tumor cells are produced. Human mono reintroduction into the Subject, or by any other means that clonal antibodies can be produced by transferring donor would allow for introduction into the desired target cell. complementarity determining regions (CDRS) from heavy and light variable chains of the donormouse immunoglobulin Generation of Antibodies into a human variable domain, and then Substituting human 0224 One of ordinary skill in the art can readily generate residues in the framework regions when required to retain antibodies which specifically bind to the disclosed ovarian affinity. The use of antibody components derived from endothelial cell tumor-associated molecules. These antibod humanized monoclonal antibodies obviates potential prob ies can be monoclonal or polyclonal. They can be chimeric or lems associated with the immunogenicity of the constant humanized. Any functional fragment or derivative of an anti regions of the donor antibody. For example, when mouse body can be used including Fab, Fab', Fab2, Fab'2, and single monoclonal antibodies are used therapeutically, the develop chain variable regions. So long as the fragment or derivative ment of human anti-mouse antibodies (HAMA) leads to retains specificity of binding for the ovarian endothelial cell clearance of the murine monoclonal antibodies and other tumor-associated molecule it can be used in the methods possible adverse events. Chimeric monoclonal antibodies, provided herein. Antibodies can be tested for specificity of with human constant regions, humanized monoclonal anti binding by comparing binding to appropriate antigen to bind bodies, retaining only murine CDRs, and “fully human” ing to irrelevant antigen or antigen mixture under a given set monoclonal antibodies made from phage libraries or trans of conditions. If the antibody binds to appropriate antigen at genic mice have all been used to reduce or eliminate the least 2, at least 5, at least 7 or 10 times more than to irrelevant murine content of therapeutic monoclonal antibodies. antigenorantigen mixture, then it is considered to be specific. 0229 Techniques for producing humanized monoclonal 0225. In an example, monoclonal antibodies are generated antibodies are described, for example, by Jones et al., Nature to the ovarian endothelial cell tumor-associated molecules 321:522, 1986; Riechmann et al., Nature 332:323, 1988; Ver disclosed in Tables 1, 2, 4 or 5 that are upregulated in ovarian hoeyen et al., Science 239:1534, 1988; Carter et al., Proc. endothelial cell tumor cells. These monoclonal antibodies Natl. Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. each include a variable heavy (V) and a variable light (V) Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150: chain and specifically bind to the specific ovarian endothelial 2844, 1993. The antibody may be of any isotype, but in US 2013/02743 15 A1 Oct. 17, 2013 22 several embodiments the antibody is an IgG, including but not polypeptide chain with a linker peptide bridging the two V limited to, IgG, IgG, IgGs and IgG4. domains. Methods for producing scFVs are known in the art 0230. In one example, the sequence of the humanized (see Whitlow et al., Methods: a Companion to Methods in immunoglobulin heavy chain variable region framework can Enzymology, Vol. 2, page 97, 1991; Bird et al., Science 242: beat least about 65% identical to the sequence of the donor 423, 1988: U.S. Pat. No. 4,946,778; Packet al., Bio/Technol immunoglobulin heavy chain variable region framework. ogy 11:1271, 1993; and Sandhu, supra). Thus, the sequence of the humanized immunoglobulin heavy 0233 Antibody fragments can be prepared by proteolytic chain variable region framework can be at least about 75%, at hydrolysis of the antibody or by expression in E. coli of DNA least about 85%, at least about 99% or at least about 95%, encoding the fragment. Antibody fragments can be obtained identical to the sequence of the donor immunoglobulin heavy by pepsin or papain digestion of whole antibodies by conven chain variable region framework. Human framework regions, tional methods. For example, antibody fragments can be pro and mutations that can be made in a humanized antibody duced by enzymatic cleavage of antibodies with pepsin to framework regions, are known in the art (see, for example, in provide a 5S fragment denoted F(ab'). This fragment can be U.S. Pat. No. 5,585,089). further cleaved using a thiol reducing agent, and optionally a 0231. Antibodies, such as murine monoclonal antibodies, blocking group for the Sulfhydryl groups resulting from chimeric antibodies, and humanized antibodies, include full cleavage of disulfide linkages, to produce 3.5S Fab' monova length molecules as well as fragments thereof. Such as Fab, lent fragments. Alternatively, an enzymatic cleavage using F(ab'), and Fv, which include a heavy chain and light chain pepsin produces two monovalent Fab' fragments and an Fc variable region and are capable of binding the epitopic deter fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat. minant. These antibody fragments retain some ability to No. 4331,647, and references contained therein; Nisonhoff selectively bind with their epitope. These fragments include: et al., Arch. Biochem. Biophy's. 89:230, 1960; Porter, Bio (1) Fab, the fragment which contains a monovalent antigen chem. J. 73:119, 1959; Edelman et al., Methods in Enzymol binding fragment of an antibody molecule, can be produced ogy, Vol. 1, page 422. Academic Press, 1967; and Coligan et by digestion of whole antibody with the enzyme papain to al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4). yield an intact light chainanda portion of one heavy chain; (2) 0234. Other methods of cleaving antibodies, such as sepa Fab', the fragment of an antibody molecule can be obtained by ration of heavy chains to form monovalent light-heavy chain treating whole antibody with pepsin, followed by reduction, fragments, further cleavage of fragments, or other enzymatic, to yield an intact light chain and a portion of the heavy chain; chemical, or genetic techniques may also be used, so long as two Fab' fragments are obtained per antibody molecule; (3) the fragments bind to the antigen that is recognized by the (Fab'), the fragment of the antibody that can be obtained by intact antibody. treating whole antibody with the enzyme pepsin without sub 0235. One of skill will realize that conservative variants of sequent reduction; F(ab') is a dimer of two Fab' fragments the antibodies can be produced. Such conservative variants held together by two disulfide bonds; (4) Fv, a genetically employed in antibody fragments, such as dsEV fragments or engineered fragment containing the variable region of the in ScFV fragments, will retain critical amino acid residues light chain and the variable region of the heavy chain necessary for correct folding and stabilizing between the V expressed as two chains; and (5) Single chain antibody (Such and the V, regions, and will retain the charge characteristics as ScFV), defined as a genetically engineered molecule con of the residues in order to preserve the low pland low toxicity taining the variable region of the light chain, the variable of the molecules. Amino acid Substitutions (such as at most region of the heavy chain, linked by a suitable polypeptide one, at most two, at most three, at most four, or at most five linker as a genetically fused single chain molecule. Methods amino acid Substitutions) can be made in the V and the V, of making these fragments are known in the art (see for regions to increase yield. Conservative amino acid substitu example, Harlow and Lane, Antibodies. A Laboratory tion tables providing functionally similar amino acids are Manual, Cold Spring Harbor Laboratory, NewYork, 1988. Fv well known to one of ordinary skill in the art. The following antibodies are typically about 25kDa and contain a complete six groups are examples of amino acids that are considered to antigen-binding site with three CDRs per each heavy chain be conservative substitutions for one another: 1)Alanine (A), and each light chain. To produce these antibodies, the V and Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid the V, can be expressed from two individual nucleic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), constructs in a host cell. If the V and the V, are expressed Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), non-contiguously, the chains of the Fv antibody are typically Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryp held together by noncovalent interactions. However, these tophan (W). chains tend to dissociate upon dilution, so methods have been 0236. In some examples, naked antibodies can be admin developed to crosslink the chains through glutaraldehyde, istered at at least 5 mg per kg every two weeks, such as at least intermolecular disulfides, or a peptide linker Thus, in one 10 mg per kg, at least 25 mg per kg, at least 30 mg per kg, at example, the Fv can be a disulfide stabilized Fv (dsEv), least 50 mg per kg (for example, 6, 7, 8, 9, 10, 15, 20, 25, 30. wherein the heavy chain variable region and the light chain 35, 40, 45, 50 mg per kg), at least once a week, at least once variable region are chemically linked by disulfide bonds. every 2 weeks, at least once every 3 weeks, or at least once 0232. In an additional example, the Fv fragments include every month depending upon the ovarian cancer. In an V, and V, chains connected by a peptide linker These single example, the antibodies are administered continuously. In chain antigen binding proteins (ScFV) are prepared by con another example, antibodies or antibody fragments conju structing a structural gene comprising DNA sequences gated to cytotoxic agents (immunotoxins) are administered at encoding the V and V, domains connected by an oligonucle at least 10 ug per kg, Such as at least 20, at least 30, at least 50, otide. The structural gene is inserted into an expression vec at least 70, at least 100g per kg, at least twice a week, at least tor, which is Subsequently introduced into a host cell Such as once a week, at least once every two weeks, at least once every E. coli. The recombinant host cells synthesize a single month depending upon the ovarian cancer. In one example, 50 US 2013/02743 15 A1 Oct. 17, 2013
ug per kg is administered twice a week for 2 to 3 weeks. In example at least 3-fold, at least 4-fold, at least 6-fold, or at other examples, the Subject is administered the therapeutic least 10-fold as compared to a control. Exemplary ovarian composition that a binding agent specific for one or more of endothelial cell tumor-associated molecules that are up-regu the disclosed ovarian endothelial cell tumor-associated mol lated in ovarian tumor endothelial cells are presented in ecules daily for a period of at least 30 days, such as at least 2 Tables 1, 2, 4 and 5. Thus, a decrease in the expression of one months, at least 4 months, at least 6 months, at least 12 or more of the molecules listed in Tables 1, 2, 4 or 5 that is months, at least 24 months, or at least 36 months. Subjects can noted as being up-regulated in ovarian tumor endothelial cells be monitored by methods known to those skilled in the art to following treatment indicates that the agent is of use for determine ovariantumorresponsiveness to the antibody treat treating the ovarian cancer. ment. The subject can be monitored by non invasive tech 0242 Exemplary ovarian endothelial cell tumor-associ niques such as CT or MRI imaging to assess tumor response. ated molecules that are down-regulated in ovarian tumor It is contemplated that additional agents can be administered, endothelial cells are presented in Table 1 with specific Such as antineoplastic agents in combination with or follow examples provided in Table 3. Thus, an increase in the expres ing treatment with the antibodies. sion of one or more of the molecules listed in Table 3 follow 0237 Methods of Evaluating the Effectiveness of an Ova ing administration indicates that the agent is effective for the rian Tumor Treatment treatment of ovarian cancer. 0238 Methods are disclosed herein for determining the 0243 In another example, an alteration can include pro effectiveness of a binding agent specific for one of the dis cesses that increase transcription of a gene or translation of closed ovarian endothelial cell tumor-associated molecules) mRNA. Gene up-regulation includes any detectable increase for the treatment of an ovarian tumor in a subject with the in the production of a gene product. In certain examples, ovarian tumor. In an example, the method includes detecting production/expression of a gene product increases by at least expression of an ovarian endothelial cell tumor-associated 2-fold, for example at least 3-fold or at least 4-fold, at least molecule in a sample from the Subject following administra 6-fold, at least 10-fold or at least 28-fold following treatment tion of the binding agent (such as an siRNA), for example at as compared to a control. least 24 hours, at least 1 week, at least 2 weeks, or at least 4 weeks following administration of the agent. The expression Detection of Ovarian Endothelial Cell Tumor-Associated of the ovarian endothelial cell tumor-associated molecule Nucleic Acids following administration can be compared to a control. Such 0244. Nucleic acids can be detected by any method known as a reference value. An alteration in the expression of the in the art. In some examples, nucleic acids are isolated, ampli ovarian endothelial cell tumor-associated molecule relative to fied, or both, prior to detection. In an example, the biological the control following administration indicates that the agent is sample can be incubated with primers that permit the ampli effective for the treatment of the ovarian cancer in the subject. fication of one or more of the disclosed ovarian endothelial 0239. In a specific example, the method includes detecting cell tumor-associated mRNAs, under conditions sufficient to and comparing the protein expression levels of the ovarian permit amplification of such products. For example, the bio endothelial cell tumor-associated molecules. In other logical sample is incubated with probes that can bind to one or examples, the method includes detecting and comparing the more of the disclosed ovarian endothelial cell tumor-associ mRNA expression levels of the ovarian endothelial cell ated nucleic acid sequences (such as cDNA, genomic DNA, tumor-associated molecules. In certain examples, the treat or RNA (such as mRNA)) under high stringency conditions. ment is considered effective if the expression levels are The resulting hybridization can then be detected using meth altered by at least 2-fold, such as by at least 3-fold, at least ods known in the art. In one example, the effectiveness of an 4-fold, at least 6-fold or at least 10-fold relative to the control. ovarian tumor treatment is identified by applying isolated 0240. In one example, the specific ovarian endothelial cell nucleic acid molecules to an array in which the isolated tumor-associated molecule is detected in a biological sample. nucleic acid molecules are obtained from a biological sample In a particular example, the biological sample is a tumor including ovarian endothelial cancer cells following treat biopsy. In another example, the ovarian endothelial cell ment with the ovarian tumor treatment. In Such example, the tumor-associated molecule is detected in a serum sample. For array includes oligonucleotides complementary to all ovarian example, the ovarian endothelial cell tumor-associated mol endothelial cell tumor-associated genes listed in Table 1. In a ecule is detected in a serum sample if the specific molecule is particular example, the array is a commercially available known to be secreted or located on a cell surface susceptible to enzymatic cleavage. array such as a U133 Plus 2.0 oligonucleotide array from AFFYMETRIXR (AFFYMETRIX(R), Santa Clara, Calif.). 0245. In an example, the isolated nucleic acid molecules Altering Ovarian Endothelial Cell Tumor-Associated are incubated with the array including oligonucleotides Molecules Activity Such as Expression complementary to the ovarian endothelial cell tumor-associ 0241. In an example, an alteration in the expression of one ated molecules that are up-regulated in ovarian tumor endot or more of the disclosed ovarian endothelial cell tumor-asso helial cells, such as those listed in Table 2, Table 3, Table 4 ciated molecules following administration includes an and/or Table 5 for a time sufficient to allow hybridization increase or decrease in production of a gene product/expres between the isolated nucleic acid molecules and oligonucle Sion, Such as RNA or protein. For example, an alteration can otide probes, thereby forming isolated nucleic acid molecule: include processes that downregulate or decrease transcription oligonucleotide complexes. The isolated nucleic acid mol of a gene or translation of mRNA whose expression or activ ecule:oligonucleotide complexes are then analyzed to ity is increased in ovarian endothelial tumor cells. Gene determine if expression of the isolated nucleic acid molecules downregulation includes any detectable decrease in the pro is altered. In Such example, an ovarian tumor treatment is duction of a gene product. In certain examples, production/ effective if a decrease in the expression of ovarian endothelial expression of a gene product decreases by at least 2-fold, for tumor-associated molecules is observed as compared to a US 2013/02743 15 A1 Oct. 17, 2013 24 control (such as a normal endothelial cell) or reference value. absence of the specific ovarian endothelial cell tumor-associ In an additional example, the array includes oligonucleotides ated molecule. In other examples, the biological sample is complementary to the ovarian endothelial cell tumor-associ analyzed by mass spectrometry for the presence or absence of ated molecules that down-regulated in ovarian tumor endot the specific ovarian endothelial cell tumor-associated mol helial cells, such as those listed in Table 3. In this example, an ecule. ovarian tumor treatment is effective if an increase in the 0250 In one example, the antibody that specifically binds expression of one or more ovarian endothelial tumor-associ an ovarian endothelial cell tumor-associated molecule (Such ated molecules is observed. as those listed in Table 1) is directly labeled with a detectable label. In another example, each antibody that specifically Gene Expression Profile binds an ovarian endothelial cell tumor-associated molecule (the first antibody) is unlabeled and a second antibody or 0246 A gene expression profile is disclosed herein that other molecule that can bind the human antibody that specifi can be used to identify the effectiveness of an ovarian tumor cally binds the respective ovarian endothelial cell tumor treatment. In an example, the gene expression profile includes associated molecule is labeled. As is well known to one of at least two of the ovarian endothelial cell tumor-associated skill in the art, a second antibody is chosen that is able to molecules listed in Table 1, such as at least 5, at least 7, at least specifically bind the specific species and class of the first 10, at least 20, at least 30, at least 40, at least 50, at least 60, antibody. For example, if the first antibody is a human IgG, at least 70, at least 80, at least 90, at least 100, at least 150, at then the secondary antibody can be an anti-human-IgG. Other least 200, at least 250, at least 300, at least 400, at least 500, molecules that can bind to antibodies include, without limi at least 600, at least 700, at least 800, at least 900, at least tation, Protein A and Protein G, both of which are available 1000, or at least 1100 molecules (for example, 2, 6, 8, 9, 10, commercially. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50,55, 0251 Suitable labels for the antibody or secondary anti 60, 65, 70, 75, 80, 85,90, 95, 100,105, 110, 120, 130, 140, body include various enzymes, prosthetic groups, fluorescent 150, 175, 225, 275,325, 350, 375,450, 550, 650, 750, 850, materials, luminescent materials, magnetic agents and radio 950, 1050 or 1149 of those listed). active materials. Non-limiting examples of Suitable enzymes 0247. In a particular example, the gene expression profile include horseradish peroxidase, alkaline phosphatase, beta includes at least 2, at least 5, at least 7, at least 10, at least 20, galactosidase, or acetylcholinesterase. Non-limiting at least 25, at least 27 molecules (for example, 2, 3, 4, 5, 6, 7, examples of Suitable prosthetic group complexes include 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, streptavidin/biotin and avidin/biotin. Non-limiting examples 27, 28 or 29 molecules) listed in Table 2, Table 4 and/or Table of suitable fluorescent materials include umbelliferone, fluo 5 that are associated with an at least six-fold increase in rescein, fluorescein isothiocyanate, rhodamine, dichlorotri expression in tumor endothelial cells. In a particular example, azinylamine fluorescein, dansylchloride orphycoerythrin. A the at least two molecules include EGFL6 and TNFAIP6. In non-limiting exemplary luminescent material is luminol; a other particular examples, the at least two ovarian endothelial non-limiting exemplary magnetic agent is gadolinium, and cell tumor-associated molecules include EGFL6, TNFAIP6, non-limiting exemplary radioactive labels include 'I. 'I, TWIST1, STC1, HOP, CSPG2, and PLXDC1. SS or H. 0248. In other particular examples, the gene expression 0252. In an alternative example, ovarian endothelial cell profile includes at least 2, at least 5, at least 7, at least 10, at tumor-associated molecules can be assayed in a biological least 13, or at least 15 molecules (for example, 2, 3, 4, 5, 6, 7, sample by a competition immunoassay utilizing ovarian 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 molecules) that are endothelial cell tumor-associated molecule standards labeled down-regulated in ovarian tumor endothelial cells as listed in with a detectable substance and unlabeled antibody that spe Table 3. For example, the profile includes the seventeen ova cifically bind to the desired ovarian endothelial cell tumor rian endothelial cell tumor-associated molecules listed in associated molecule. In this assay, the biological sample Table 3. (such as serum, tissue biopsy, or cells isolated from a tissue biopsy), the labeled ovarian endothelial cell tumor-associated Detecting Ovarian Endothelial Cell Tumor-Associated molecule standards and the antibody that specifically binds to Proteins ovarian endothelial cell tumor-associated molecule are com 0249. As an alternative to analyzing the sample for the bined and the amount of labeled ovarian endothelial cell presence of nucleic acids, the presence of proteins can be tumor-associated molecule standard bound to the unlabeled determined. Proteins can be detected by any method known in antibody is determined. The amount of ovarian endothelial the art. In some examples, proteins are purified prior to detec cell tumor-associated molecule in the biological sample is tion. For example, the effect of an ovarian tumor treatment inversely proportional to the amount of labeled ovarian endot can be determined by incubating the biological sample with helial cell tumor-associated molecule standard bound to the one or more antibodies that specifically binds to one of the antibody that specifically binds the ovarian endothelial cell disclosed ovarian endothelial cell tumor-associated proteins tumor-associated molecule. encoded by the genes listed in Tables 1, Table 2, Table 3, Table 4 or Table 5 to detect expression. The primary antibody can Identifying Agents to Treat Ovarian Cancer include a detectable label. For example, the primary antibody 0253 Methods are provided herein for identifying agents can be directly labeled, or the sample can be subsequently to treat an ovarian cancer. For example, agents that decrease incubated with a secondary antibody that is labeled (for expression or activity of a gene that is upregulated in ovarian example with a fluorescent label). The label can then be endothelial tumor cells (such as those listed in Tables 2 and 4), detected, for example by microscopy, ELISA, flow cytom as well as agents that increase activity of a gene that is down etery, or spectrophotometry. In another example, the biologi regulated in ovarian endothelial tumor cells (such as those cal sample is analyzed by Western blotting for the presence or listed in Table 3), can be identified using these methods. In an US 2013/02743 15 A1 Oct. 17, 2013
example, the method includes contacting an ovarian tumor ovarian endothelial cell tumor-associated protein encoded by endothelial cell with one or more test agents under conditions one of the genes listed in any of Tables 1, 2, 4 or 5 that are sufficient for the one or more test agents to alter the activity of upregulated in ovarian endothelial tumor cells. In a particular at least one ovarian endothelial cell tumor-associated mol example, the antibody is directed to an ovarian endothelial ecule listed in any of Tables 1-5. It is contemplated that cell tumor-associated protein encoded by one of the genes several doses of the agent can be tested and then expression listed in Tables 2, 4 or 5 that is upregulated in ovarian endot levels of nucleic acids or proteins can be determined. The helial tumor cells and which is involved in angiogenesis, Such method also includes detecting the activity or expression of as a gene that is involved in at least one of cell proliferation, the at least one ovarian endothelial cell tumor-associated tube formation or cell motility. In another example, the test molecule in the presence and absence of the one or more test agent is a nucleic acid encoding one or more of the proteins agents. The activity or expression of the at least one ovarian listed in Table 3. For example, the nucleic acid can be part of endothelial cell tumor-associated molecule in the presence of a vector Suitable for gene therapy. the one or more test agents is then compared to the activity or expression of the at least one ovarian endothelial cell tumor Altering Ovarian Endothelial Cell Tumor-Associated associated molecule in the absence of Such agents to deter Molecules Activity mine if there is differential expression of the at least one ovarian endothelial cell tumor associated molecule. In several 0258. In an example, an alteration in the activity of one or examples, differential expression of the ovarian endothelial more of the disclosed ovarian endothelial cell tumor-associ cell tumor-associated molecule in the presence of the agent ated molecules includes an increase or decrease in production (as compared to expression in the absence of the agent) indi of a gene product, such as RNA or protein. For example, an cates that the one or more test agents is of use to treat the alteration can include processes that downregulate or ovarian tumor. decrease transcription of a gene or translation of mRNA. 0254. In an example, determining whether there is differ Gene downregulation includes any detectable decrease in the ential expression of one or more ovarian endothelial cell production of a gene product. In certain examples, produc tumor-associated molecules includes generating a gene tion/expression of a gene product decreases by at least 2-fold, expression profile for the Subject. For example, a gene expres for example at least 3-fold, at least 4-fold, at least 6-fold, or at least 10-fold as compared to a control (such as a reference sion profile for the Subject can be generated by using an array value or a normal endothelial cell). For example, a decrease in of molecules including an ovarian endothelial cell tumor one or more of the disclosed ovarian endothelial cell tumor associated expression profile. associated molecules up-regulated in ovarian tumor endothe Ovarian Endothelial Cell Tumor-Associated Molecules lial cells (such as those listed in Tables 2, 0259 4 and 5), is indicative of an agent that is effective at 0255 Ovarian endothelial cell tumor-associated mol treating ovarian cancer. ecules can include nucleic acid sequences (such as DNA, 0260. In another example, an alteration can include pro cDNA, or mRNAs) and proteins. In a specific example, cesses that increase transcription of a gene or translation of detecting differential expression of the ovarian endothelial mRNA. Gene up-regulation includes any detectable increase cell tumor-associated molecules includes detecting differen in the production of a gene product. In certain examples, tial mRNA expression of the disclosed ovarian endothelial production/expression of a gene product increases by at least cell tumor-associated molecules. For example, Such differen 2-fold, for example at least 3-fold or at least 4-fold, at least tial expression can be measured by real time quantitative 6-fold, at least 10-fold or at least 28-fold as compared to a polymerase chain reaction or microarray analysis or other control. For example, an increase in one or more of the dis methods known in the art. In another example, detecting closed ovarian endothelial cell tumor-associated molecules differential expression of the ovarian endothelial cell tumor down-regulated in ovarian tumor endothelial cells (such as associated molecules includes detecting differential protein those listed in Table 3) is indicative of an agent that is effective expression of the disclosed ovarian endothelial cell tumor at treating ovarian cancer. associated molecules. For example, protein differential expression is measured by Western blot analysis or a protein Detection of Ovarian Endothelial Cell Tumor-Associated microarray. Nucleic Acids Test Agents 0261 Nucleic acids can be detected by any method known in the art, Such as those described above. In one example, a 0256 The one or more test agents can be any substance, therapeutic agent is identified by applying isolated nucleic including, but not limited to, a protein (Such as an antibody), acid molecules to an array in which the isolated nucleic acid a nucleic acid molecule (such as a siRNA), an organic com molecules are obtained from a biological sample including pound, an inorganic compound, a small molecule or any other ovarian endothelial cancer cells following treatment with the molecule of interest. In a particular example, the test agent is one or more test agents. In Such example, the array includes a siRNA that reduces or inhibits the activity (such as the oligonucleotides complementary to all ovarian endothelial expression) of one of the ovarian endothelial cell tumor cell tumor-associated genes listed in Table 1. In a particular associated molecules listed in Tables 2, 4 or 5. For example, example, the array is a commercially available array Such as the siRNA is directed to an ovarian endothelial cell tumor a U133 Plus 2.0 oligonucleotide array from AFFYMETRIX(R) associated molecule listed in Table 2, 4 or 5 which is involved (AFFYMETRIX(R), Santa Clara, Calif.). in angiogenesis, such as a molecule that is involved in at least 0262. In an example, the isolated nucleic acid molecules one of cell proliferation, tube formation or cell motility. are incubated with the array including oligonucleotides 0257. In other examples, the test agent is an antibody. For complementary to the ovarian endothelial cell tumor-associ example, the antibody is directed to specifically bind to an ated molecules listed in Table 2, 4 and/or 5 for a time suffi US 2013/02743 15 A1 Oct. 17, 2013 26 cient to allow hybridization between the isolated nucleic acid can be directly labeled, or the sample can be subsequently molecules and oligonucleotide probes, thereby forming iso incubated with a secondary antibody that is labeled (for lated nucleic acid molecule:oligonucleotide complexes. The example with a fluorescent label). The label can then be isolated nucleic acid molecule: oligonucleotide complexes detected, for example by microscopy, ELISA, flow cytom are then analyzed to determine if expression of the isolated etery, or spectrophotometry. In another example, the biologi nucleic acid molecules is altered. In Such example, an agent is cal sample is analyzed by Western blotting for the presence or considered is effective if the test agent decreases expression absence of the specific ovarian endothelial cell tumor-associ of ovarian endothelial tumor-associated molecules upregu ated molecule. In some examples, the biological sample is lated in ovarian endothelial cell tumors relative to the absence analyzed by mass spectrometry. of the agent (such as a decrease of at least 2-, 3-, 4-, 5- or 0267 In one example, the antibody that specifically binds 10-fold). Similarly, an agent is considered is effective if the an ovarian endothelial cell tumor-associated molecule (Such test agent increases expression of ovarian endothelial tumor as those listed in Table 1) is directly labeled with a detectable associated molecules downregulated in ovarian endothelial label. In another example, each antibody that specifically cell tumors relative to the absence of the agent (such as an binds an ovarian endothelial cell tumor-associated molecule increase of at least 2-, 3-, 4-, 5- or 10-fold). (the first antibody) is unlabeled and a second antibody or other molecule that can bind the human antibody that specifi Gene Expression Profile cally binds the respective ovarian endothelial cell tumor associated molecule is labeled. As is well known to one of 0263. The disclosed gene profile (as described above) can skill in the art, a second antibody is chosen that is able to also be used to identify agents to treat an ovarian tumor. Such specifically bind the specific species and class of the first as a cancer, in a subject. In an example, the gene expression antibody. For example, if the first antibody is a human IgG, profile includes at least two of the ovarian endothelial cell then the secondary antibody can be an anti-human-IgG. Other tumor-associated molecules listed in Table 1. Such as at least molecules that can bind to antibodies include, without limi 5, at least 7, at least 10, at least 20, at least 30, at least 40, at tation, Protein A and Protein G, both of which are available least 50, at least 60, at least 70, at least 80, at least 90, at least commercially. 100, at least 150, at least 200, at least 250, at least 300, at least 0268 Suitable labels for the antibody or secondary anti 400, at least 500, at least 600, at least 700, at least 800, at least body include various enzymes, prosthetic groups, fluorescent 900, at least 1000, or at least 1100 molecules (for example, 2. materials, luminescent materials, magnetic agents and radio 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, active materials. Non-limiting examples of suitable enzymes 45, 50,55, 60, 65,70, 75, 80, 85,90, 95, 100,105, 110, 120, include horseradish peroxidase, alkaline phosphatase, beta 130, 140, 150, 175, 225, 275,325, 350, 375, 450, 550, 650, galactosidase, or acetylcholinesterase. Non-limiting 750, 850, 950, 1050 or 1149 of those listed). examples of Suitable prosthetic group complexes include 0264. In a particular example, the gene expression profile streptavidin/biotin and avidin/biotin. Non-limiting examples includes at least 2, at least 5, at least 7, at least 10, at least 20, of suitable fluorescent materials include umbelliferone, fluo at least 25, or at least 27 molecules (for example, 2, 3, 4, 5, 6, rescein, fluorescein isothiocyanate, rhodamine, dichlorotri 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 25, 26, azinylamine fluorescein, dansylchloride orphycoerythrin. A 27, 28 or 29 molecules) listed in Table 2, 4 and/or 5 that are non-limiting exemplary luminescent material is luminol; a associated with an at least six-fold increase in expression in non-limiting exemplary magnetic agent is gadolinium, and tumor endothelial cells. In a particular example, the at least non-limiting exemplary radioactive labels include I, I, two molecules include EGFL6 and TNFAIP6. In other par SS or H. ticular examples, the at least two ovarian endothelial cell 0269. In an alternative example, ovarian endothelial cell tumor-associated molecules include EGFL6, TNFAIP6, tumor-associated molecules can be assayed in a biological TWIST1, STC1, HOP, CSPG2, and PLXDC1. sample by a competition immunoassay utilizing ovarian 0265. In other particular examples, the gene expression endothelial cell tumor-associated molecule standards labeled profile includes at least 2, at least 5, at least 7, at least 10, at with a detectable substance and an unlabeled antibody that least 13, or at least 15 molecules (for example, 2, 3, 4, 5, 6, 7, specifically binds the desired ovarian endothelial cell tumor 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 molecules) that are associated molecule. In this assay, the biological sample down-regulated in ovarian tumor endothelial cells as listed in (such as serum, tissue biopsy, or cells isolated from a tissue Table 3. For example, the profile includes the seventeen ova biopsy), the labeled ovarian endothelial cell tumor-associated rian endothelial cell tumor-associated molecules listed in molecule standards and the antibody that specifically binds Table 3. the desired ovarian endothelial cell tumor-associated mol ecule are combined and the amount of labeled ovarian endot Detecting Ovarian Endothelial Cell Tumor-Associated helial cell tumor-associated molecule standard bound to the Proteins unlabeled antibody is determined. The amount of ovarian 0266. As an alternative to analyzing the sample for the endothelial cell tumor-associated molecule in the biological presence of nucleic acids, the presence of proteins can be sample is inversely proportional to the amount of labeled determined using any method known in the art. In some ovarian endothelial cell tumor-associated molecule standard examples, proteins are purified before detection. For bound to the antibody that specifically binds the ovarian example, the effect of one or more test agents on an ovarian endothelial cell tumor-associated molecule. tumor can be determined by incubating the biological sample with an antibody that specifically binds to one of the disclosed Methods of Diagnosing and Prognosing an Ovarian ovarian endothelial cell tumor-associated proteins encoded Tumor by the genes listed in Tables 1-5. The primary antibody can 0270 Metastasis is a major complication in the pathogen include a detectable label. For example, the primary antibody esis of tumors. Such as ovarian cancer, and is typically indica US 2013/02743 15 A1 Oct. 17, 2013 27 tive of poor prognosis. It is also known that angiogenesis is a of such molecules. For example, the relative or absolute quan crucial factor in the progression of Solid tumors and tity of the at least one angiogenic ovarian endothelial cell metastases, including ovarian cancer. The formation of the tumor-associated molecule in a sample can be determined. vascular stroma plays an important role in the pathophysiol 0275. The activity such as the expression level of the dis ogy of malignancy. For instance, in the absence of vascular closed pro-angiogenic ovarian endothelial cell tumor-associ Support tumors may become necrotic, or even apoptotic. In ated molecules in a sample obtained from a subject is com contrast, the onset of angiogenesis marks a phase of rapid pared to a control. In one example, an increase in expression proliferation, local invasion, and ultimately metastasis. of one or more of the angiogenic ovarian endothelial cell 0271. Without wishing to be bound to a particular theory, tumor-associated molecules upregulated in ovarian tumor it is proposed that an alteration in the expression of the dis endothelial cells (such as those listed in Table 2) as compared closed ovarian endothelial tumor-associated molecules asso to a non-tumor control or reference value indicates the pres ciated with angiogenesis, such as molecules involved in cell ence of an ovarian tumor, the ovarian tumor is metastatic, the proliferation, cell motility or tube formation, including those ovariantumor is likely to become metastatic, a poor prognosis disclosed in FIG. 5 (such as EZH2), is related to enhanced or a combination thereof. In some examples, a decrease in ovarian tumor cell metastasis and a poor clinical outcome. expression of one or more of the angiogenic ovarian endot Thus, methods of diagnosing or prognosing an ovarian tumor helial cell tumor-associated molecules that is downregulated that overexpresses at least one pro-angiogenic ovarian endot in ovarian tumor endothelial cells (such as those listed in helial cell tumor-associated molecule (such as those listed in Table 3 or VASH1) as compared to a non-tumor control or Tables 1, 2, 4 and 5 that are upregulated in ovarian endothelial reference value indicates the presence of an ovarian tumor, tumor cells; e.g., EZH2) or underexpresses at least one proan the ovarian tumor is metastatic, the ovarian tumor is likely to giogenic ovarian endothelial cell tumor associated molecule become metastatic, a poor prognosis or a combination (such as those listed in Tables 1 and 3 that are downregulated thereof. in ovarian endothelial tumor cells), are disclosed. In some 0276 For example, the level of the angiogenic ovarian examples, such methods can be used to identify those Subjects endothelial cell tumor-associated molecules, such as the level that will benefit from the disclosed treatment methods. For of EZH2, detected can be compared to a non-tumor control or example, such diagnostic methods can be performed prior to reference value. Such as a value that represents a level of the Subject undergoing the treatments described above. In angiogenic ovarian endothelial cell tumor-associated mol other examples, these methods are utilized to predict the ecules expected if an ovarian tumor is or is not metastatic or metastatic potential of the ovarian cancer, a poor prognosis, or is a low grade tumor or early stage tumor. In one example, the combinations thereof. In one particular example, these meth angiogenic ovarian endothelial cell tumor-associated mol ods are utilized to predicta poor prognosis, such as to indicate ecules detected in a tumor sample are compared to the level of a decreased Survival time. Such molecules detected in a sample obtained from a subject 0272. In an example, the method includes detecting that does not have an ovarian tumor or has a non-metastatic expression of at least one angiogenic ovarian endothelial cell ovarian tumor. In certain examples, detection of at least a tumor-associated molecule listed in Tables 1-5. Such as at 2-fold, such as by at least 3-fold, at least 4-fold, at least 6-fold least two, at least 5, at least 7, at least 10, at least 20, at least or at least 10-fold alteration in the relative amount of the 30, at least 40, at least 50, at least 60, at least 70, at least 80, pro-angiogenic ovarian endothelial cell tumor-associated at least 90, at least 100, at least 150, at least 200 ovarian molecules in a tumor sample, as compared to the relative endothelial cell tumor-associated molecules related to angio amount of Such molecules in a control indicates that the genesis (for example, 1,2,3,4,5,6,7,8,9, 10, 12, 15, 17, 20, Subject has a tumor with metastatic potential, has a tumor that 25, 30, 35, 40, 45,50, 55, 60, 65,70, 75, 80, 85,90, 100,125, has metastasized, has a poor prognosis, or combinations 150, 175,200, or 250 listed in Tables 1-5) in a sample from the thereof. In some examples, detection of statistically similar Subject exhibiting one or more symptoms associated with relative amounts of pro-angiogenic ovarian endothelial cell ovarian cancer. tumor-associated molecules observed in a tumor sample, as compared to the relative amount of such molecules in a con 0273. In an example, the specific angiogenic ovarian trol sample that is not metastatic, indicates that that Subject endothelial cell tumor-associated molecule, such as EZH2, is does not have a tumor with metastatic potential, does not have detected in a biological sample. In a particular example, the a tumor that has metastasized, has a good prognosis, or com biological sample is a sample taken from a subject with ova binations thereof. rian epithelial cancer. In a particular example, the biological 0277. In a specific example, the method includes detecting sample is a tumor biopsy. In another example, the angiogenic and comparing the nucleic acid expression levels of the pro ovarian endothelial cell tumor-associated molecule is angiogenic ovarian endothelial cell tumor-associated mol detected in a serum sample. For example, the ovarian endot ecules such as DNA, cDNA, or mRNAs. In a specific helial cell tumor-associated molecule is detected in a serum example, the method includes detecting and comparing the sample if the specific molecule is secreted or located on a cell mRNA expression levels of the pro-angiogenic ovarian Surface Susceptible to enzymatic cleavage. endothelial cell tumor-associated molecules. For example, 0274. In one example, detection of at least one angiogenic Such expression can be measured by real time quantitative ovarian endothelial cell tumor-associated molecule listed in polymerase chain reaction or microarray analysis. In a par any of Tables 1, 2, 3, 4 or 5, such as detection of EZH2, in a ticular example, the disclosed gene expression profile is uti biological sample from the Subject is used to diagnose or lized to diagnosis and/or prognosis an ovarian tumor. prognose an ovarian tumor. Methods of detecting Such mol ecules in a sample are known in the art and are routine. In Detection of Ovarian Endothelial Cell Tumor-Associated Some examples, the relative amount of pro-angiogenic ova Nucleic Acids rian endothelial cell tumor-associated molecules present is 0278 Nucleic acids can be detected by any method known determined, for example by quantitating the expression level in the art. In some examples, nucleic acids are isolated, ampli US 2013/02743 15 A1 Oct. 17, 2013 28 fied, or both, prior to detection. In an example, the biological Table 3. For example, the profile includes the seventeen ova sample can be incubated with primers that permit the ampli rian endothelial cell tumor-associated molecules listed in fication of one or more of the disclosed ovarian endothelial Table 3. cell tumor-associated mRNAs, under conditions sufficient to permit amplification of such products. For example, the bio Detecting Ovarian Endothelial Cell Tumor-Associated logical sample is incubated with probes that can bind to one or Proteins more of the disclosed ovarian endothelial cell tumor-associ ated nucleic acid sequences (such as cDNA, genomic DNA, 0283 As an alternative to analyzing the sample for the or RNA (such as mRNA)) under high stringency conditions. presence of nucleic acids, alterations in protein expression The resulting hybridization can then be detected using meth can be measured by methods known in the art such as Western ods known in the art. In one example, a therapeutic agent is blot analysis, mass spectrometry, immunoassay or a protein identified by applying isolated nucleic acid molecules to an microarray (as described above). For example, the metastatic array in which the isolated nucleic acid molecules are potential of an ovarian tumor can be determined by using a obtained from a biological sample including ovarian endot protein array that includes one or more capture agents, such as helial cancer cells for example following treatment with the antibodies that are specific for the one or more disclosed one or more test agents. In Such example, the array includes ovarian endothelial tumor-associated molecules that are oligonucleotides complementary to all ovarian endothelial related to angiogenesis, such as molecules that play a role in cell tumor-associated genes listed in Table 1. In a particular cell proliferation, cell motility or tube formation, such as example, the array is a commercially available array Such as EZH2. 0284. The disclosure is further illustrated by the following a U133 Plus 2.0 oligonucleotide array from AFFYMETRIX(R) non-limiting Examples. (AFFYMETRIX(R), Santa Clara, Calif.). 0279. In an example, the isolated nucleic acid molecules Example 1 are incubated with the array including oligonucleotides complementary to the ovarian endothelial cell tumor-associ Materials and Methods for Examples 2-7 ated molecules listed in Tables 2, 3, 4 and/or 5 for a time sufficient to allow hybridization between the isolated nucleic Sample Preparation. acid molecules and oligonucleotide probes, thereby forming isolated nucleic acid molecule:oligonucleotide complexes. In 0285 Fresh tissue samples (5 normal ovaries and 10 epi a particular example, the isolated nucleic acid molecules are thelial high-grade, stage III or IV invasive serous ovarian incubated with the array including oligonucleotides comple cancers) were obtained from patients undergoing primary mentary to at least EZH2. The isolated nucleic acid molecule: surgical exploration at the M.D. Anderson Cancer Center. The oligonucleotide complexes are then analyzed to determine if minced tissue was digested with collagenase A, elastase and expression of the isolated nucleic acid molecules is altered. DNase 1 at 37° C. for 90 minutes to yield a single cell Suspension. A number of negative selections followed includ Gene Expression Profile ing removal of platelets and red blood cells (RBCs) by Percoll separation, removal of epithelial cells using M450 beads, 0280. The disclosed gene profile (as described above) can which are prebound to BerEP4 antibody, removal of leuko also be used in the diagnosis and prognosis of an ovarian cytes using anti-CD-14, CD-45, and CD-64 beads (Dynal tumor in a Subject. In an example, the gene expression profile Biotech, Brown Deer, Wis.). Positive selection was per includes at least two of the ovarian endothelial cell tumor formed with P1H12 (CD 146) immunobeads (P1H12 anti associated molecules listed in Table 1. Such as at least 5, at body was from Chemicon, Temecula, Calif.), and the beads least 7, at least 10, at least 20, at least 30, at least 40, at least linked to secondary antibody were from Dynal Biotech. 50, at least 60, at least 70, at least 80, at least 90, at least 100, Immunostaining was then performed using von Willebrand at least 150, at least 200, at least 250, at least 300, at least 400, factor and 4,6-diamidino-2-phenylindole nuclear staining to at least 500, at least 600, at least 700, at least 800, at least 900, confirm the purification of endothelial cells. at least 1000, or at least 1100 molecules (for example, 2, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, Total RNA Amplification for AFFYMETRIX GENECHIPR 50, 55,60, 65,70, 75, 80, 85,90, 95, 100,105,110, 120, 130, Hybridization and Image Acquisition. 140, 150, 175, 225, 275,325, 350, 375, 450, 550, 650, 750, 0286 To successfully generate sufficient labeled cRNA 850, 950, 1050 or 1149 of those listed). for microarray analysis from 25 ng of total RNA, two rounds 0281. In a particular example, the gene expression profile of amplification were necessary. For the first round synthesis includes at least 1, at least 3, at least 5, at least 7, at least 10, of double-stranded cDNA, 25 ng of total RNA was reverse at least 20, at least 25, or at least 27 molecules (for example, transcribed using the Two-Cycle cDNA Synthesis Kit (AF 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, FYMETRIX(R), Santa Clara, Calif.) and oligo-dT24-T7 (SEQ 22, 24, 25, 26, 27, 28 or 29 molecules) listed in Table 2, 4 ID NO:1: 5'-GGCCAGTGAATTGTAATACG ACTCAC and/or 5 that are associated with angiogenesis, such as mol TATAGGGAGGCGG-3") primer according to the manufac ecules involved in cell proliferation, cell motility and/or tube turer's instructions followed by amplification with the formation. In a particular example, the at least one molecule MEGA script T7 Kit (Ambion, Inc., Austin, Tex.). After includes EZH2. cleanup of the cRNA with a GENECHIPR) Sample Cleanup 0282. In other particular examples, the gene expression Module IVT column (AFFYMETRIX(R), second round profile includes at least 2, at least 5, at least 7, at least 10, at double stranded cDNA was amplified using the IVT Labeling least 13, or at least 15 molecules (for example, 2, 3, 4, 5, 6, 7, Kit (AFFYMETRIX(R). A 15.0 g aliquot of labeled product 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 molecules) that are was fragmented by heat and ion-mediated hydrolysis at 94° down-regulated in ovarian tumor endothelial cells as listed in C. for 35 minutes in 24 ul HO and 6 ul of 5x Fragmentation US 2013/02743 15 A1 Oct. 17, 2013 29
Buffer (AFFYMETRIX(R). The fragmented cRNA was lated in tumor-associated endothelial cell isolates. This was hybridized for 16 hr at 45° C. in a Hybridization Oven 640 to accomplished using Pathway Assist version 3.0 software (Io a U133 plus 2.0 oligonucleotide array (AFFYMETRIX(R). bion Informatics LLC, La Jolla, Calif.). This software pack Washing and staining of the arrays with phycoerythrin-con age contains over 500,000 documented protein interactions jugated Streptavidin (Molecular Probes, Eugene, Oreg.) was acquired from MedLine using the natural language process completed in a Fluidics Station 450 (AFFYMETRIX(R). The ing algorithm MEDSCAN. The proprietary database can be arrays were then scanned using a confocal laser GENE used to develop a biological association network (BAN) to CHIPR) Scanner 3000 and GENECHIPR Operating Software identify putative co-regulated signaling pathways using (AFFYMETRIX(R). expression data. qRT-PCR Validation. Data Normalization and Filtering. 0290 Quantitative real-time PCR (qRT-PCR) was per 0287 Global normalization at a target value of 500 was formed on 100 ng of double-amplified product from the 15 applied to all 15 of the arrays under consideration using specimens using primer sets specific for 23 select genes, and GENECHIPR) Operating Software (AFFYMETRIX(R). Nor the housekeeping genes GAPDH, GUSB, and cyclophilin. An malized data were uploaded into the National Cancer Insti iCycler iQ Real-Time PCR Detection System (BIORADR) tute's Microarray Analysis Database (mAdb) for quality con Laboratories, Hercules, Calif.) was used in conjunction with trol screening and collation prior to downstream analyses. the QuantiTect SYBR Green RT-PCR Kit (QIAGENR, Inc., Biometric Research Branch (BRB) ArrayTools version 3.2.2 Valencia, Calif.) according to previously described cycling software developed by Drs. Richard Simon and Amy Peng conditions (Donninger et al., Oncogene 23: 8065-8077. Lam of the Biometrics Research Branch of the National Can 2004). To calculate the relative expression for each gene, the cer Institute was used to filter and complete the statistical 2-method was used averaging the C values for the three analysis of the array data. BRB-ArrayTools is a multifunc housekeeping genes for a single reference gene value (Livak tional Excel add-in that contains utilities for processing and and Schmittgen, Methods 25: 402-408, 2001). analyzing microarray data using the R version 2.0.1 environ ment (R Development Core Team, 2004). Of the 47,000 tran Scripts represented on the array, hybridization control probe Immunohistochemical Staining. sets and probe sets scored as absent at C.1=0.05 or marginal 0291 Paraffin sections were stained for the following anti (M) at O2=0.065 were excluded. In addition, only those tran bodies: rabbit anti-Fyn at 1:400 (Santa Cruz, Biotechnology, scripts present in greater than 50% of the arrays and display Inc., Santa Cruz, Calif.), or rabbit anti-Fak at 1:50, mouse ing a variance in the top 50th percentile were evaluated. anti-MMP-9 at 1:40 (Oncogene-Research Products, Boston, Mass.), anti-f2-Arrestin at 1:200 (Santa Cruz Biotechnology, Class Comparison Analysis. Inc.), anti-PLXDC1 at 1:200 (Abcam, Inc., Cambridge, 0288 Differentially expressed genes were identified for Mass.), or anti-Jagged 1 at 1:200 (Santa Cruz, Biotechnology, tumor and normal endothelial cell specimens using a multi Inc.) diluted in PBS at 4° C. After three washes in PBS, variate permutation test in BRB-ArrayTools (Simon et al., sections were incubated with secondary antibody for 1 hr at “Design and Analysis of DNA Microarray Investigations' room temperature. Positive reactions were rendered visible Springer-Verlag, 2003). A total of 2000 permutations were by incubating the slides with stable 3.3-diaminobenzidine for completed to identify the list of probe sets with a false dis 5-10 min. The sections were rinsed with distilled water, coun covery rate less than 10% at a confidence of 95%. Differential terstained with Gill's hematoxylin for 30s, and mounted with expression was considered significant at a p<0.001. A ran Universal Mount (Research Genetics, Huntsville, Ala.). The dom-variance t-test was selected to permit the sharing of intensity of protein expression in the endothelial cells was information among probe sets within class variation without evaluated using OPTIMAS 6.5 software and the mean optical assuming that all of the probe sets possess the same variance density (OD) was calculated from 5 normal ovarian and 5 (Wright et al., Bioinformatics 19:2448-2455, 2003). A global ovarian cancer samples. Ten vessels were selected randomly assessment of whether expression profiles were different from each sample for the measurements. between classes was also performed. During each permuta Small Interfering RNA (siRNA). tion the class labels were reassigned randomly and the p-value for each probe set recalculated. The proportion of 0292. The small interfering RNA (siRNA) constructs were permutations yielding at least as many significant genes as the purchased from QIAGENR (Germantown, Md.): a control sequence with no homology to any human mRNA (as deter actual data set at a p-values0.001 was reported as the signifi mined by BLAST search), and separate sequences designed cance level of the global test. to target EZH2, Jagged 1, or PTK2 mRNA. The Jagged 1 Pathway Analysis. siRNA target sequence is SEQ ID NO: 2 (5'-CTGCATT TAGGGAGTATTCTA-3). The EZH2 siRNA was targeted to 0289 Differentially regulated genes identified in a series the region corresponding to residues 85-106 of human EZH2 of 48 late-stage (III and IV) high-grade (Hurwitz et al., N. (Gene accession No. NM004456; 5'-AACCATGTTTA Engl. J. Med. 350: 2335-2342, 2004) microdissected papil CAACTATCAA-3'; SEQID NO:3). The EZH2 siRNA sense lary serous ovarian carcinomas, as compared to 10 normal sequence was 5'-CCAUGUUUACAACUAUCAAtt-3'; SEQ ovarian Surface epithelial brushings (Bonome et al., Cancer ID NO: 4) and the antisense sequence was 3'-ttGGUA Res. 65: 10602-10612, 2005), were categorized by cellular CAAAUGUUGAUAGUU-5'; SEQ ID NO. 5). For in vitro component according to the Gene Ontology (GO) ontological delivery, siRNA (5ug) was incubated with 30 ul, RNAi Fect hierarchy. Epithelial genes associated with the cell mem transfection reagent (QIAGENR) for 10 min at room tem brane, extracellular matrix, and extracellular region were perature and added to cells in culture at 80% confluence in 35 used as central nodes to identify signaling pathways modu mm culture plates. US 2013/02743 15 A1 Oct. 17, 2013 30
Cell Migration Assay. 0296. According to the methods described in Example 1, 0293 Unstimulated motility was determined in mem samples were immunostained with endothelial cell markers brane invasion culture system chambers containing polycar P1H12 and von Willebrand factor to determine endothelial bonate filter (with 10 Limpores) that had been soaked in 0.1% cell purity. Immunostaining revealed that the employed puri gelatin, as described previously (Sood et al., Am. J. Pathol. fication technique yielded endothelial cell purity of >95% in 165: 1087-1095, 2004). HUVECs (1x105) were seeded in all samples. Thus, the disclosed isolation technique resulted each upper well, allowed to incubate at 37° C. for 6 hr in in a highly pure population of endothelial cells. Dulbecco's modified Eagle's medium (DMEM) containing Example 3 15% serum, and subsequently processed as described for the invasion assay. Development of Gene Expression Profile for Ovarian Tumor-Endothelial Cells Tube Formation Assay. 0297. This example provides a gene expression profile for 0294. Matrigel (12.5 mg/ml) was thawed at 4°C. and 50 ul ovarian tumor endothelial cells. were quickly added to each well of a 96-well plate and 0298. According to the methods described in Example 1, allowed to solidify for 10 min at 37°C. The wells were then gene expression differences in purified endothelial cells from incubated for 6hat 37°C. with HUVECs (20,000 cells/well), 10 invasive epithelial ovarian cancers and 5 normal (non which had previously been treated for 18 h with the indicated tumor) ovaries were determined by using the AFFYME siRNA. The formation of capillary-like structures was exam TRIX(R) Human U133 Plus 2.0 Gene Chip platform. The ined microscopically and photographs (50x) were taken nucleic acid sequence of each AFFYMETRIX(R) probe listed using a RETIGAR 1300 camera and a ZEISS.R. Axiovert in the tables below is herein incorporated by reference, and is S100 microscope. The extent to which capillary-like struc available from the AFFYMETRIXR website. As illustrated in tures formed in the gel was quantified by analysis of digitized Table 1, 1149 genes were identified as being differentially images to determine the thread length of the capillary-like regulated 2-fold in endothelium derived from epithelial ova network, using a commercially available image analysis pro rian cancers compared to normal ovarian tissue. A positive gram (Northern Eclipse, North Tonawanda, N.Y.). fold change indicates the gene was upregulated in ovarian endothelial tumor sample and a negative fold change indi Example 2 cates the gene was downregulated in Such sample. A multi variate permutation t-test (p<0.001) providing 95% confi Purity of Isolated Endothelial Cells dence that the number of false discoveries did not exceed 10% 0295) This example illustrates the purity of the endothelial of the complete gene list identified. In addition, global analy cell samples utilized in the disclosed microarray analyses. sis of the gene list returned ap value(5x10'. TABLE 1 Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 17 at O.OOOS215 4.4 HSPA6 heat shock 70 kDa protein 6 1q23 (HSP7OB') (HSPA6), mRNA. 552365 at O.OOO8711 6.6 SCIN scinderin (SCIN), mRNA. 7p21.3 552767 a at 0.0002277 -6.5 HS6ST2 heparan Sulfate 6-O- Xq26.2 sulfotransferase 2 (HS6ST2), mRNA. 552790 a. at 0.0009635 -2.2 TLOC1 translocation protein 1 3q26.2 (TLOC1), mRNA. 5.52889 a. at 0.0003238 2.5 XTP7 protein 7 transactivated by 19q13.32 hepatitis B virus X antigen (HBxAg) (XTP7), mRNA. 553185 at O.OOO128 4.6 RASEF RAS and EF-hand domain 9q21.32 containing 553186 X at 3.33E-05 5.3 RASEF RAS and EF-hand domain 9q21.32 containing 553407 at O.OOO3912 2.5 MACF1 Glycine-rich protein (GRP3S) 1p32-p31 553538. S. at 0.000594 2.2 Unknown 553569 at O.OOO1391 2.6 Unknown 553.570 x at O.OOO2337 2.5 Unknown 5.53575 at 2.8OE-06 4.6 Unknown 553909 x at 0.0005681 3.8 C10orf6 Chromosome 10 open reading 10q24.32 rame 6 553959 a. at O.OOO9778 2.3 B3GALT6 UDP-Gal:beta Gal beta1,3- 1p36.33 galactosyltransferase polypeptide 6 (B3GALT6), mRNA. 553983 at O.OOO152 2.5 DTYMK deoxythymidylate kinase 2a37.3 (thymidylate kinase) (DTYMK), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 31
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 554168 a at 0.0003894 2.1 SH3KBP1 SH3-domain kinase binding Xp22.1-p21.3 protein 1 (SH3KBP1), transcript variant 2 mRNA. 554.309 at O.OOO6114 2.3 EIF4G3 Eukaryotic translation initiation 1p36.12 factor 4 gamma, 3 554334 a at 7.15E-05 5.2 DNAJA4 DnaJ (Hsp40) homolog, 15q25.1 Subfamily A, member 4 (DNAJA4), mRNA. 554455 at O.OOO7696 2.4 LINS1 lines homolog 1 (Drosophila) 15q26.3 (LINS1), transcript variant 2, mRNA. 554464 a at 0.000263 -2.8 CRTAP cartilage associated protein 3p22.3 (CRTAP), mRNA. 554595 at O.OOOS225 2.6 SYMPK Symplekin 19q13.3 554640 at O.OOO4537 2.6 PALM2-AKAP2 Paralemmin 2 9q31-q33 554678 s at 0.0002435 -2 HNRPDL Heterogeneous nuclear 4q13-q21 ribonucleoprotein D-like 554703 at O.OOO8778 2.1 ARHGEF10 Rho guanine nucleotide 8p23 exchange factor (GEF) 10 555014 x at 0.0004322 3.5 OKASW-cl.92 555.241 at O.OOO1337 3.2 Hypothetical gene Supported by 8q21.2 BCOSSO92 555.243 x at 0.0001753 2.8 Hypothetical gene Supported by 8q21.2 BCOSSO92 555374 at O.OOO3108 4 TTL Tubulin tyrosine ligase 2d 13 555823 at O.OOO1833 -2 BS 3076 14 556126 s at 1.04E-05 3.5 GPATC2 G patch domain containing 2 1q41 5561.38 a. at 0.0001589 2.9 COLSA1 Collagen, type V, alpha 1 9q34.2-q34.3 556185 a. at 0.0002463 2.6 CDNA clone IMAGE: 5260162 7 556242 a. at 3.82E-05 2.1 Homo sapiens, clone 8 IMAGE: 3885623, mRNA 556,316 s at 0.0005561 3.3 MIF Macrophage migration 22d 11.23 inhibitory factor (glycosylation inhibiting factor) 556499 s at 0.0005246 2.5 COL1A1 collagen, type I, alpha 1 17q21.3-q22.1 (COL1A1), mRNA. 556835 S. at 0.0002018 2.2 Transcribed locus 11 557432 at O.OOO1321 3.6 RASAL2 RAS protein activator like 2 1q24 557527 at O.OOO3572 2.5 RUNX1 Runt-related transcription factor 21q22.3 (acute myeloid leukemia 1: aml1 oncogene) 558O19 at 2.09E-OS -2.9 DST Dystonin 6p12-p11 558048 x at 2.01E-05 4.1 Unknown 558292 s at 0.0007105 2 PIGW phosphatidylinositol glycan, 17q12 class W (PIGW), mRNA. 558426 X at 0.000189 2.9 Chromosome 7 open reading 7 rame 19 558487 a. at 0.0002657 -2.6 TMED4 Transmembrane emp24 protein 7p13 transport domain containing 4 558836 a S.35E-05 3.2 MRNA, cDNA 2 DKFZp667A182 (from clone DKFZp667A182) 559060 a. at 5.16E-05 3 KIAA1961 KIAA1961 gene 5q23.3 559078 a 6.58E-OS 4.6 BCL11A B-cell CLLlymphoma 1 1A 2p16.1 (Zinc finger protein) 559101 a 9.12E-OS 2.6 FYN FYN oncogene related to SRC, 6q21 FGR, YES 5594.10 a 4.66E-OS 3.6 Unknown 559436 x at 4.54E-05 4.8 ARRB2 Arrestin, beta 2 17p13 559585 a. O.OOO489 3.2 FLJ31033 Hypothetical protein FLJ31033 4q32.3 559593 a at 0.0005238 2.9 CRSP7 Cofactor required for Sp1 19p 13.11 transcriptional activation, subunit 7, 70 kDa 560817 a O.OOO1564 2.5 MOV10 Mov10, Moloney leukemia virus 1p13.2 0, homolog (mouse) 562062 a O.OOO114 4 Homo sapiens transcribed sequence with weak similarity to protein ref: NP O55301.1 (H. Sapiens) neuronal thread protein Homo sapiens US 2013/02743 15 A1 Oct. 17, 2013 32
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 1562063 x at 0.0003481 3.4 Homo sapiens transcribed sequence with weak similarity to protein ref: NP O55301.1 (H. Sapiens) neuronal thread protein Homo sapiens 562270 at O.OOO382 4 ARHGEF7 Rho guanine nucleotide 13q34 exchange factor (GEF) 7 562271 x at 1.81E-05 3.9 ARHGEF7 Rho guanine nucleotide 13q34 exchange factor (GEF) 7 562456 at O.OOO8617 2.3 MRNA, cDNA 11 DKFZp566C0924 (from clone DKFZp566C0924) 563357 at O.OOO2718 3.4 SERPINB9 Serpin peptidase inhibitor, clade 6p25 B (ovalbumin), member 9 (SERPINB9), mRNA. 565579 at O.OOO3108 3.7 TATDN2 TatD DNase domain containing 2 3p25.3 565823 at 1.91E-05 4.8 7-Sep septin 7 (SEPT7), transcript 7p14.3-p14.1 variant 2 mRNA. 565974 at O.OOO8723 2.8 SUV42OH1 Suppressor of variegation 4-20 11 q13.2 homolog 1 (Drosophila) 56.6887 X at 8.32E-05 4.3 KIAAO284 KIAAO284 14q32.33 568619 S at 0.0002665 2.2 LOC162O73 Hypothetical protein 16p12.3 LOC162O73 568954 S at 0.0001751 2.2 Unknown 56.9519 at O.OOO61.99 2.5 FL21272 hypothetical protein FLJ21272 1q21.2 56.9872 a. at 0.0004892 2.4 Homo sapiens, clone 16 MAGE: 5242623 570061 at 8.83E-OS 3.2 CDNA clone IMAGE: 4555.030 3 570143 at 0.0005727 2.4 Homo sapiens, clone 8 MAGE: 3932570, mRNA 57O185 at O.OOO3O81 3 Homo sapiens, clone 10 MAGE: 5766850, mRNA 200005 at O.OOOSSO9 -2 EIF3S7 eukaryotic translation initiation 22d 13.1 actor 3, subunit 7 zeta, 66/67 kDa (EIF3S7), mRNA. 200010 at O.OOO1997 -2.2 RPL11 Ribosomal protein L11 1p36.1-p35 200013 at 2. SOE-OS -2.6 RPL24 ribosomal protein L24 (RPL24), 3q12 mRNA. 200021 at O.OOO1O3S 2.5 CFL1 cofilin 1 (non-muscle) (CFL1), 11 q13 mRNA. 200022 at O.OOO2003 -2.1 RPL18 ribosomal protein L18 (RPL18), 19q13 mRNA. 200023 s at 1.61E-05 -2.4 EIF3S5 eukaryotic translation initiation 11 p 15.4 factor 3, Subunit 5 epsilon, 47 kDa (EIF3S5), mRNA. 200024 at O.OOO4964 -2.5 RPS5 ribosomal protein S5 (RPS5), 19q13.4 mRNA. 20007.4 S at 0.0001721 -2.2 RPL14 ribosomal protein L14 (RPL14), 3p22-p21.2 mRNA. 200081 s at 6.2OE-06 -3.3 RPS6 ribosomal protein S6 (RPS6), 9p21 mRNA. 200642 at O.OOO7047 -2 SOD1 Superoxide dismutase 1, Soluble 21q22.11 (amyotrophic lateral sclerosis 1 (adult)) (SOD1), mRNA. 200651 at O.OOO1597 -2.3 GNB2L1 guanine nucleotide binding 5q.35.3 protein (G protein), beta polypeptide 2-like 1 (GNB2L1), mRNA. 200665 s at 0.00039 2.7 SPARC Secreted protein, acidic, 5q31.3-q32 cysteine-rich (osteonectin) (SPARC), mRNA. 200676 s at 0.0007037 -2.5 UBE2IL3 ubiquitin-conjugating enzyme 22d 11.21 E2L 3 (UBE2IL3), transcript variant 1 mRNA. 200700 s at 4.15E-05 2.3 KDELR2 KDEL (Lys-Asp-Glu-Leu) 7p22.1 endoplasmic reticulum protein retention receptor 2 (KDELR2), mRNA. 200734 S. at 6.61E-05 2.5 ARF3 ADP-ribosylation factor 3 12q13 (ARF3), mRNA. US 2013/02743 15 A1 Oct 17, 2013 33
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 20073S X at 9.63E-05 -2.1 NACA nascent-polypeptide-associated 12q23-q24.1 complex alpha polypeptide (NACA), mRNA. 200755 s at 0.0007055 2.1 CALU calumenin (CALU), mRNA. 7q32 200760 S at 5.53E-05 -2.6 ARL6IPS ADP-ribosylation-like factor 6 3p14 interacting protein 5 (ARL6IP5), mRNA. 200806 s at O.OOO815S -2 HSPD1 heat shock 60 kDa protein 1 2a33.1 (chaperonin) (HSPD1), nuclear gene encoding mitochondrial protein, transcript variant 2, mRNA. 200811 at 1.2SE-OS -3.6 CIRBP cold inducible RNA binding 9p13.3 protein (CIRBP), mRNA. 200827 at O.OOO4906 2.3 PLOD1 procollagen-lysine 1,2- p36.3-p36.2 oxoglutarate 5-dioxygenase 1 (PLOD1), mRNA. 2008.66 s at 0.0004.451 -2.1 PSAP prosaposin (variant Gaucher Oq21-q22 disease and variant metachromatic leukodystrophy) (PSAP), mRNA. 200883 at 7.19E-OS -3.2 UQCRC2 ubiquinol-cytochrome c 6p12 reductase core protein II (UQCRC2), mRNA. 200906 S. at 0.0002674 -3.2 KIAAO992 palladin (KIAA0992), mRNA. 4q32.3 200920 S. at 0.0001341 -2.3 BTG B-cell translocation gene 1, anti- 2q22 proliferative (BTG1), mRNA. 200937 S at 5.83E-05 -2.4 RPL5 ribosomal protein L5 (RPL5), p22.1 mRNA. 200951 S. at 0.000314 4.1 CCND2 cyclin D2 (CCND2), mRNA. 2p13 200953 s at 0.0001997 2.6 CCND2 cyclin D2 (CCND2), mRNA. 2p13 200965 S at 0.000112 -5.9 ABLIM1 actin binding LIM protein 1 Oq25 (ABLIM1), transcript variant 4, mRNA. 200999 s at 0.0009198 2 CKAP4 cytoskeleton-associated protein 2d23.3 4 (CKAP4), mRNA. 201008 S. at 0.0009045 -2.7 TXNIP thioredoxin interacting protein q21.1 (TXNIP), mRNA. 201009 s at 0.0004645 -2.8 TXNIP thioredoxin interacting protein q21.1 (TXNIP), mRNA. 201018 at O.OOO7096 -2.3 EIF1AX eukaryotic translation initiation Xp22.12 factor 1A, X-linked (EIF1AX), mRNA. 201023 at 2.69E-OS -2.4 TAF7 TAF7 RNA polymerase II, 5q31 TATA box binding protein (TBP)-associated factor, 55 kDa (TAF7), mRNA. 201030 x at 2.40E-05 -2.2 LDHB lactate dehydrogenase B 12p12.2-p12.1 (LDHB), mRNA. 201036 s at 0.0005734 -3.7 HADHSC L-3-hydroxyacyl-Coenzyme A 4q22-q26 dehydrogenase, short chain (HADHSC), mRNA. 201054 at 1.41E-05 -2.2 HNRPAO heterogeneous nuclear 5q31 ribonucleoprotein AO (HNRPAO), mRNA. 201076 at 182E-05 -2.4 NHP2L1 NHP2 non-histone chromosome 22d 13.2-q13.31 protein 2-like 1 (S. cerevisiae) (NHP2L1), transcript variant 2, mRNA. 201085 S at 0.000222 -2.3 SON SON DNA binding protein 21q22.11 (SON), transcript varianta, mRNA. 201088 at 9.1OE-06 2.7 KPNA2 karyopherin alpha 2 (RAG 17q23.1-q23.3 cohort 1, importin alpha 1) (KPNA2), mRNA. 201101 s at 3.64E-05 -2.9 BCLAF1 BCL2-associated transcription 6q22-q23 factor 1 (BCLAF1), mRNA. 201129 at O.OOO2614 -2.6 SFRS7 splicing factor, arginine?serine- 2p22.1 rich 7, 35 kDa (SFRS7), transcript variant 1, mRNA. 201133 s at 0.0001686 -2.5 PA2 praja 2, RING-H2 motif 5q21.3 containing (PJA2), mRNA. US 2013/02743 15 A1 Oct 17, 2013 34
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 201154 X at 0.0001159 -2.2 RPL4 ribosomal protein L4 (RPL4), 15q22 mRNA. 201163 s at 0.0003882 2.2 IGFBP7 insulin-like growth factor 4q12 binding protein 7 (IGFBP7), mRNA. 201193 at O.OOO4945 -2.9 IDEH1 isocitrate dehydrogenase 1 2a33.3 (NADP+), soluble (IDH1), mRNA. 201204 s at 1.43E-05 2.2 RRBP1 Ribosome binding protein 1 20p12 homolog 180 kDa (dog) 201206 s at 0.0002058 2.5 RRBP1 ribosome binding protein 1 20p12 homolog 180 kDa (dog) (RRBP1), mRNA. 201250 S at 0.0001573 3.1 SLC2A1 solute carrier family 2 1p35-p31.3 (facilitated glucose transporter), member 1 (SLC2A1), mRNA. 201261 x at 0.0003831 4.3 BGN biglycan (BGN), mRNA. Xq28 201302 at 1.57E-OS -3.1 ANXA4 annexin A4 (ANXA4), mRNA. 2p13 201370 S. at 0.0002857 -2.3 CUL3 cullin 3 (CUL3), mRNA. 2a36.3 201376 s at O.OOO1535 -2.4 HNRPF heterogeneous nuclear 10q11.21-q11.22 ribonucleoprotein F (HNRPF), mRNA. 201408 at 3.9SE-OS -3.2 PPP1CB protein phosphatase 1, catalytic 2p23 subunit, beta isoform (PPP1CB), transcript variant 2, mRNA. 201425 at 18OE-06 -2.9 ALDH2 aldehyde dehydrogenase 2 12q24.2 family (mitochondrial) (ALDH2), nuclear gene encoding mitochondrial protein, mRNA. 201427 s at 0.0004665 -2.5 SEPP1 Selenoprotein P. plasma, 1 5q31 (SEPP1), mRNA. 201431 S. at 0.0003957 -4.5 DPYSL3 dihydropyrimidinase-like 3 5q.32 (DPYSL3), mRNA. 201432 at 2.09E-OS -3.3 CAT catalase (CAT), mRNA. 11 p.13 2014.55 S at 0.0004393 -2.7 NPEPPS aminopeptidase puromycin 17q21 sensitive (NPEPPS), mRNA. 2014.82 at O.OOO4221 -2.9 QSCN6 quiescin Q6 (QSCN6), transcript 1q24 variant 2 mRNA. 201484 at O.OOO1619 -2.2 SUPT4H1 Suppressor of Ty 4 homolog 1 17q21-q23 (S. cerevisiae) (SUPT4H1), mRNA. 201487 at O.OOO2325 3.1 CTSC cathepsin C (CTSC), transcript 11 q14.1-q14.3 variant 1 mRNA. 201496 X at 0.0005336 -6.1 MYH11 myosin, heavy polypeptide 11, 16p13.13-p13.12 Smooth muscle (MYH11), transcript variant SM2, mRNA. 201506 at O.OOO3278 3.3 TGFBI transforming growth factor, 5q31 beta-induced, 68 kDa (TGFBI), mRNA. 201529 S. at 0.0001633 -3 RPA1 replication protein A1, 70 kDa 17p13.3 (RPA1), mRNA. 201535 at 6.72E-OS -2.3 UBL3 ubiquitin-like 3 (UBL3), 13q12-q13 mRNA. 201554 X at 0.0003257 -2.5 GYG glycogenin (GYG), mRNA. 3q24-q25.1 201579 at O.OOO7798 2.6 FAT FAT tumor Suppressor homolog 4q34-q35 1 (Drosophila) (FAT), mRNA. 201581 at 2.11E-05 -2.4 DJ971N18.2 Hypothetical protein 20p12 DJ971N18.2 201584 S at 0.0001736 2.3 DDX39 DEAD (Asp-Glu-Ala-Asp) box 19p 13.12 polypeptide 39 (DDX39), transcript variant 1, mRNA. 201596 x at 1.62E-05 4.3 KRT18 keratin 18 (KRT18), transcript 12q13 variant 2 mRNA. 201600 at 3.48E-05 -2.3 PHB2 prohibitin 2 (PHB2), mRNA. 12p13 201666 at O.OOO4812 3.4 TIMP1 TIMP metallopeptidase inhibitor Xp11.3-p11.23 1 (TIMP1), mRNA. 201674 S at 0.0001795 -3 AKAP1 A kinase (PRKA) anchor protein 17q21-q23 1 (AKAP1), nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA. US 2013/02743 15 A1 Oct. 17, 2013 35
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 201696 at O.OOO121 -2 SFRS4 splicing factor, arginine?serine- 1p35.3 rich 4 (SFRS4), mRNA. 201697 s a O.OOO12S6 2.4 DNMT1 DNA (cytosine-5-)- 19p 13.2 methyltransferase 1 (DNMT1), mRNA. 201712 s a O.OOO8422 -2.2 RANBP2 RAN binding protein 2 2d 12.3 (RANBP2), mRNA. 201737 s a 5.1OE-06 -3.1 6-Mar membrane-associated ring finger 5p15.2 (C3HC4) 6 (MARCH6), mRNA. 201756 at O.OOO3S33 -2 RPA2 replication protein A2, 32 kDa 1p35 (RPA2), mRNA. 20181.0 s a O.OOO1904 -3.2 SH3BP5 SH3-domain binding protein 5 3p24.3 (BTK-associated) (SH3BP5), transcript variant 2, mRNA. 201816 s a O.OOO4168 -2.2 GBAS glioblastoma amplified sequence 7p12 (GBAS), mRNA. 201871 s a O.OOO1348 -2 LOCS1035 ORF (LOC51035), mRNA. 11 q12.3 201891 s a 0.0007795 2 B2M beta-2-microglobulin (B2M), 15q21-q22.2 mRNA. 201893 x at 0.0006272 -3.1 DCN decorin (DCN), transcript 12q21.33 variant B, mRNA. 201911 s a O.OOO1467 3.4 FARP1 FERM, RhoGEF (ARHGEF) 13q32.2 and pleckstrin domain protein 1 (chondrocyte-derived) (FARP1), transcript variant 1, mRNA. 201922 at O.OOO1538 -2 TINP1 TGFbeta-inducible nuclear 5q13.3 protein 1 (TINP1), mRNA. 201960 S at 0.0002203 -2 MYCBP2 MYC binding protein 2 13q22 (MYCBP2), mRNA. 201973 s at 0.0004504 2 C7orf28A chromosome 7 open reading 7p22.1 rame 28A (C7orf28A), mRNA. 202016 at O.OOO1808 3.2 MEST mesoderm specific transcript 7q32 homolog (mouse) (MEST), transcript variant 3, mRNA. 202028 s at 0.0002331 3 RPL38 ribosomal protein L38 (RPL38), 17q23-q25 mRNA. 202029 X at 1.93E-05 -2.1 RPL38 ribosomal protein L38 (RPL38), 17q23-q25 mRNA. 202037 s at 0.0002558 -3.1 SFRP1 secreted frizzled-related protein 8p12-p11.1 1 (SFRP1), mRNA. 202068. S. at 0.0001171 -4.1 LDLR low density lipoprotein receptor 19p13.3 (familial hypercholesterolemia) (LDLR), mRNA. 202073 at 4.49E-05 -2.6 OPTN optineurin (OPTN), transcript 10p13 variant 2 mRNA. 202105 at 3.OOE-07 -3.2 IGBP1 immunoglobulin (CD79A) Xq13.1-q13.3 binding protein 1 (IGBP1), mRNA. 202119 S at 0.0002779 -3.2 CPNE3 copine III (CPNE3), mRNA. 8q21.3 202139 at O.OOO4872 -2.1 AKR7A2 aldo-keto reductase family 7, p35.1-p36.23 member A2 (aflatoxin aldehyde reductase) (AKR7A2), mRNA. 202148 S. at 0.0006205 2.2 PYCR1 pyrroline-5-carboxylate 7q25.3 reductase 1 (PYCR1), transcript variant 2 mRNA. 202156 S at 0.0004765 -2.8 CUGBP2 CUG triplet repeat, RNA Op13 binding protein 2 (CUGBP2), transcript variant 2, mRNA. 202157 s at 9.00E-07 -6.1 CUGBP2 CUG triplet repeat, RNA Op13 binding protein 2 (CUGBP2), transcript variant 2, mRNA. 20215.8 s at 2.99E-05 -4.2 CUGBP2 CUG triplet repeat, RNA Op13 binding protein 2 (CUGBP2), transcript variant 2, mRNA. 202172 at O.OOO6154 -2.4 ZNF161 Zinc finger protein 161 7q23.2 (ZNF161), mRNA. 2022.02 s at 0.0002996 3.4 LAMA4 aminin, alpha 4 (LAMA4), 6q21 mRNA. 202214 s at 0.0005849 -2 CUL4B Cullin 4B Xq23 202232 S at 0.0002955 -2.2 lf-BS dendritic cell protein (hfl-B5), 11 p.13 mRNA. US 2013/02743 15 A1 Oct 17, 2013 36
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 202259 s at 3.74E-OS -3.2 PFAAP5 phosphonoformate immuno associated protein 5 (PFAAP5), mRNA. 202260 s at O.OOOS909 -2.2 STXBP1 Syntaxin binding protein 1 (STXBP1), transcript variant 2, mRNA. 202286 s at O.OOO3361 5.5 TACSTD2 tumor-associated calcium signal 1p32-p31 transducer 2 (TACSTD2), mRNA. 202292 X at O.OOO1802 2.4 LYPLA2 lysophospholipase II (LYPLA2), 1p36.12-p35.1 mRNA. 202297 s at 6.2SE-OS 2.2 RER1 RER1 retention in endoplasmic 1pter-q24 reticulum 1 homolog (S. cerevisiae) (RER1), mRNA. 202314 at O.OOO1115 -2.6 cytochrome P450, family 51, Subfamily A, polypeptide 1 (CYP51A1), mRNA. 202350 s at O.OOO2121 -3.7 MATN2 matrilin 2 (MATN2), transcript variant 2 mRNA. 202364 at O.OOO2246 -2.5 MAX interactor 1 (MXI1), transcript variant 3, mRNA. 202378 s at 6.45E-OS -2.2 LEPROT leptin receptor overlapping 1p31.2 transcript (LEPROT), mRNA. 202404 S at O.OOO452 2.9 COL1A2 collagen, type I, alpha 2 (COL1A2), mRNA. 202429 s at O.OOO12SS -2.4 PPP3CA protein phosphatase 3 (formerly 2B), catalytic Subunit, alpha isoform (calcineurin Aalpha) (PPP3CA), mRNA. 202464 S at O.OOO9823 3.5 6-phosphofructo-2- 10p14-p15 kinase? fructose-2,6- biphosphatase 3 2O2465 at 4.8 PCOLCE procollagen C-endopeptidase enhancer (PCOLCE), mRNA. 202468 s at 7.84E-OS -2.5 CTNNAL1 catenin (cadherin-associated protein), alpha-like 1 (CTNNAL1), mRNA. 2025.02 at ACADM acyl-Coenzyme A 1p31 dehydrogenase, C-4 to C-12 straight chain (ACADM), nuclear gene encoding mitochondrial protein, mRNA. 20251.0 s at 1.14E-05 3.7 TNFAIP2 tumor necrosis factor, alpha induced protein 2 (TNFAIP2), mRNA. 202512 s at 6.24E-OS -2.2 APGSL APG5 autophagy 5-like (S. cerevisiae) (APGSL), mRNA. 202536 at O.OOO2234 CHMP2B chromatin modifying protein 2B (CHMP2B), mRNA. 202546 at O.OOO4572 S.6 WAMP8 vesicle-associated membrane 2p12-p11.2 protein 8 (endobrevin) (VAMP8), mRNA. 202547 s at 6.98E-OS ARHGEF7 Rho guanine nucleotide exchange factor (GEF) 7 (ARHGEF7), transcript variant 1, mRNA. 202573 at O.OOO2474 casein kinase 1, gamma 2 19p13.3 (CSNK1G2), mRNA. 202581 at O.OOO2675 2.8 heat shock 70 kDa protein 1B 6p21.3 (HSPA1B), mRNA. 202630 at 6.64E-OS -2.7 APPBP2 amyloid beta precursor protein (cytoplasmic tail) binding protein 2 (APPBP2), mRNA. 202665 s at O.OOO3645 2.9 WASPIP Wiskott-Aldrich syndrome 2a31.1 protein interacting protein (WASPIP), mRNA. 20272.2 s at O.OOO2949 2.1 glutamine-fructose-6-phosphate 2p13 transaminase 1 (GEPT1), mRNA. 202723 s at 2.2OE-06 FOXO1A forkhead box O1A 13q14.1 (rhabdomyosarcoma) (FOXO1A), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 37
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 202724 S at 6.00E-07 -3.3 FOXO1A forkhead box O1A 13q14.1 (rhabdomyosarcoma) (FOXO1A), mRNA. 202731 at O.OOO8149 -2.6 PDCD4 programmed cell death 4 10q24 (neoplastic transformation inhibitor) (PDCD4), transcript variant 1 mRNA. 202733 at 0.0005751 3 P4HA2 procollagen-proline, 2- 5q31 oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide II (P4HA2), transcript variant 3, mRNA. 202746 at O.OOOS864 -6.4 ITM2A integral membrane protein 2A Xq13.3-Xq21.2 (ITM2A), mRNA. 202749 at O.OOO9431 -2.2 WRB Tryptophan rich basic protein 21q22.3 202761 s at 0.0003908 -2.3 SYNE2 spectrin repeat containing, 14q23.2 nuclear envelope 2 (SYNE2), transcript variant 4, mRNA. 202780 at O.OOO9309 -2 OXCT1 3-oxoacid CoA transferase 1 5p13.1 (OXCT1), nuclear gene encoding mitochondrial protein, mRNA. 202820 at 1.90E-OS 3.3 AHR aryl hydrocarbon receptor 7p15 (AHR), mRNA. 202825 at O.OOO4002 -2.1 SLC25A4 solute carrier family 25 4q35 (mitochondrial carrier; adenine nucleotide translocator), member 4 (SLC25A4), nuclear gene encoding mitochondrial protein, mRNA. 202888 s at 0.0005702 4.7 ANPEP alanyl (membrane) 15q25-q26 aminopeptidase (aminopeptidase N, aminopeptidase M, microsomal aminopeptidase, CD13, p15O) (ANPEP), mRNA. 202899 s at 5.55E-05 -2.8 SFRS3 splicing factor, arginine?serine- 6p21 rich 3 (SFRS3), mRNA. 202908 at S.90E-06 -3.4 WFS1 Wolfram syndrome 1 4p16 (wolframin) (WFS1), mRNA. 202911 at O.OOO8837 -2.1 MSH6 mutS homolog 6 (E. coli) 2p16 (MSH6), mRNA. 202920 at 4.6OE-06 -4.2 ANK2 ankyrin 2, neuronal (ANK2), 4q25-q27 transcript variant 2, mRNA. 202952 S at 2.37E-05 7.6 ADAM12 ADAM metallopeptidase 10q26.3 domain 12 (meltrin alpha) (ADAM12), transcript variant 1, mRNA. 202954 at O.OOO6434 2.8 UBE2C ubiquitin-conjugating enzyme 20d 13.12 E2C (UBE2C), transcript variant 1, mRNA. 202957 at O.OOO374 2.7 HCLS1 hematopoietic cell-specific Lyn 3q13 Substrate 1 (HCLS1), mRNA. 202968 S. at 0.0001551 2.3 DYRK2 dual-specificity tyrosine-(Y)- 12q15 phosphorylation regulated kinase 2 (DYRK2), transcript variant 1 mRNA. 202975 S. at 2.87E-05 -3.6 RHOBTB3 Rho-related BTB domain 5q15 containing 3 (RHOBTB3), mRNA. 202992 at O.OOOS 19 -5.4 C7 complement component 7 (C7), 5p13 mRNA. 202998 s at 0.0006468 3 LOXL2 lysyl oxidase-like 2 (LOXL2), 8p21.3-p21.2 mRNA. 2O3O88 at O.OOO8436 -4.5 FBLNS fibulin 5 (FBLN5), mRNA. 14q32.1 203156 at 3.37E-OS -2.4 AKAP11 A kinase (PRKA) anchor protein 13q14.11 11 (AKAP11), transcript variant 1, mRNA. 203166 at O.OOO1939 -2.3 CFDP1 craniofacial development protein 16q22.2-q22.3 1 (CFDP1), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 38
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 203178 at O.OOO2428 -3.3 GATM glycine amidinotransferase (L- 15q21.1 arginine:glycine amidinotransferase) (GATM), mRNA. 203249 at O.OOO4O75 -2.5 EZH1 enhancer of Zeste homolog 1 17q21.1-q21.3 (Drosophila) (EZH1), mRNA. 203297 s at 4.33E-05 2.4 ARID2 Jumonji, AT rich interactive 6p24-p23 domain 2 (JARID2), mRNA. 203298 s at 0.0005468 2.1 ARID2 Jumonji, AT rich interactive 6p24-p23 domain 2 (JARID2), mRNA. 203349 s at 7.26E-05 2.8 ETV5 ets variant gene 5 (ets-related 3q28 molecule) (ETV5), mRNA. 203356 at 7.77E-05 -2.6 CAPN7 calpain 7 (CAPN7), mRNA. 3p24 203358. S. at 2.44E-05 2.9 EZH2 enhancer of Zeste homolog 2 7q35-q36 (Drosophila) (EZH2), transcript variant 2 mRNA. 203401 at O.OOO3991 -3.8 PRPS2 phosphoribosyl pyrophosphate Xp22.3-p22.2 synthetase 2 (PRPS2), mRNA. 203423 at O.OOO8061 -3.9 RBP1 retinol binding protein 1, 3q23 cellular (RBP1), mRNA. 203424 S at 0.0007037 -3.8 GFBP5 insulin-like growth factor 2a33-q36 binding protein 5 (IGFBP5), mRNA. 203427 at 7. OSE-OS -2.6 ASF1A ASF1 anti-silencing function 1 6q22.31 homolog A (S. cerevisiae) (ASF1A), mRNA. 203450 at O.OOO7322 -2.1 PGEA1 PKD2 interactor, golgi and 22q12 endoplasmic reticulum associated 1 (PGEA1), transcript variant 1 mRNA. 203455 s at 0.0005583 2 SAT spermidine?spermine N1- Xp22.1 acetyltransferase (SAT), mRNA. 203459 s at 8.97E-05 2.1 VPS16 vacuolar protein sorting 16 20p13-p12 (yeast) (VPS16), transcript variant 2 mRNA. 203468 at O.OOO9911 2.3 CDK10 cyclin-dependent kinase (CDC2- 16q24 like) 10 (CDK10), transcript variant 2 mRNA. 203476 at 1.6SE-OS 2.6 TPBG trophoblast glycoprotein 6q14-q15 (TPBG), mRNA. 203493 s at 0.0009.017 -2.1 PIG8 translokin (PIG8), mRNA. 11q21 203494 S at 0.0001.224 -2.2 PIG8 translokin (PIG8), mRNA. 11q21 203505 at O.OOO1234 2.7 ABCA1 ATP-binding cassette, Sub- 9q31.1 family A (ABC1), member 1 203549 s at 0.0001406 3.9 LPL lipoprotein lipase (LPL), 8p22 mRNA. 203599 s at 0.0004137 -2 WBP4. WW domain binding protein 4 13q14.11 (formin binding protein 21) (WBP4), mRNA. 203640 at O.OOO2347 -2 MBNL2 muscleblind-like 2 (Drosophila) 13q32.1 (MBNL2), transcript variant 3, mRNA. 203657 s at 0.0007798 -2.3 CTSF cathepsin F (CTSF), mRNA. 11 q13 203680 at 9.OOE-07 -6.1 PRKAR2B protein kinase, cAMP- 7q22 dependent, regulatory, type II, beta (PRKAR2B), mRNA. 2036.92 s at 0.0005974 2.2 E2F3 E2F transcription factor 3 6p22 (E2F3), mRNA. 203695 s at O.OOO9082 -2.2 DFNAS deafness, autosomal dominant 5 7p15 (DFNA5), mRNA. 203758 at O.OOO2672 -2 CTSO cathepsin O (CTSO), mRNA. 4q31-q32 203762 s at 0.0003113 -2.1 D2LIC dynein 2 light intermediate chain 2p25.1-p24.1 (D2LIC), transcript variant 1, mRNA. 203799 at 1.06E-05 -3.3 CD302 CD302 antigen (CD302), 2d24.2 mRNA. 203803 at 3.79E-05 -3.9 PCYOX1 prenylcysteine oxidase 1 2p13.3 (PCYOX1), mRNA. 203845 at O.OOO1562 -2.8 PCAF p300/CBP-associated factor 3p24 (PCAF), mRNA. US 2013/02743 15 A1 Oct 17, 2013 39
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 203878. S. at 2.11E-05 3.7 MMP11 matrix metallopeptidase 11 22d 11.23 (stromelysin 3) (MMP11), mRNA. 203888 at O.OOO8536 THEBD thrombomodulin (THBD), 20p12-cen mRNA. 203897 at 7.35E-05 -2.2 LOCS7149 hypothetical protein A-211C6.1 16p11.2 (LOC57149), mRNA. 203908 at 2.7 Solute carrier family 4, Sodium bicarbonate cotransporter, member 4 (SLC4A4), mRNA. 203936 s at 9.4 MMP9 matrix metallopeptidase 9 20d 11.2-q13.1 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) (MMP9), mRNA. 204004 at 1SOE-06 -3.2 PAWR PRKC, apoptosis, WT1, regulator 204029 at O.OOOS472 2.4 CELSR2 cadherin, EGF LAG seven-pass G-type receptor 2 (flamingo homolog, Drosophila) (CELSR2), mRNA. 204041 at -6.4 MAOB Monoamine oxidase B Xp11.23 204045 at -4.5 TCEAL1 Transcription elongation factor Xq22.1 A (SII)-like 1 204078 at O.OOO1645 2.6 SC65 synaptonemal complex protein 17q21.2 SC65 (SC65), mRNA. 204082 at O.OOO2318 -2.8 PBX3 pre-B-cell leukemia transcription factor 3 (PBX3), mRNA. 204109 s at O.OOO6487 2.5 NFYA nuclear transcription factorY. 6p21.3 alpha (NFYA), transcript variant 2, mRNA. 204136 at 7.18E-OS 3.4 COL7A1 collagen, type VII, alpha 1 3p21.1 (epidermolysis bullosa, dystrophic, dominant and recessive) (COL7A1), mRNA. 20418.4 S at 9.26E-OS 3 ADRBK2 adrenergic, beta, receptor kinase 22q12.1 2 (ADRBK2), mRNA. 204235 s at 2.4SE-OS -4.3 GULP GULP, engulfment adaptor PTB domain containing 1 (GULP1), mRNA. 204237 at 1.32E-OS -3.2 GULP GULP, engulfment adaptor PTB domain containing 1 (GULP1), mRNA. 204256 at O.OOO8289 2.1 ELOVL6 ELOVL family member 6, elongation of long chain fatty acids (FEN1/Elo2, SUR4/Elo3 like, yeast) (ELOVL6), mRNA. 204285 S at 1.2OE-06 7.7 PMAIP1 phorbol-12-myristate-13 18q21.32 acetate-induced protein 1 (PMAIP1), mRNA. 204286 s at 3. SOE-06 8.5 PMAIP1 phorbol-12-myristate-13 18q21.32 acetate-induced protein 1 (PMAIP1), mRNA. 20438.7 x at 7.88E-OS 2.6 MRP63 mitochondrial ribosomal protein 13p11.1-q11 63 (MRP63), nuclear gene encoding mitochondrial protein, mRNA. 204454 at -2.6 LDOC1 leucine Zipper, down-regulated in cancer 1 (LDOC1), mRNA. 204473 s at O.OOO9569 ZNF592 Zinc finger protein 592 15q25.3 (ZNF592), mRNA. 204493 at 2.7 BID BH3 interacting domain death 22q11.1 agonist (BID), transcript variant 3, mRNA. 204495 s at O.OOO6319 2.8 DKFZP434H132 DKFZP434H132 protein 204531 s at O.OOO4972 2.5 BRCA1 breast cancer 1, early onset (BRCA1), transcript variant BRCA1-delta 9-11, mRNA. 204595 S. at STC1 stanniocalcin 1 (STC1), mRNA. 204597 x at STC1 stanniocalcin 1 (STC1), mRNA. 204619 s at O.OOOSS99 4.6 CSPG2 Chondroitin sulfate proteoglycan 2 (versican) US 2013/02743 15 A1 Oct 17, 2013 40
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 204639 at 1.47E-OS 4.2 ADA adenosine deaminase (ADA), 20d 12-q13.11 mRNA. 204641 at O.OOO6499 4.6 NEK2 NIMA (never in mitosis genea)- related kinase 2 (NEK2), mRNA. 20466.9 s at O.OOO8759 RNF24 ring finger protein 24 (RNF24), 20p13-p12.1 mRNA. 204731 at O.OOO2926 -3.2 transforming growth factor, beta receptor III (betaglycan, 300 kDa) (TGFBR3), mRNA. 204735 at 3.38E-OS 3.5 PDE4A phosphodiesterase 4A, cAMP 19p 13.2 specific (phosphodiesterase E2 dunce homolog, Drosophila) (PDE4A), mRNA. 204749 at O.OOOS462 NAP1L3 nucleosome assembly protein 1 ike 3 (NAP1L3), mRNA. 204786 s at 1.59E-OS IFNAR2 interferon (alpha, beta and 21q22.11 omega) receptor 2 (IFNAR2), transcript variant 1, mRNA. 204793 at 1.31E-OS GPRASP1 G protein-coupled receptor Xq22.1 associated sorting protein 1 (GPRASP1), mRNA. 204939 s at -5.7 PLN phospholamban (PLN), mRNA. 6q22.1 204994 at 4.5 MX2 myxovirus (influenza virus) 21q22.3 resistance 2 (mouse) (MX2), mRNA. 205068 s at 2.4OE-06 3.8 ARHGAP26 Rho GTPase activating protein 26 (ARHGAP26), mRNA. 205079 s at O.OOO3985 MPDZ multiple PDZ domain protein (MPDZ), mRNA. 205226 at O.OOO4657 PDGFRL platelet-derived growth factor 8p22-p21.3 receptor-like (PDGFRL), mRNA. 205231 s at O.OOO8847 epilepsy, progressive myoclonus type 2A, Lafora disease (laforin) (EPM2A), transcript variant 1, mRNA. 205241 at 4.2OE-05 4.5 SCO2 SCO cytochrome oxidase 22d 13.33 deficient homolog 2 (yeast) (SCO2), nuclear gene encoding mitochondrial protein, mRNA. 205259 at -2.5 nuclear receptor subfamily 3, 4q31.1 group C, member 2 (NR3C2), mRNA. 205269 at 4.OOE-06 8.7 LCP2 ymphocyte cytosolic protein 2 (SH2 domain containing eukocyte protein of 76 kDa) (LCP2), mRNA. 205270 s at O.OOO6743 6.7 LCP2 ymphocyte cytosolic protein 2 (SH2 domain containing eukocyte protein of 76 kDa) (LCP2), mRNA. 205304 is at O.OOO7499 2.4 KCN8 potassium inwardly-rectifying 12p11.23 channel, Subfamily J, member 8 (KCNJ8), mRNA. 205353 S at 3.92E-OS -3.3 PBP prostatic binding protein (PBP), 12q24.23 mRNA. 205367 at O.OOO1487 2.7 APS adaptor protein with pleckstrin homology and Src homology 2 domains (APS), mRNA. 205370 X at O.OOO4381 2.9 DBT dihydrolipoamide branched 1p31 chain transacylase E2 (DBT), mRNA. 205381 at O.OOO1503 -5.4 LRRC17 leucine rich repeat containing 17 (LRRC17), transcript variant 1, mRNA. 2054.06 s at 3.91E-OS 2.5 SPA17 sperm autoantigenic protein 17 11q24.2 (SPA17), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 41
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 205412 at 6.3OE-06 -2.7 ACAT1 acetyl-Coenzyme A 11q22.3-q23.1 acetyltransferase 1 (acetoacetyl Coenzyme A thiolase) (ACAT1), nuclear gene encoding mitochondrial protein, mRNA. 205463 s (OOO255 3.1 PDGFA Platelet-derived growth factor alpha polypeptide 205466 s 617E-05 heparan Sulfate (glucosamine) 3 O-sulfotransferase 1 (HS3ST1), mRNA. 205479 s (OOO306 3.8 PLAU plasminogen activator, 10q24 urokinase (PLAU), mRNA. 205483 s (OOO1248 6.9 interferon, alpha-inducible 1p36.33 protein (clone IFI-15K) (G1P2), mRNA. 205532 S ()004946 2.8 CDH6 cadherin 6, type 2, K-cadherin 5p15.1-p14 (fetal kidney) (CDH6), mRNA. 205572 at O.OOO3915 5.4 ANGPT2 angiopoietin 2 (ANGPT2), 8p23.1 mRNA. 205687 at 0.000463 UBPH similar to ubiquitin binding 16p12 protein (UBPH), mRNA. 205771 s (OOO9058 -2.3 AKAP7 A kinase (PRKA) anchor protein 7 (AKAP7), transcript variant alpha, mRNA. 205812 S 90OE-06 2.4 TMED9 transmembrane emp24 protein transport domain containing 9 (TMED9), mRNA. 205849 s ()005492 UQCRB ubiquinol-cytochrome c reductase binding protein (UQCRB), mRNA. 205862 at 7.65E-05 -8.6 GREB1 GREB1 protein (GREB1), 2p25.1 transcript varianta, mRNA. 205871 at 0.0003369 3.1 PLGLB1 plasminogen-like B1 (PLGLB1), mRNA. 205943 at 0.0004032 4.4 TDO2 tryptophan 2,3-dioxygenase (TDO2), mRNA. 205961 s ()002574 -2.9 PSIP1 PC4 and SFRS1 interacting protein 1 (PSIP1), transcript variant 2 mRNA. 206026 s pat 29.1 TNFAIP6 tumor necrosis factor, alpha 2d23.3 induced protein 6 (TNFAIP6), mRNA. 20615.8 s ()006831 -2.2 Zinc finger protein 9 (a cellular retroviral nucleic acid binding protein) (ZNF9), mRNA. 206169 x (a)0084.28 3.9 ROXaN Zinc finger CCCH-type 22d 13.2 containing 7B 206211 at 0.0002057 -8.9 SELE selectin E (endothelial adhesion 1q22-q25 molecule 1) (SELE), mRNA. 206247 at 9.38E-05 3.3 MICB MHC class I polypeptide-related 6p21.3 sequence B (MICB), mRNA. 206359 at 0.0004232 -3.5 SOCS3 Suppressor of cytokine signaling 3 17q25.3 206377 at 2.4OE-06 FOXF2 forkhead box F2 (FOXF2), 6p25.3 mRNA. 206435 at 0.0009321 3.2 GALGT UDP-N-acetyl-alpha-D- 12q13.3 galactosamine:(N- acetylneuraminyl)- galactosylglucosylceramide N acetylgalactosaminyltransferase (GalNAc-T) (GALGT), mRNA. 206483 at 0.0009412 LRRC6 leucine rich repeat containing 6 8q24.22 (LRRC6), mRNA. 206,551 X (a)001129 2.7 DRE1 DRE1 protein 3q27.1 206571 S. (ht)00131 2.7 MAP4K4 mitogen-activated protein kinase 2d 11.2-q12 kinase kinase kinase 4 (MAP4K4), transcript variant 1, mRNA. 206621 S ()002653 -2.3 WBSCR1 Williams-Beuren syndrome chromosome region 1 (WBSCR1), transcript variant 2, mRNA. US 2013/02743 15 A1 Oct 17, 2013 42
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 206637 at 4.8 purinergic receptor P2Y, G protein coupled, 14 (P2RY14), mRNA. 206792 x at 6.82E-OS 3.7 phosphodiesterase 4C, cAMP 19p 13.11 specific (phosphodiesterase E1 dunce homolog, Drosophila) (PDE4C), mRNA. 206809 s at O.OOO4239 -2.8 Heterogeneous nuclear 10q11.21 ribonucleoprotein A3 pseudogene 1 206.857 s at O.OOO2413 2.5 FK506 binding protein 1B, 12.6 kDa 2p23.3 (FKBP1 B), transcript variant 1 mRNA. 206874 S at -2.9 SLK STE20-like kinase (yeast) 10q25.1 206927 s at 3.3 GUCY1A2 guanylate cyclase 1, Soluble, 11q21-q22 alpha 2 (GUCY1A2), mRNA. 207040 S at 1.56E-OS -2.6 ST13 Suppression of tumorigenicity 13 22d 13.2 (colon carcinoma) (Hsp70 interacting protein) (ST13), mRNA. 207132 X at O.OOO2292 -2.5 prefoldin 5 (PFDN5), transcript variant 1 mRNA. 207147 at O.OOO7438 3.3 distal-less homeo box 2 (DLX2), mRNA. 207170 S. at 1.76E-OS -2.6 LETMD1 LETM1 domain containing 1 12q13.12 (LETMD1), transcript variant 3, mRNA. 207239 s at O.OOO2121 2.2 PCTK1 PCTAIRE protein kinase 1 Xp11.3-p11.23 (PCTK1), transcript variant 1, mRNA. 207365 x at 6.06E-OS 3.7 USP34 Ubiquitin specific protease 34 207386 at O.OOO9685 2.7 CYP7B1 cytochrome P450, family 7, Subfamily B, polypeptide 1 (CYP7B1), mRNA. 207598 x at O.OOO1463 3.5 X-ray repair complementing defective repair in Chinese hamster cells 2 (XRCC2), mRNA. 2O7688 s at O.OOO6478 2.4 LOC387933 PREDICTED: Similar to 13 heterogeneous nuclear ribonucleoprotein A3 (LOC387933), mRNA. 2O7730 X at O.OOO2746 3.1 hypothetical protein FLJ20700 19p13.3 207761 s at 1.38E-OS -4.2 DKFZP586 AO522 protein 12q13.12 (DKFZP586A0522), mRNA. 207961 X at O.OOO6679 -3.8 MYH11 myosin, heavy polypeptide 11, 16p13.13-p13.12 Smooth muscle (MYH11), transcript variant SM1 mRNA. 207974 S at O.OOO1511 -2.1 SKP1A S-phase kinase-associated protein 1A (p19A) (SKP1A), transcript variant 2, mRNA. 207983 s at O.OOO9431 -2.3 STAG2 stromal antigen 2 (STAG2), Xq25 mRNA. 208092 s at O.OOO41.78 2.9 FAM49A amily with sequence similarity 2p24.3-p24.2 49, member A (FAM49A), mRNA. 208137 X at O.OOO8157 2.6 ZNF611 Zinc finger protein 611 19q13.41 208.238 x at O.OOO7.093 2.4 LZLP eucine Zipper-like protein 11 q13.1 208.246 x at 8.09E-OS 3.6 FL2OOO6 hypothetical protein FLJ20006 16q23.1 208.248 x at S.35E-05 -3.5 APLP2 amyloid beta (A4) precursor-like 11q24 protein 2 (APLP2), mRNA. 208.540 x at O.OOO7507 S100A14 S100 calcium binding protein 7q22-q31.1 A14 (calgizZarin) 208.626 s at O.OOOSO9 -2.1 WAT1 vesicle amine transport protein 1 homolog (T californica) (VAT1), mRNA. 208.631 s at O.OOO3804 -2.3 HADHA hydroxyacyl-Coenzyme A 2p23 dehydrogenase 3-ketoacyl Coenzyme A thiolasetenoyl Coenzyme A hydratase (trifunctional protein), alpha subunit (HADHA), mRNA. US 2013/02743 15 A1 Oct 17, 2013 43
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 208635 X at 8.57E-OS -2 NACA nascent-polypeptide-associated 12q23-q24.1 complex alpha polypeptide (NACA), mRNA. 208.643 s at 2.41E-05 -2.9 XRCCS X-ray repair complementing defective repair in Chinese hamster cells 5 (double-strand break rejoining; Ku autoantigen, 80 kDa) (XRCC5), mRNA. 208647 at O.OOO4172 -2.1 FDFT1 farnesyl-diphosphate 8p23.1-p22 farnesyltransferase 1 (FDFT1), mRNA. 208653 s at O.OOO2993 CD164 CD164 antigen, sialomucin (CD164), mRNA. 208655 at O.OOO1893 -2.8 CCNI Cyclin I 208.658 at O.OOOS959 PDIA4 protein disulfide isomerase family A, member 4 (PDIA4), mRNA. 208662 s at O.OOO9682 -2.1 TTC3 tetratricopeptide repeat domain 21q22.2 3 (TTC3), transcript variant 2, mRNA. 208.666 s at S.OOE-07 Suppression of tumorigenicity 13 22d 13.2 (colon carcinoma) (Hsp70 interacting protein) (ST13), mRNA. 208.667 s at 4.53E-OS -2.8 ST13 Suppression of tumorigenicity 13 22d 13.2 (colon carcinoma) (Hsp70 interacting protein) (ST13), mRNA. 208671 at TDE2 tumor differentially expressed 2 (TDE2), mRNA. 208673 s at O.OOOSOO1 -2.2 SFRS3 splicing factor, arginine?serine rich 3 (SFRS3), mRNA. 208697 s at 4.8SE-OS -2.4 eukaryotic translation initiation actor 3, subunit 6 48 kDa (EIF3S6), mRNA. 2087.03 s at O.OOO2231 -3.7 amyloid beta (A4) precursor-like 11q24 protein 2 (APLP2), mRNA. 208704 x at 4.90E-OS -3.6 amyloid beta (A4) precursor-like 11q24 protein 2 (APLP2), mRNA. 208740 at -2.2 sin3-associated polypeptide, 13q12.11 18 kDa (SAP18), mRNA. 208760 at 7.17E-05 -2.7 Ubiquitin-conjugating enzyme 16p13.3 E2I (UBC9 homolog, yeast) 20877O S at O.OOOS681 eukaryotic translation initiation actor 4E binding protein 2 (EIF4EBP2), mRNA. 208771 s at 2.1OE-05 -2.6 eukotriene A4 hydrolase (LTA4H), mRNA. 208781 X at O.OOO6007 -2.2 sorting nexin 3 (SNX3), transcript variant 1, mRNA. 208791 at O.OOO1028 CLU clusterin (complement lysis inhibitor, SP-40.40, sulfated glycoprotein 2, testosterone repressed prostate message 2, apolipoprotein J) (CLU), transcript variant 1 mRNA. 208792 S at O.OOO1175 CLU clusterin (complement lysis inhibitor, SP-40.40, sulfated glycoprotein 2, testosterone repressed prostate message 2, apolipoprotein J) (CLU), transcript variant 1 mRNA. 208794 S at 1.47E-OS 2.4 SMARCA4 SWI/SNF related, matrix 19p 13.2 associated, actin dependent regulator of chromatin, Subfamily a member 4 (SMARCA4), mRNA. 208796 S. at O.OOO1639 -2.6 CCNG1 cyclin G1 (CCNG1), transcript variant 2 mRNA. 208848 at 1SOE-06 -3.7 ADHS alcohol dehydrogenase 5 (class III), chi polypeptide (ADH5), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 44
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 20886O is at O.OOO1719 -2 ATRX alpha thalassemiamental Xq13.1-q21.1 retardation syndrome X-linked (RAD54 homolog, S. cerevisiae) (ATRX), transcript variant 2, mRNA. 208873 s at -2.1 CSOrf18 chromosome 5 open reading frame 18 (C5orf18), mRNA. 208920 at O.OOO3111 -2.3 SRI Sorcin (SRI), transcript variant 2, mRNA. 208925 at O.OOOS429 -2.3 chromosome 3 open reading frame 4 (C3orf4), mRNA. 208950 s at O.OOO4293 -2.3 ALDHAA1 aldehyde dehydrogenase 7 family, member A1 (ALDH7A1), mRNA. 208951 at 4.2OE-05 -2.8 ALDHAA1 aldehyde dehydrogenase 7 family, member A1 (ALDH7A1), mRNA. 208962 s at -2.4 FADS1 fatty acid desaturase 1 (FADS1), 11 q12.2-q13.1 mRNA. 208990 s. at S.40 E-OS -2.2 HNRPH3 heterogeneous nuclear 10q22 ribonucleoprotein H3 (2H9) (HNRPH3), transcript variant 2H9A, mRNA. 209009 at 1. SOE ESD esterase D formylglutathione 13q14.1-q14.2 hydrolase (ESD), mRNA. 209030 s at 3.07E IGSF4 immunoglobulin Superfamily, 11q23.2 member 4 (IGSF4), mRNA. 209034 at 3.2OE PNRC1 proline-rich nuclear receptor 6q15 coactivator 1 (PNRC1), mRNA. 209068 at 1.OOE HNRPDL heterogeneous nuclear ribonucleoprotein D-like (HNRPDL), transcript variant 2, mRNA. 209081 s at COL18A1 collagen, type XVIII, alpha 1 21q22.3 (COL18A1), transcript variant 2, mRNA. 209082 s at 4.04E S.1 COL18A1 collagen, type XVIII, alpha 1 21q22.3 (COL18A1), transcript variant 2, mRNA. 209137 s at O.OOO7917 -2.1 USP10 ubiquitin specific peptidase 10 16q24.1 (USP10), mRNA. 209143 s at O.OOO9179 -2.1 CLNS1A chloride channel, nucleotide 11 q13.5-q14 sensitive, 1A (CLNS1A), mRNA. 209146 at O.OOO2317 -3.2 SC4MOL sterol-C4-methyl oxidase-like (SC4MOL), transcript variant 2, mRNA. 2091.69 at O.OOO3677 4.2 glycoprotein M6B (GPM6B), Xp22.2 transcript variant 1, mRNA. 209170 s at O.OOO14 5.8 glycoprotein M6B (GPM6B), Xp22.2 transcript variant 4, mRNA. 209243 s at 3.90E-06 -7.7 PEG3 paternally expressed 3 (PEG3), 19q13.4 mRNA. 2093.05 s at O.OOO1816 -3.7 GADD45B growth arrest and DNA-damage 19p13.3 inducible, beta (GADD45B), mRNA. 209337 at 4.01E-OS PSIP1 PC4 and SFRS1 interacting 9p22.3 protein 1 (PSIP1), transcript variant 2 mRNA. 209357 at -3.3 CITED2 Cbp/p300-interacting transactivator, with Glu/Asp rich carboxy-terminal domain, 2 (CITED2), mRNA. 209360 s at 7.44E-05 4.8 RUNX1 runt-related transcription factor 21q22.3 1 (acute myeloid leukemia 1: aml1 oncogene) (RUNX1), transcript variant 1, mRNA. 209384 at -2 PROSC proline synthetase co-transcribed 8p11.2 homolog (bacterial) (PROSC), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 45
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 209385 S at 3.63E-OS -2.6 PROSC proline synthetase co-transcribed 8p11.2 homolog (bacterial) (PROSC), mRNA. 209447 at 3.84E-05 -2.5 SYNE1 spectrin repeat containing, nuclear envelope 1 (SYNE1), transcript variant alpha, mRNA. 209512 at 3.90E-06 -3.9 HSDL2 hydroxysteroid dehydrogenase like 2 (HSDL2), mRNA. 209513 s at 1.94E-05 -3.9 HSDL2 hydroxysteroid dehydrogenase like 2 (HSDL2), mRNA. 209596 at 4.8OE-06 6.9 MXRAS matrix-remodelling associated 5 (MXRA5), mRNA. 209605 at O.OOO4157 -2.4 TST thiosulfate sulfurtransferase 22d 13.1 (rhodanese) (TST), nuclear gene encoding mitochondrial protein, mRNA. 20961.2 s at O.OOO1328 ADH1B alcohol dehydrogenase IB (class ), beta polypeptide (ADH1B), mRNA. 209613 s at O.OOO3092 -5.8 ADH1B alcohol dehydrogenase IB (class ), beta polypeptide (ADH1B), mRNA. 209633 at O.OOO6246 -2.2 protein phosphatase 2 (formerly 3q22.1 2A), regulatory subunit B", alpha (PPP2R3A), transcript variant 2 mRNA. 209657 s at O.OOO3701 -2.1 heat shock transcription factor 2 6q22.31 (HSF2), mRNA. 209685 S at 1.1OE-OS 3.7 PRKCB1 protein kinase C, beta 1 16p11.2 (PRKCB1), transcript variant 2, mRNA. 209733 at 164E-05 -2.7 LOC286440 Hypothetical protein LOC28644O 209737 at -2.2 MAGI2 membrane associated guanylate kinase, WW and PDZ domain containing 2 (MAGI2), mRNA. 209875 s at 9.4OE-06 9.5 SPP1 Secreted phosphoprotein 1 (Osteopontin, bonesialoprotein early T-lymphocyte activation ) (SPP1), mRNA. 2098.94 at O.OOO1625 LEPR eptin receptor (LEPR), 1p31 transcript variant 2, mRNA. 209897 s at 2.5 SLIT2 slit homolog 2 (Drosophila) 4p15.2 (SLIT2), mRNA. 209969 s at 3.7 STAT1 signal transducer and activator 2a32.2 of transcription 1, 91 kDa (STAT1), transcript variant beta, mRNA. 210.048 at O.OOO8484 2.2 NAPG N-ethylmaleimide-sensitive 18p11.22 factor attachment protein, gamma (NAPG), mRNA. 210.069 at 3.07E-OS 2.8 carnitine palmitoyltransferase 22d 13.33 1B (muscle) (CPT1B), nuclear gene encoding mitochondrial protein, transcript variant 3, mRNA. 210365 at O.OOO2181 2.6 RUNX1 Runt-related transcription factor 21q22.3 1 (acute myeloid leukemia 1: aml1 oncogene) 210438 x at O.OOO3684 -2.7 TROVE2 TROVE domain family, member 2 (TROVE2), mRNA. 210621 s at -2.1 RASA1 RAS p21 protein activator (GTPase activating protein) 1 (RASA1), transcript variant 2, mRNA. 210664 S at O.OOO6973 2.4 TFPI tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) (TFPI), transcript variant 1, mRNA. US 2013/02743 15 A1 Oct 17, 2013 46
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 210665 at O.OOO3981 3.1 TFPI issue factor pathway inhibitor 231-q32.1 (lipoprotein-associated coagulation inhibitor) (TFPI), transcript variant 2, mRNA. 210679 X at 0.000265 3.4 BCL7A B-cell CLLlymphoma 7A 12q24.13 210788 s at 1.26E-05 -3.1 DHRS7 dehydrogenase/reductase (SDR 14q23.1 amily) member 7 (DHRS7), mRNA. 210800 at O.OOO97O6 4.7 MGC12262 hypothetical protein MGC12262 210809 S at 0.0002365 6.7 POSTN periostin, osteoblast specific 13q13.3 actor (POSTN), mRNA. 210944 s at 0.0009253 2.3 CAPN3 calpain 3, (p94) (CAPN3), 15q15.1-q21.1 transcript variant 7, mRNA. 210950 s at 1.38E-OS -3.3 FDFT1 airnesyl-diphosphate 8p23.1-p22 arnesyltransferase 1 (FDFT1), mRNA. 211040 x at 0.0006364 2.4 GTSE1 G-2 and S-phase expressed 1 22d 13.2-q13.3 (GTSE1), mRNA. 211276 at 8.52E-OS -5.6 TCEAL2 transcription elongation factor A Xq22.1-q22.3 (SII)-like 2 (TCEAL2), mRNA. 211423 s at 0.000164 -2.8 SCSDL sterol-C5-desaturase (ERG3 11q23.3 delta-5-desaturase homolog, ungal)-like (SCSDL), transcript variant 1 mRNA. 211445 X at 0.0007153 3.6 FKSG17 FKSG17 8q22.3 211452 x at 5.23E-05 3.4 LRRFIP1 eucine rich repeat (in FLII) 2a37.3 interacting protein 1 (LRRFIP1), mRNA. 211454 X at 0.0006917 3.7 211569 is a 2.66E-05 -5.7 HADHSC L-3-hydroxyacyl-Coenzyme A 4q22-q26 dehydrogenase, short chain (HADHSC), mRNA. 211597 s a 1.OOE-07 13.1 HOP homeodomain-only protein 4q11-q12 (HOP), transcript variant 2, mRNA. 211623 s a O.OOO8386 -2.2 FBL fibrillarin (FBL), mRNA. 19q13.1 211666 X at 9.6OE-06 -2.6 RPL3 ribosomal protein L3 (RPL3), 22d 13 mRNA. 211673 s a S.6SE-OS 3.5 MOCS1 Molybdenum cofactor synthesis 1 6p21.3 211710 X at 0.0001136 -2.2 RPL4 ribosomal protein L4 (RPL4), 15q22 mRNA. 211725 s a O.OOO6346 2.4 BID BH3 interacting domain death 22q11.1 agonist (BID), transcript variant 3, mRNA. 211727 s a O.OOO4584 -2.5 COX11 COX11 homolog, cytochrome c 17q22 oxidase assembly protein (yeast) (COX11), nuclear gene encoding mitochondrial protein, mRNA. 211749 s at 7.41E-05 -2.6 WAMP3 vesicle-associated membrane 1p36.23 protein 3 (cellubrevin) (VAMP3), mRNA. 211762 s at 0.0002377 2.3 KPNA2 karyopherin alpha 2 (RAG 17q23.1-q23.3 cohort 1, importin alpha 1) (KPNA2), mRNA. 211769 X at 4.67E-05 -2.4 TDE1 tumor differentially expressed 1 20131-13.3 (TDE1), transcript variant 1, mRNA. 211813 X at 1.69E-05 -4.8 DCN decorin (DCN), transcript 12q21.33 variant D, mRNA. 211896 S. at 0.0006287 -3.7 DCN decorin (DCN), transcript 12q21.33 variant C. mRNA. 211937 at O.OOO1012 -3.2 EIF4B eukaryotic translation initiation 12q13.13 factor 4B (EIF4B), mRNA. 211938 at 3.OOE-07 -3.2 EIF4B eukaryotic translation initiation 12q13.13 factor 4B (EIF4B), mRNA. 211941 s at 2.01E-05 -2.3 PBP prostatic binding protein (PBP), 12q24.23 mRNA. 211942 X at 7.02E-05 -2.8 RPL13A Ribosomal protein L13a 1913.3 211964 at O.OOO6935 3.4 COL4A2 Collagen, type IV, alpha 2 1334 211980 at O.OOOS493 3.9 COL4A1 collagen, type IV, alpha 1 1334 (COL4A1), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 47
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 211986 at 3.42E-05 -3.3 AHNAK AHNAKnucleoprotein 11 q12.2 (desmoyokin) (AHNAK), transcript variant 1, mRNA. 211988 at 1.3OE-OS -2.2 SMARCE1 SWI/SNF related, matrix 17q21.2 associated, actin dependent regulator of chromatin, Subfamily e, member 1 (SMARCE1), mRNA. 211994 at -2 Transcribed locus, strongly 12 similar to XP 508919.1 PREDICTED: similar to protein kinase, lysine deficient 1; kinase deficient protein Pan troglodytes 211997 X at -2.5 H3 histone, family 3B (H3.3B) 17q25 (H3F3B), mRNA. 211998 at -3 H3 histone, family 3B (H3.3B) 17q25 (H3F3B), mRNA. 212037 at O.OOO1271 -2.7 PNN Pinin, desmosome associated 14q21.1 protein 212044 S at 9.OOE-07 3.4 RPL27A ribosomal protein L27a 11 p 15 (RPL27A), mRNA. 212052 s at O.OOOS363 KIAAO676 KIAA0676 protein (KIAAO676), transcript variant 2, mRNA. 212094 at O.OOO1189 PEG10 PREDICTED: paternally expressed 10 (PEG10), mRNA. 212096 s at O.OOO2831 MTUS1 mitochondrial tumor suppressor (MTUS1), nuclear gene encoding mitochondrial protein, transcript variant 5, mRNA. 212131 at 4.28E-OS FAM61A amily with sequence similarity 19q13.11 61, member A (FAM61A), mRNA. 212134 at PHILDB1 pleckstrin homology-like 11q23.3 domain, family B, member 1 (PHLDB1), mRNA. 212144 at O.OOOS431 -2.1 UNC84B unc-84 homolog B (C. elegans) 22d 13.1 (UNC84B), mRNA. 212148 at 9.2OE-06 -2.9 PBX1 Pre-B-cell leukemia 1q23 transcription factor 1 212151 at O.OOO1748 -2.5 PBX1 Pre-B-cell leukemia 1q23 transcription factor 1 212171 x at O.OOO6474 3.3 VEGF vascular endothelial growth actor (VEGF), transcript variant 6, mRNA. 212179 at O.OOO3184 -2.5 Chromosome 6 open reading rame 111 212188 at O.OOO184 -2.9 KCTD12 potassium channel 13q22.3 etramerisation domain containing 12 (KCTD12), mRNA. 212195 at O.OOO2328 -2.1 interleukin 6 signal transducer (gp130, oncostatin M receptor) 212199 at O.OOO1637 -2.5 MRFAP1L1 Morfa family associated protein 4p16.1 -like 1 (MRFAP1L1), transcript variant 2, mRNA. 212215 at O.OOO1021 -2.8 PREPL prolyl endopeptidase-like (PREPL), mRNA. 212224 at 1.18E-05 ALDH1A1 aldehyde dehydrogenase 1 amily, member A1 (ALDH1A1), mRNA. 2 2236 X at O.OOO2768 5.3 KRT17 keratin 17 (KRT17), mRNA. 212254 s at 4.OOE-06 -2.7 DST dystonin (DST), transcript variant lea, mRNA. 212256 at O.OOO7645 -2.9 GALNT10 UDP-N-acetyl-alpha-D- galactosamine:polypeptide N acetylgalactosaminyltransferase O (GalNAc-T10) 212323 s at 2.11E-05 2.5 VPS13D vacuolar protein sorting 13D 1p36.22-p36.21 (yeast) (VPS13D), transcript variant 2 mRNA. US 2013/02743 15 A1 Oct. 17, 2013 48
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 212351 a 8.OOE-06 2.2 EIF2B5 eukaryotic translation initiation 3q27.1 factor 2B, subunit 5 epsilon, 82 kDa (EIF2B5), mRNA. 212353 a O.OOO225 3.6 SULF1 Sulfatase 1 (SULF1), mRNA. 8q13.2-q13.3 212354 a O.OOO2219 2.9 SULF1 Sulfatase 1 (SULF1), mRNA. 8q13.2-q13.3 212365 a O.OOO3347 4.1 MYO1B myosin IB (MYO1 B), mRNA. 2012-q34 212368 a O.OOO2867 -2.3 ZNF292 PREDICTED: zinc finger 6 protein 292 (ZNF292), mRNA. 212408 a 1.41E-05 -2.6 TOR1AIP1 torsin A interacting protein 1 1q24.2 (TOR1AIP1), mRNA. 212413 a O.OOOS848 2 6-Sep septin 6 (SEPT6), transcript Xq24 variant II, mRNA. 212414 S at 0.0001527 2.4 6-Sep septin 6 (SEPT6), transcript Xq24 variant II, mRNA. 212435 a. O.OOO3489 -2.3 TRIM33 tripartite motif-containing 33 1p13.1 (TRIM33), transcript variant b, mRNA. 212468 a O.OOO1247 -2 SPAG9 Sperm associated antigen 9 17q21.33 212498 a 1SOE-06 -2.7 MARCH-VI Membrane-associated ring 5p15.2 finger (C3HC4) 6 212510 a O.OOO4844 -3.1 GPD1 L, glycerol-3-phosphate 3p24.1 dehydrogenase 1-like (GPD1L), mRNA. 212520 S. at 3.25E-05 3.1 SMARCA4 SWI/SNF related, matrix 19p 13.2 associated, actin dependent regulator of chromatin, Subfamily a member 4 (SMARCA4), mRNA. 212526 at 6.11E-OS -2.3 SPG20 spastic paraplegia 20, spartin 13q13.3 (Troyer syndrome) (SPG20), mRNA. 212549 at 6.64E-OS -2.4 STATSB signal transducer and activator 17q11.2 of transcription 5B (STATSB), mRNA. 212556 at O.OOO3S6S 2.8 SCRIB scribbled homolog (Drosophila) 8q24.3 (SCRIB), transcript variant 2, mRNA. 212586 at O.OOO4909 -2.1 CAST calpastatin (CAST), transcript 5q15-q21 variant 2 mRNA. 212595 s a O.OOO7053 -2 DAZAP2 DAZ associated protein 2 12q12 (DAZAP2), mRNA. 212609 s a 1.8SE-OS -2.4 AKT3 V-akt murine thymoma viral 1q43-q14 oncogene homolog 3 (protein kinase B, gamma) 212624 S a 5.4OE-06 6.9 CHN1 chimerin (chimaerin) 1 (CHN1), 231-q32.1 transcript variant 2, mRNA. 212632 at O.OOO8287 -2 STX7 Syntaxin 7 6q23.1 212638 s a O.OOO7524 -2.2 WWP1 WW domain containing E3 8q21 ubiquitin protein ligase 1 (WWP1), mRNA. 212644 S a O.OOO1184 -2.3 C14orf32 chromosome 14 open reading 14q22.2-q22.3 frame 32 (C14orf32), mRNA. 212646 at O.OOO3295 2.6 RAFTLIN raft-linking protein (RAFTLIN), 3p25.1-p24.3 mRNA. 212653 s a 3.OOE-07 -3.8 EHBP1 EH domain binding protein 1 2p15 (EHBP1), mRNA. 212675 s a O.OOO3318 -3.5 KIAAOS82 KIAAOS82 2p14 212730 at O.OOO67O6 -3.4 DMN desmuslin (DMN), transcript 15p26.3 variant B, mRNA. 212731 at O.OOO1 OS -3.3 ANKRD46 ankyrin repeat domain 46 8q22.3 (ANKRD46), mRNA. 212751 at O.OOO3336 -2.7 UBE2IN ubiquitin-conjugating enzyme 12q22 E2N (UBC13 homolog, yeast) (UBE2N), mRNA. 212769 at O.OOO184 2.7 TLE3 Transducin-like enhancer of 15q22 split 3 (E(Sp1) homolog, Drosophila) 212776 s at 0.0004769 -3 KIAAO657 PREDICTED: KIAAO657 2 protein (KIAAO657), mRNA. 212779 at O.OOOS/62 -2 KIAA1109 PREDICTED: hypothetical 4 protein KIAA1109 (KLAA1109), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 49
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 212798. S. at 3.32E-05 -2 ANKMY2 ankyrin repeat and MYND 7p21 domain containing 2 (ANKMY2), mRNA. 212809 at 7.32E-OS 2.6 NFATC2IP nuclear factor of activated T- 16p11.2 cells, cytoplasmic, calcineurin dependent 2 interacting protein (NFATC2IP), mRNA. 212841 s at 0.0004662 2.6 PPFIBP2 PTPRF interacting protein, 11 p 15.4 binding protein 2 (liprin beta 2) (PPFIBP2), mRNA. 212943 at O.OOO9511 -2.2 KIAAOS28 KIAA0528 gene product 12p12.1 (KIAAO528), mRNA. 212971 at O.OOO4892 2.2 CARS Cysteinyl-tRNA synthetase 11 p 15.5 213002 at 7.97E-05 3.8 MARCKS myristoylated alanine-rich 6q22.2 protein kinase C Substrate (MARCKS), mRNA. 213005 s at 0.0003887 -2.3 ANKRD15 ankyrin repeat domain 15 9p24.3 (ANKRD15), transcript variant 1, mRNA. 213027 at O.OOO1503 -2.1 SSA2 TROVE domain family, 1q31 member 2 213029 at 6.18E-OS -2.4 NFIB Nuclear factor IB 9p24.1 213047 X at 0.0003826 -2.6 SET SET translocation (myeloid 9q34 eukemia-associated) (SET), mRNA. 213074 at O.OOO2338 -2.6 PHIP Pleckstrin homology domain 6q14 interacting protein 213085 S at 0.0005289 3.8 KIBRA KIBRA protein (KIBRA), 5q34 mRNA. 213093 at 18OE-05 -2.8 PRKCA protein kinase C, alpha 17q22-q23.2 (PRKCA), mRNA. 213110 S. at 5.00E-06 -3.9 COL4A5 collagen, type IV, alpha 5 Xq22 (Alport syndrome) (COL4A5), transcript variant 1, mRNA. 213111 a O.OOO6152 -2 PIPSK3 phosphatidylinositol-3- 2a34 phosphate:phosphatidylinositol 5-kinase, type III (PIP5K3), transcript variant 1, mRNA. 213117 a O.OOO8783 -2 KLHL9 kelch-like 9 (Drosophila) 9p22 (KLHL9), mRNA. 213139 a O.OOO8306 2.6 SNAI2 Snail homolog 2 (Drosophila) 8q11 (SNAI2), mRNA. 213146 a O.OOO12S 2.9 KIAAO346 KIAA0346 protein 17p13.1 213224 S at 0.0005954 -2 LOC92482 PREDICTED: hypothetical 10 protein LOC92482 (LOC92482), mRNA. 213227 a O.OOO1677 -2 PGRMC2 Progesterone receptor membrane 4q26 component 2 213248 a O.OOO6637 2.4 LOC221362 Hypothetical protein 6p12.3 LOC221362 213258 a 3.1OE-OS 2.5 TFPI Tissue factor pathway inhibitor 231-q32.1 (lipoprotein-associated coagulation inhibitor) 213272 s at 6.8OE-06 -4 LOCS7146 promethin (LOC57146), mRNA. 16p12 213344 S at 3.91E-05 2.7 H2AFX H2A histone family, member X 11q23.2-q23.3 (H2AFX), mRNA. 213350 at 1.2OE-05 6.1 RPS11 ribosomal protein S11 (RPS11), 19q13.3 mRNA. 213364 S at 4.00E-07 -3.7 SNX1 sorting nexin 1 (SNX1), 15q22.31 transcript variant 2, mRNA. 213370 S. at 0.0001347 2.1 SFMBT1 Scm-like with four mbt domains 3p21.1 1 (SFMBT1), transcript variant 3, mRNA. 213397 X at 4.3OE-06 -5.2 RNASE4 ribonuclease, RNase A family, 4 14q11.1 (RNASE4), transcript variant 3, mRNA. 213405 at O.OOO663 -2.4 RAB22A RAB22A, member RAS 20d 13.32 oncogene family (RAB22A), mRNA. 213413 at O.OOOS89 -2.4 SBLF stoned B-like factor (SBLF), 2p16.3 mRNA. US 2013/02743 15 A1 Oct. 17, 2013 50
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 213418 at 3.79E-05 6.8 HSPA6 heat shock 70 kDa protein 6 1q23 (HSP7OB') (HSPA6), mRNA. 213464 at 0.0005718 2.7 SHC2 SHC (Src homology 2 domain 19p13.3 containing) transforming protein 2 213479 at 2.75E-05 4.3 NPTX2 neuronal pentraxin II (NPTX2), 7q21.3-q22.1 mRNA. 213523 at 5.86E-05 2.6 CCNE1 cyclin E1 (CCNE1), transcript 19q12 variant 2 mRNA. 213560 at 0.0005733 2.4 GADD45B growth arrest and DNA-damage- 19p13.3 inducible, beta (GADD45B), mRNA. 213574 s 1a)OE-06 -2.4 KPNB1 Karyopherin (importin) beta 1 17q21.32 213605 S ()009961 2.5 FLJ40092 FLJ40092 protein 5q13.2 213661 at 8.00E-07 6.2 DKFZP586H2123 regeneration associated muscle 11 p.13 protease (DKFZP586H2123), transcript variant 2, mRNA. 213687 S (OOO9746 -2.1 RPL3SA ribosomal protein L35a 3q29-qter (RPL35A), mRNA. 213693 S. 45E-05 6.2 MUC1 mucin 1, transmembrane q21 (MUC1), transcript variant 4, mRNA. 213778 x (a)00467 2.2 ZFP276 Zinc finger protein 276 homolog 6q24.3 (mouse) (ZFP276), mRNA. 213790 at 7.51E-05 4.4 ADAM12 A disintegrin and Oq26.3 metalloproteinase domain 12 (meltrin alpha) 213803 at p <1e-O7 -3.6 KPNB1 Karyopherin (importin) beta 1 7q21.32 213836 s (OOO1261 2.2 WIPI49 WD40 repeat protein Interacting 7q24.2 with phosphoInositides of 49 kDa (WIPI49), mRNA. 213848 at 2.OOE-07 3.1 DUSP7 Dual specificity phosphatase 7 3p21 213869 x (a)002103 4.6 THY1 Thy-1 cell Surface antigen 1q22.3-q23 (THY1), mRNA. 213895 at 0.0004485 -2.5 EMP1 Epithelial membrane protein 1 2p12.3 213900 at 3.89E-05 -2.7 C9orf61 chromosome 9 open reading 9q13-q21 frame 61 (C9orf61), mRNA. 213905. X (a)001976 4.6 BGN Biglycan Xq28 213943 at 4.00E-07 19 TWIST1 twist homolog 1 7p21.2 (acrocephalosyndactyly 3: Saethre-Chotzen syndrome) (Drosophila) (TWIST1), mRNA. 213979 s (OOO2226 3.3 CTBP1 C-terminal binding protein 1 4p16 (CTBP1), transcript variant 1, mRNA. 214023 x (a)00646 2.8 MGC8685 Tubulin, beta polypeptide 6p25 paralog 214041 x (a)00362 2.8 RPL37A Ribosomal protein L37a 2a35 214052 x (a)005.361 2.7 BAT2D1 BAT2 domain containing 1 1q23.3 (BAT2D1), mRNA. 214057 at 0.0005414 2.3 MCL1 myeloid cell leukemia sequence 1q21 1 (BCL2-related) (MCL1), transcript variant 2, mRNA. 214081 at 4.OOE-07 10.2 PLXDC1 plexin domain containing 1 17q21.1 (PLXDC1), mRNA. 214097 at 2.29E-05 -2.3 RPS21 Ribosomal protein S21 20d 13.3 214110 S (OOO1953 3 ESTs, Highly similar to A43542 lymphocyte-specific protein 1 H. Sapiens 214140 at 0.0004058 2 SLC25A16 solute carrier family 25 10q21.3 (mitochondrial carrier; Graves disease autoantigen), member 16 (SLC25A16), nuclear gene encoding mitochondrial protein, mRNA. 214149 s (OOO3428 3.4 ATP6VOE ATPase, H+ transporting, 5q.35.1 lysosomal 9 kDa, VO subunite 214177 s ()001027 -2.2 PBXIP1 pre-B-cell leukemia 1q22 transcription factor interacting protein 1 (PBXIP1), mRNA. 214264 s (at)002244 2.1 C14orf143 chromosome 14 open reading 14q32.11 frame 143 (C14orf143), mRNA. US 2013/02743 15 A1 Oct 17, 2013 51
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 214316 x at 7.97E-05 2.4 CALR Calreticulin 19p13.3-p13.2 214359 is at O.OOO1495 -3 HSPCB heat shock 90 kDa protein 1, beta 6p12 (HSPCB), mRNA. 214426 x at O.OOO8417 2.1 CHAF1A chromatin assembly factor 1, 19p13.3 subunit A (p150) (CHAF1A), mRNA. 214435 X at 0.0005753 2.8 RALA v-ral simian leukemia viral oncogene homolog A (ras related) (RALA), mRNA. 214438 at O.OOO956S 3.4 H2.0-like homeo box 1 (Drosophila) (HLX1), mRNA. 214527 s at 4.2OE-06 -2.3 PQBP1 polyglutamine binding protein 1 Xp11.23 (PQBP1), transcript variant 5, mRNA. 214594 X at 0.0002968 2.9 ATP8B1 ATPase, Class I, type 8B, member 1 2 4707 X at O.OOO3366 2.8 ALMS1 Alstrom syndrome 1 214715 x at O.OOO2241 4.3 ZNF160 Zinc finger protein 160 (ZNF160), transcript variant 1, mRNA. 214724 at O.OOO6038 -2.1 DIXDC1 DIX domain containing 1 (DDXDC1), mRNA. 214737 X at 0.0004018 -2.2 HNRPC heterogeneous nuclear 4q11.2 ribonucleoprotein C (C1/C2) (HNRPC), transcript variant 2, mRNA. 214855 s at 4.03E-05 -2.6 GARNL1 GTPase activating Rap/RangAP 4q13.2 domain-like 1 (GARNL1), transcript variant 2, mRNA. 214862 X at 0.0009591 -2.8 MRNA, cDNA DKFZp564G 1162 (from clone DKFZp564G 1162) 214924 S at 2.90E-06 3.8 OIP106 OGT(O-Glc-NAc transferase)- 3p25.3-p24.1 interacting protein 106 KDa (OIP106), mRNA. 215016 X at 1.5OE-05 -2.3 DST dystonin (DST), transcript variant 1 mRNA. 215046 at O.OOO6908 -2.2 FLU23861 hypothetical protein FLJ23861 (FLJ23861), mRNA. 215073 s at 3.83E-05 -2.1 nuclear receptor subfamily 2, group F, member 2 (NR2F2), mRNA. 215076 s at 0.000876 COL3A1 collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant) (COL3A1), mRNA. 215179 x at 6.8OE-05 4.5 PGF Placental growth factor, vascular 14q24-q31 endothelial growth factor-related protein 215203 at O.OOO626S 2.2 GOLGA4 Golgi autoantigen, golgin 3p22-p21.3 Subfamily a 4 215206 at O.OOO1618 3.1 EXT1 Exostoses (multiple) 1 8q24.11-q24.13 2 5208 X at O.OOO6292 3.4 RPL3SA Ribosomal protein L35a 215294 is at 8.91E-OS -2.9 SMARCA1 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, Subfamily a member 1 (SMARCA1), transcript variant 2, mRNA. 215306 at O.OOO8848 -3.4 LHCGR. Luteinizing 2p21 hormone? choriogonadotropin receptor 215336 at 9.2iSE-OS 2.7 AKAP11 A kinase (PRKA) anchor protein 13q14.11 11 (AKAP11), transcript variant 2, mRNA. 215373 X at 0.0004506 2.8 SET8 PR/SET domain containing 12q24.31 protein 8 215383 x at 0.0009763 2.6 SPG21 Spastic paraplegia 21 (autosomal recessive, Mast syndrome) US 2013/02743 15 A1 Oct. 17, 2013 52
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 215404 x a O.OOOS104 3.2 FGFR1 Fibroblast growth factor 8p11.2-p11.1 receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome) 215467 x a O.OOO4682 3.9 DHX9 DEAH (Asp-Glu-Ala-His) box 1q25 polypeptide 9 215504 x a O.OOO6038 2.5 ANKRD10 Ankyrin repeat domain 10 13q34 215529 X a O.OOO7444 2.6 C21orf106 Chromosome 21 open reading 21q22.3 rame 106 215566 x a O.OOO7954 2.2 LYPLA2 ysophospholipase II (LYPLA2), 1p36.12-p35.1 mRNA. 215577 at S.29E-OS 2.4 UBE2E1 Ubiquitin-conjugating enzyme 3p24.2 E2E 1 (UBC45 homolog, yeast) 215588. X a 6.38E-OS 3.7 RIOK3 RIOkinase 3 (yeast) 18q11.2 215599 at 4.1OE-06 3.9 SMA4 SMA4 5q13 215600 x a O.OOOS929 3.3 FBXW12 F-box and WD-40 domain 3p21.31 protein 12 215604 x a O.OOO3643 3.8 UBE2D2 Ubiquitin-conjugating enzyme 5q31.2 E2D2 (UBC45 homolog, yeast) 215628 x a O.OOOSOOS 2.7 PPP2CA Protein phosphatase 2 (formerly 5q31.1 2A), catalytic Subunit, alpha isoform 215978 X a 9.04E-05 S.6 LOC152719 ATP-binding cassette, Sub- 4p16.3 amily A (ABC1), member 11 (pseudogene) 216035 X a O.OOO632 -2 TCF7L2 Transcription factor 7-like 2 (T. 10q25.3 cell specific, HMG-box) 216051 X a 5.83E-OS 5 KIAA1217 KIAA1217 10p12.31 21618.7 x a O.OOO1456 3.7 XRCC3 X-ray repair complementing 14q32.3 defective repair in Chinese hamster cells 3 216215 s at 7.67E-05 -2.1 RBM9 RNA binding motif protein 9 22d 13.1 216221 S at 0.0007062 -2 PUM2 pumilio homolog 2 (Drosophila) 2p22-p21 (PUM2), mRNA. 216241 s at 0.0005875 -2.1 TCEA1 transcription elongation factor A 8q11.2 (SII), 1 (TCEA1), transcript variant 2 mRNA. 216246 at O.OOO4251 2.7 RPS2O ribosomal protein S20 (RPS20), 8q12 mRNA. 216274 S at 0.0003437 -2.3 SEC11L1 SEC11-like 1 (S. cerevisiae) 15q25.3 (SEC11L1), mRNA. 216733 s at 0.0005519 -3.3 GATM glycine amidinotransferase (L- 15q21.1 arginine:glycine amidinotransferase) (GATM), mRNA. 216858 x at 0.0002005 4.1 216859 X at 0.0004599 3.7 216944 s at 0.0001923 -2.7 ITPR1 inositol 14,5-triphosphate 3p26-p25 receptor, type 1 (ITPR1), mRNA. 217028 at O.OOO384 3.4 CXCR4 chemokine (C X—C motif) 2d21 receptor 4 (CXCR4), transcript variant 2 mRNA. 217118 s at 2.15E-05 2.2 C22Orf chromosome 22 open reading 22d 13.31 frame 9 (C22orf), transcript variant 2 mRNA. 217466 x at O.OOO9002 -2.2 RPS2 Ribosomal protein S2 16p13.3 217497 at O.OOO2299 3.3 ECGF1 Endothelial cell growth factor 1 22d 13 (platelet-derived) 217579 x at O.OOO6875 2.5 ARL6IP2 ADP-ribosylation factor-like 6 2p22.2-p22.1 interacting protein 2 217586 X at 0.000568 2.9 ESTs 217679 x at 7.86E-OS 4.8 ESTs, Weakly similar to hypothetical protein FLJ20489 Homo sapiens H. Sapiens 217713 x at O.OOO1301 3.4 ESTs, Weakly similar to ALU6 HUMAN ALU SUBFAMILY SP SEQUENCE CONTAMINATION WARNING ENTRY H. Sapiens US 2013/02743 15 A1 Oct. 17, 2013 53
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 217715 x at O.OOO2336 3.9 ESTs 217773 s at O.OOO2007 -2.4 NDUFA4 NADH dehydrogenase 7p21.3 (ubiquinone) 1 alpha Subcomplex, 4, 9 kDa (NDUFA4), nuclear gene encoding mitochondrial protein, mRNA. 217774 s at O.OOO7057 -2.1 HSPC152 hypothetical protein HSPC152 11 q13.1 (HSPC152), mRNA. 217781 s at O.OOO3457 -2.5 ZFP106 Zinc finger protein 106 homolog 15q15.1 (mouse) (ZFP106), mRNA. 217787 s at 8.07E-OS 2.7 GALNT2 UDP-N-acetyl-alpha-D- galactosamine:polypeptide N acetylgalactosaminyltransferase 2 (GalNAc-T2) (GALNT2), mRNA. 217795. S. at O.OOO1341 -2.1 TMEM43 transmembrane protein 43 3p25.1 (TMEM43), mRNA. 217814 at O.OOO4705 -2.2 GKOO1 GKOO1 protein (GKO01), 17q23.3 mRNA. 217833 at O.OOOS95 -2.5 SYNCRIP Synaptotagmin binding, cytoplasmic RNA interacting protein 217862 at -2.6 PIAS1 Protein inhibitor of activated 15q. STAT, 1 217864 S at O.OOO4053 -2.2 PIAS1 protein inhibitor of activated 15q. STAT, 1 (PIAS1), mRNA. 21788.8 s at 2.2 ARFGAP1 ADP-ribosylation factor GTPase 20d 13.33 activating protein 1 (ARFGAP1), transcript variant , mRNA. 217915 S. at 9.2iSE-OS -2.2 C1 Sorf15 chromosome 15 open reading rame 15 (C15orf15), mRNA. 217989 at 2.94E-05 -2.7 DHRS8 dehydrogenase/reductase (SDR 4q22.1 amily) member 8 (DHRS8), mRNA. 217992 s at O.OOO3726 2.8 EFEHD2 EF-hand domain family, 1p36.21 member D2 (EFHD2), mRNA. 218018 at 3.6SE-OS 2.2 pyridoxal (pyridoxine, vitamin 21q22.3 B6) kinase (PDXK), mRNA. 218025 s at 2.2OE-06 PECI peroxisomal D3.D2-enoyl-CoA 6p24.3 isomerase (PECI), transcript variant 1 mRNA. 218031 is at 4.OOE-07 CHES1 checkpoint Suppressor 1 14q24.3-q32.11 (CHES1), mRNA. 218113 at O.OOO146 TMEM2 transmembrane protein 2 9q13-q21 (TMEM2), mRNA. 218130 at O.OOO4049 MGC4368 hypothetical protein MGC4368 17q25.3 (MGC4368), mRNA. 218131 s at 2.87E-OS 2.9 GATAD2A GATA zinc finger domain 19p 13.11 containing 2A (GATAD2A), mRNA. 218151 X at O.OOOSS94 2.2 GPR172A G protein-coupled receptor 8q24.3 72A (GPR172A), mRNA. 21815S X at 2.6 FLJ10534 hypothetical protein FLJ10534 17p13.3 (FLJ10534), mRNA. 21815.8 s at O.OOO1389 -2.7 APPL adaptor protein containing pH 3p21.1-p14.3 domain, PTB domain and eucine zipper motif 1 (APPL), mRNA. 218167 at 7.25E-05 -2.4 AMZ2 archaemetZincins-2 (AMZ2), mRNA. 218191 s at 6.81E-OS -2.8 LMBRD1 LMBR1 domain containing 1 (LMBRD1), mRNA. 218193 s at O.OOO2316 2.8 GOLT1B golgi transport 1 homolog B (S. cerevisiae) 12p12.1 (GOLT1B), mRNA. 218204 s at 8.33E-OS -2.3 FYCO1 FYVE and coiled-coil domain 3p21.31 containing 1 (FYCO1), mRNA. 218311 at O.OOO1916 -2.3 MAP4K3 mitogen-activated protein kinase 2p22.1 kinase kinase kinase 3 (MAP4K3), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 54
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 218373 at O.OOO1922 -2.3 FTS used toes homolog (mouse) 16q12.2 (FTS), transcript variant 2, mRNA. 218383 at O.OOO3931 -2.7 C14orf4 chromosome 14 open reading 14q11.2 rame 94 (C14orf)4), mRNA. 218432 at O.OOO3175 -2.1 FBXO3 F-box protein 3 (FBXO3), 11 p.13 transcript variant 1, mRNA. 218450 at O.OOO2984 -2.5 HEBP1 heme binding protein 1 12p13.1 (HEBP1), mRNA. 218504 at O.OOOSS44 -2 FAHD2A umarylacetoacetate hydrolase 2p24.3-p11.2 domain containing 2A (FAHD2A), mRNA. 218528 S. at 4.23E-05 -2.3 RNF38 ring finger protein 38 (RNF38), 9p13-p12 transcript variant 4, mRNA. 2186.38 S. at 0.0008018 4 SPON2 spondin 2, extracellular matrix 4p16.3 protein (SPON2), mRNA. 218730 S. at 0.0002624 -6.8 OGN osteoglycin (osteoinductive 9q22 actor, mimecan) (OGN), transcript variant 3, mRNA. 218739 at O.OOO2299 2.6 ABHDS abhydrolase domain containing 3p21 5 (ABHD5), mRNA. 218804 at 1.58E-OS 3.3 TMEM16A transmembrane protein 16A 11 q13.3 (TMEM16A), mRNA. 218817 at O.OOOSS21 2 SPCS3 signal peptidase complex 4q34.2 Subunit 3 homolog (S. cerevisiae) (SPCS3), mRNA. 218820 at O.OOO7651 -3 C14orf132 chromosome 14 open reading 14q32.2 frame 132 (C14orf132), mRNA. 218831 s at 7.99E-05 -2.6 FCGRT Fc fragment of IgG, receptor, 19q13.3 transporter, alpha (FCGRT), mRNA. 218856 at 2.2OE-06 6.6 TNFRSF21 tumor necrosis factor receptor 6p21.1-12.2 Superfamily, member 21 (TNFRSF21), mRNA. 218902 at 4.33E-OS 2.5 NOTCH1 Notch homolog 1, translocation- 9q34.3 associated (Drosophila) (NOTCH1), mRNA. 218919 at O.OOO1794 -3.1 ZFAND1 Zinc finger, AN1-type domain 1 8q21.13 (ZFAND1), mRNA. 218929 at 3.34E-OS -2.1 CARF collaborates, cooperates with 4q35.1 ARF (alternate reading frame) protein (CARF), mRNA. 218961 S. at 0.0007861 2 PNKP polynucleotide kinase 3'- 19q13.3-q13.4 phosphatase (PNKP), mRNA. 219023 at 7.64E-OS -3.1 C4orf16 chromosome 4 open reading 4q25 rame 16 (C4orf16), mRNA. 219025 at 5.55E-05 4.3 CD248 CD248 antigen, endosialin 11 q13 (CD248), mRNA. 219033 at O.OOO7081 2.3 PARP8 poly (ADP-ribose) polymerase 5q11.1 amily, member 8 (PARP8), mRNA. 219054 at 4.17E-OS -2.6 FLJ14054 hypothetical protein FLJ14054 5p13.3 (FLJ14054), mRNA. 219092 S at 2.14E-05 2.2 C9orf12 chromosome 9 open reading 9q21.33-q22.31 rame 12 (C9orf12), mRNA. 219099 at 9.73E-OS 2 C12Orfs chromosome 12 open reading 12p13.3 rame 5 (C12orf5), mRNA. 219102 at 1.21E-05 3 RCN3 reticulocalbin 3, EF-hand 19q13.33 calcium binding domain (RCN3), mRNA. 219105 X at 9.00E-07 3.8 ORC6L origin recognition complex, 16q12 Subunit 6 homolog-like (yeast) (ORC6L), mRNA. 219117 s at 0.0002973 2.4 FKBP11 FK506 binding protein 11, 19 kDa 12q13.12 (FKBP11), mRNA. 219263 at S.92E-OS 4.2 RNF128 ring finger protein 128 Xq22.3 (RNF128), transcript variant 2, mRNA. 219279 at O.OOO2O23 2.7 DOCK10 dedicator of cytokinesis 10 2a36.3 (DOCK10), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 55
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 219289 at O.OOO1862 2.3 FL2O718 hypothetical protein FLJ20718 16q12.1 (FLJ20718), transcript variant 1, mRNA. 219290 x at 0.0009817 3.8 DAPP1 dual adaptor of phosphotyrosine 4q25-q27 and 3-phosphoinositides (DAPP1), mRNA. 21935.8 s at 16OE-06 6.1 CENTA2 centaurin, alpha 2 (CENTA2), 17q11.2 mRNA. 219359 at 4.OOE-06 4.4 FLJ2263S hypothetical protein FLJ22635 11 p 15.5 (FLJ22635), mRNA. 219368 at O.OOO9624 -2.9 NAP1L2 nucleosome assembly protein 1- Xq13 ike 2 (NAP1L2), mRNA. 219392 X at 3.14E-05 4.3 FLJ11029 hypothetical protein FLJ11029 17q23.2 (FLJ11029), mRNA. 219407 s at 0.0009809 4.1 LAMC3 aminin, gamma 3 (LAMC3), 9q31-q34 mRNA. 219449 s at O.OOO6528 -2.5 TMEM70 transmembrane protein 70 8q21.11 (TMEM70), mRNA. 219454 at 4.OOE-07 36.8 EGFL6 EGF-like-domain, multiple 6 Xp22 (EGFL6), mRNA. 219493 at O.OOO4597 2.6 SHCBP1 SHC SH2-domain binding 16q11.2 protein 1 (SHCBP1), mRNA. 219511 S. at 7.7OE-06 -4.1 SNCAIP Synuclein, alpha interacting 5q23.1-q23.3 protein (synphilin) (SNCAIP), mRNA. 219522 at 1.OOE-07 7.7 FJX1 our jointed box 1 (Drosophila) 1p13 (FJX1), mRNA. 219549 s at 0.0004199 -2.2 RTN3 reticulon 3 (RTN3), transcript 1q13 variant 4, mRNA. 219582 at O.0004924 3 OGFRL1 opioid growth factor receptor- 6q13 like 1 (OGFRL1), mRNA. 219634 at 8.1OE-06 3.8 CHST11 carbohydrate (chondroitin 4) 2q Sulfotransferase 11 (CHST11), mRNA. 219641 at O.OOO33O8 -2.1 DET1 de-etiolated homolog 1 5q25.3 (Arabidopsis) (DET1), mRNA. 219700 at 1.1OE-06 7.7 PLXDC1 plexin domain containing 1 7q21.1 (PLXDC1), mRNA. 219764 at 9.OOE-07 6.7 FZD10 rizzled homolog 10 2d24.33 (Drosophila) (FZD10), mRNA. 21993.9 s at 2.10E-06 -3.5 CSDE1 cold shock domain containing p22 E1, RNA-binding (CSDE1), transcript variant 2, mRNA. 219958 at O.OOO873 3.2 C20orfA6 chromosome 20 open reading 20p13 rame 46 (C20orf46), mRNA. 21996.1 s at 0.0003792 -2.5 C20orf19 chromosome 20 open reading 20pter-q11.23 rame 19 (C20orf19), mRNA. 220014 at 1.38E-OS 4.6 LOCS1334 mesenchymal stem cell protein 5q23.1 DSC54 (LOC51334), mRNA. 220094 S at 0.0002242 2 C6orf79 chromosome 6 open reading 6p24.3-p23 rame 79 (C6orf79), transcript variant 1 mRNA. 22O113 x at 0.0004.047 3.1 POLR1B polymerase (RNA) I polypeptide 2d 13 B, 128 kDa (POLR1B), mRNA. 22O167 s at 0.0008187 2 TP53TG3 TP53TG3 protein (TP53TG3), 16p13 transcript variant 2, mRNA. 220232 at 4.54E-OS 4.8 SCD5 stearoyl-CoA desaturase 5 4q21.3 (SCD5), mRNA. 220242 X at 0.0001347 2.2 ZNF701 Zinc finger protein 701 19q13.41 (ZNF701), mRNA. 220266 s at 1.44E-05 -3.3 KLF4 Kruppel-like factor 4 (gut) 9q31 (KLF4), mRNA. 220301 at 1.76E-OS 5.8 C18orf14 chromosome 18 open reading 18q22.1 frame 14 (C18orf14), mRNA. 220327 at 8.86E-OS -2.7 WGL-3 vestigial-like 3 (VGL-3), 3p12.1 mRNA. 220334 at O.OOO3O43 3.1 RGS17 regulator of G-protein signalling 6q25.3 17 (RGS17), mRNA. 220432 s at 0.000473 -3.2 CYP39A1 cytochrome P450, family 39, 6p21.1-p11.2 Subfamily A, polypeptide 1 (CYP39A1), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 56
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 220575 at 4.48E-OS 3.4 FLJ11800 hypothetical protein FLJ1 1800 7p11.2 (FLJ11800), mRNA. 220603 s at 0.0001617 3.9 MCTP2 Multiple C2-domains with two 5q26.2 transmembrane regions 2 220720 x at 0.0005432 3.3 FLJ14346 Hypothetical protein FLJ14346 2d21.1 220796 X at 0.0002356 4.1 SLC3SE1 Solute carrier family 35, 9p 13.11 member E1 220817 at 4.5OE-06 4.1 TRPC4 transient receptor potential 3q13.1-q13.2 cation channel, Subfamily C, member 4 (TRPC4), mRNA. 220952 s a O.OOO293 -2.1 PLEKHAS pleckstrin homology domain 2p12 containing, family A member 5 (PLEKHA5), mRNA. 220992 s a O.OOO3763 -2.1 C1orf25 Chromosome 1 open reading q25.2 rame 25 221012 s a O.OOO4784 2 TRIM8 tripartite motif-containing 8 Oq24.3 (TRIM8), mRNA. 22105.9 s a 2.2OE-06 4.1 COTL1 coactosin-like 1 (Dictyostelium) 6q24.1 (COTL1), mRNA. 221127 s a O.OOO454 3.7 DKK3 dickkopfhomolog 3 (Xenopus 1p 15.2 laevis) (DKK3), transcript variant 3, mRNA. 221222 S a O.OOOS338 2.2 C1orf56 chromosome 1 open reading q21.2 frame 56 (C1orf56), mRNA. 221476 s a 4.74E-OS -2.4 RPL15 ribosomal protein L15 (RPL15), 3p24.2 mRNA. 221486 at O.OOO4058 -2 ENSA endosulfine alpha (ENSA), q21.2 transcript variant 7, mRNA. 221538 s a O.OOO4626 2.4 PLXNA1 plexin A1 (PLXNA1), mRNA. 3q21.3 221558 s a 186E-05 4.6 LEF1 lymphoid enhancer-binding 4q23-q25 factor 1 (LEF1), mRNA. 221577 X at 0.0006888 4.5 GDF15 growth differentiation factor 15 19p 13.1-13.2 (GDF15), mRNA. 221588 X at 0.0001441 -2.4 ALDH6A1 aldehyde dehydrogenase 6 14q24.3 family, member A1 (ALDH6A1), nuclear gene encoding mitochondrial protein, mRNA. 221589 s at 1.2OE-06 -4.3 ALDH6A1 Aldehyde dehydrogenase 6 14q24.3 family, member A1 221590 S. at 0.0008671 -2.3 ALDH6A1 Aldehyde dehydrogenase 6 14q24.3 family, member A1 221689 s at 0.000155 -2 DSCRS Down syndrome critical region 21q22.2 gene 5 (DSCR5), transcript variant 2 mRNA. 221 691 x at 1.46E-05 -3.2 NPM1 nucleophosmin (nucleolar 5q.35 phosphoprotein B23, numatrin) (NPM1), mRNA. 221725 at 3.6OE-OS -2.1 WASF2 WAS protein family, member 2 1p36.11-p34.3 221726 at 1.93E-OS -2.9 RPL22 ribosomal protein L22 (RPL22), 1p36.3-p36.2 mRNA. 221727 at O.OOO1534 -2.3 PC4 Activated RNA polymerase II 5p13.3 transcription cofactor 4 221729 at O.OOO4594 3.1 COLSA2 collagen, type V, alpha 2 214-q32 (COL5A2), mRNA. 221730 at O.OOO7972 3.4 COLSA2 collagen, type V, alpha 2 214-q32 (COL5A2), mRNA. 221731 x at 2.57E-05 10.4 CSPG2 chondroitin Sulfate proteoglycan 5q14.3 2 (versican) (CSPG2), mRNA. 221747 at O.OOO3412 -2.2 TNS Tensin 1 2a35-q36 221771 S. at 0.000498 -2.1 HSMPP8 M-phase phosphoprotein, mpp8 13q12.11 221840 at O.OOO2221 3.2 PTPRE protein tyrosine phosphatase, 10q26 receptor type, E (PTPRE), transcript variant 2, mRNA. 221882 s at 0.0003013 2.2 TMEM8 transmembrane protein 8 (five 16p13.3 membrane-spanning domains) (TMEM8), mRNA. 221943 x at 0.0002266 2.4 RPL38 ribosomal protein L38 (RPL38), 17q23-q25 mRNA. 221988 at 1.59E-OS -2.3 MGC2747 Hypothetical protein MGC2747 19p 13.11 222108 at 5.57E-05 -4.4 AMIGO2 adhesion molecule with Ig-like 12q13.11 domain 2 (AMIGO2), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 57
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 222207 X at 0.0003362 3.2 CDNA: FLJ20949 fis, clone 7 ADSEO1902 222212 s at O.OOO1439 -2.3 LASS2 LAG1 longevity assurance 1q21.2 homolog 2 (S. cerevisiae) (LASS2), transcript variant 3, mRNA. 222252 x at 5.29E-05 4 LRRCS1 eucine rich repeat containing 51 11 q13.4 (LRRC51), mRNA. 222253 S at 0.000969 4.6 DKFZP434P211 POM121-like protein 22d 11.22 222358 x at 7.05E-05 3.1 ESTs, Weakly similar to hypothetical protein FLJ20378 Homo sapiens H. sapiens 222372 a O.OOO6654 2.5 BALAP1 Membrane associated guanylate 3p 14.1 kinase, WW and PDZ domain containing 1 222379 a 1.13E-OS 3.5 KCNE4 Potassium voltage-gated 2a36.3 channel, Isk-related family, member 4 222394 a 1.44E-05 -2.8 PDCD6IP programmed cell death 6 3p23 interacting protein (PDCD6IP), mRNA. 222423 a 9.81E-OS -2.8 NDFIP1 Nedd4 family interacting protein 1 5q31.3 222431 a O.OOO2108 -2.2 SPIN Spindlin 9q22.1-q22.3 222437 s at O.OOO6141 -2 VPS24 vacuolar protein sorting 24 2p24.3-p24.1 (yeast) (VPS24), transcript variant 2 mRNA. 222449 a 7.8OE-06 4.1 TMEPAI transmembrane, prostate 20d 13.31-q13.33 androgen induced RNA (TMEPAI), transcript variant 3, mRNA. 2224.53 a 4.19E-05 -2.7 CYBRD1 cytochrome b reductase 1 2a31.1 (CYBRD1), mRNA. 222482 a 1.8SE-OS -2.4 SSBP3 Single stranded DNA binding 1p32.3 protein 3 222486 s at 5.8OE-06 -4.9 ADAMTS1 ADAM metallopeptidase with 21q21.2 thrombospondin type 1 motif, 1 (ADAMTS1), mRNA. 222488 S. at 9.1OE-05 -2.4 DCTN4 dynactin 4 (p62) (DCTN4), 5q31-q32 mRNA. 222494 at 4.OOE-06 -2.8 CHES1 checkpoint Suppressor 1 14q24.3-q32.11 (CHES1), mRNA. 2225.03 s at O.OOO3949 2.5 WDR41 WD repeat domain 41 5q13.3 (WDR41), mRNA. 222533 at O.OOO1299 -2.1 CRBN cereblon (CRBN), mRNA. 3p26.2 2225.38 S. at 0.0007304 -3.2 APPL adaptor protein containing pH 3p21.1-p14.3 domain, PTB domain and leucine zipper motif 1 (APPL), mRNA. 222605 at 164E-05 -3.1 RCOR3 REST corepressor 3 (RCOR3), 1q32.3 mRNA. 222722 at 1.2SE-OS -8.7 OGN osteoglycin (osteoinductive 9q22 factor, mimecan) (OGN), transcript variant 3, mRNA. 222753 s at 0.0001335 2.1 SPCS3 signal peptidase complex 4q34.2 Subunit 3 homolog (S. cerevisiae) (SPCS3), mRNA. 222791 at 1.2OE-06 -3.4 RSBN1 round spermatid basic protein 1 1p13.2 (RSBN1), mRNA. 222834 S. at 0.0006523 -2.1 GNG12 guanine nucleotide binding 1p31.2 protein (G protein), gamma 12 (GNG12), mRNA. 222968 at S.98E-OS 2.9 C6orf248 chromosome 6 open reading 6p21.3 frame 48 (C6orf248), mRNA. 222975 S. at 9.41E-05 -2.9 CSDE1 cold shock domain containing 1p22 E1, RNA-binding (CSDE1), transcript variant 2, mRNA. 223007 s at 0.0007305 -2.7 C9orfs chromosome 9 open reading 9q31 frame 5 (C9orf5), mRNA. 223010 S. at 0.0005426 -2.2 OCLAD1 OCIA domain containing 1 4p11 (OCLAD1), mRNA. 223.01.1 s at 0.0008498 -2 OCLAD1 OCIA domain containing 1 4p11 (OCLAD1), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 58
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 223050 S at 0.0003276 2 FBXW5 F-box and WD-40 domain 9q34.3 protein 5 (FBXW5), transcript variant 1 mRNA. 223O82 at O.OOO3668 2.1 SH3KBP1 SH3-domain kinase binding Xp22.1-p21.3 protein 1 (SH3KBP1), transcript variant 1 mRNA. 223170 at O.OOO1428 -2.6 DKFZP564K1964 DKFZP564K1964 protein 17q11.2 (DKFZP564K1964), mRNA. 223189 X at 9.46E-05 -2.4 MLLS myeloid/lymphoid or mixed- 7q22.1 ineage leukemia 5 (trithorax homolog, Drosophila) (MLL5), mRNA. 223208 at O.OOO9547 2 KCTD10 potassium channel 12q24.11 etramerisation domain containing 10 (KCTD10), mRNA. 223227 at O.OOO6403 -2.2 BBS2 Bardet-Biedl syndrome 2 16q21 (BBS2), mRNA. 223263 s at 0.0003743 -2.1 FGFR1OP2 FGFR1 oncogene partner 2 12p11.23 (FGFR1OP2), mRNA. 223276 at S.1OE-OS 2.9 NID67 putative Small membrane protein 5q33.1 NID67 (NID67), mRNA. 223283 s at 0.0009337 -2.5 SDCCAG33 Serologically defined colon 18q22.3 cancer antigen 33 (SDCCAG33), mRNA. 223306 at 6.02E-OS -2.4 EBPL emopamil binding protein-like 13q12-q13 (EBPL), mRNA. 223366 at O.OOO847S -2.9 CDNA FLJ16218 fis, clone 8 CTONG3001501, highly similar to Mus musculus glucocorticoid induced gene 1 mRNA 223384 S at 0.0009265 -2.1 TRIM4 tripartite motif-containing 4 7q22-q31.1 (TRIM4), transcript variant beta, mRNA. 223395 at 9.3SE-OS -6.6 ABI3BP ABI gene family, member 3 3q12 (NESH) binding protein (ABI3BP), mRNA. 223437 at O.OOO4419 -2.2 PPARA peroxisome proliferative 22d 13.31 activated receptor, alpha (PPARA), transcript variant 3, mRNA. 223464 at O.OOO4952 2.3 OSBPLS oxysterol binding protein-like 5 11 p 15.4 (OSBPL5), transcript variant 2, mRNA. 223501 at O.OOO44O1 4.3 TNFSF13B Tumor necrosis factor (ligand) 13q32-34 Superfamily, member 13b 223538 at O.OOO8098 2 SERF1A Small EDRK-rich factor 1A 5q12.2-q13.3 (telomeric) 223566 s at O.OOO7174 -2.7 BCOR BCL6 co-repressor (BCOR), Xp21.2-p11.4 transcript variant 1, mRNA. 223617 X at 3.01E-05 2.6 ATAD3B ATPase family, AAA domain 1p36.33 containing 3B (ATAD3B), mRNA. 223629 at O.OOO8805 -2.4 PCDHB5 protocadherin beta 5 5q31 (PCDHB5), mRNA. 223672 at O.OOO10O2 4.8 SGIP1 SH3-domain GRB2-like 1p31.2 (endophilin) interacting protein 1 (SGIP1), mRNA. 223697 x at 0.0001465 4.6 C9orf64 chromosome 9 open reading 9q21.32 frame 64 (C9orf64), mRNA. 223991 S. at 2.42E-05 2.6 GALNT2 UDP-N-acetyl-alpha-D- 1q41-q42 galactosamine:polypeptide N acetylgalactosaminyltransferase 2 (GalNAc-T2) (GALNT2), mRNA. 224254 X at 2.71E-05 5.5 TF Transferrin 3q22.1 224445 S. at 111E-05 -3 ZFYVE21 Zinc finger, FYVE domain 14q32.33 containing 21 (ZFYVE21), mRNA. 224.549 X at 6.5OE-06 8 US 2013/02743 15 A1 Oct 17, 2013 59
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 224,598 at 3.68E-OS 2.4 MGAT4B mannosyl (alpha-1,3-)- 5q.35 glycoprotein beta-1,4-N- acetylglucosaminyltransferase, isoenzyme B (MGAT4B), transcript variant 1, mRNA. 224605 a O.OOO2S48 -2.2 LOC4O1152 HCV F-transactivated protein 1 4q26 (LOC4O1152), mRNA. 224612 s at 6.79E-05 2.2 DNAJCS DnaJ (Hsp40) homolog, 20d 13.33 Subfamily C, member 5 224618 a O.OOO1475 2 ROD1 ROD1 regulator of 9q32 differentiation 1 (S. pombe) 224660 a O.OOO8567 -2.1 MGC14156 hypothetical protein MGC14156 4q22.1 (MGC14156), mRNA. 224664 a O.OOO3695 -2.3 C10orf104 chromosome 10 open reading 10q22.1 rame 104 (C10orf104), mRNA. 224665 a. O.OOO4O75 -2.2 C10orf104 chromosome 10 open reading 10q22.1 rame 104 (C10orf104), mRNA. 224667 X at 0.0002844 3.1 Transcribed locus 224689 a O.OOO1149 -2 MANBAL mannosidase, beta A, lysosomal- 20d 11.23-q12 ike (MANBAL), transcript variant 2 mRNA. 224734 a O.OOO1511 -2.9 HMGB1 High-mobility group box 1 13q12 224741 x at 0.0001144 -2.5 GASS Growth arrest-specific 5 1q23.3 224754 a O.OOO2O6 -2.2 SP1 Sp1 transcription factor (SP1), 12q13.1 mRNA. 224755 a. O.OOO1441 -2.2 SMBP SM-1104.4 binding protein 10q24.1 224763 a 5.3OE-06 -3.9 RPL37 ribosomal protein L37 (RPL37), 5p13 mRNA. 224780 a O.OOO8827 -2 RBM17 RNA binding motif protein 17 10p15.1 (RBM17), mRNA. 224812 a O.OOO1992 -2.3 HIBADH 3-hydroxyisobutyrate 7p15.2 dehydrogenase (HIBADH), mRNA. 224841 x at 0.0001078 -2.6 RNU47 PREDICTED: RNA U47 small 1 nuclear (RNU47), misc RNA. 224856 a O.OOO4439 -2.6 FKBP5 FK506 binding protein 5 6p21.3-21.2 (FKBP5), mRNA. 224893 a O.OOO3585 -2.8 DKFZP564JO863 DKFZP564JO863 protein 11 q13.1 224895 a O.OOO3O7 -2.6 YAP1 Yes-associated protein 1, 65 kDa 11 q13 (YAP1), mRNA. 224901 a 1.7OE-OS -3.5 SCD4 Stearoyl-CoA desaturase 5 4q21.3 224.950 a O.OOO9862 2.2 PTGFRN prostaglandin F2 receptor 1p13.1 negative regulator (PTGFRN), mRNA. 224967 a S.98E-OS 2.7 UGCG UDP-glucose ceramide 9q31 glucosyltransferase 224970 a 5.88E-OS -2.5 NFIA Nuclear factor IFA 1p31.3-p31.2 225050 a 4.8SE-OS -2.5 ZNF512 Zinc finger protein 512 2p23 (ZNF512), mRNA. 225,060 a O.OOO1496 -2.4 LRP11 ow density lipoprotein receptor- 6q25.1 related protein 11 (LRP11), mRNA. 225,078 a O.OOO2343 -3.4 EMP2 Epithelial membrane protein 2 16p13.2 225,098 a O.OOO1021 -2 ABI2 Abl interactor 2 2a33 225106 s at 0.0005784 2.9 FLJ10826 hypothetical protein FLJ10826 16q12.2 (FLJ10826), transcript variant 2, mRNA. 225123 a 4.OOE-07 -3.1 SESN3 Sestrin 3 11q21 225.125 a. 3. SOE-OS -3.1 TMEM32 transmembrane protein 32 Xq26.3 (TMEM32), mRNA. 225132 a. O.OOO1306 -2 FBXL3 F-box and leucine-rich repeat 13q22 protein 3 (FBXL3), mRNA. 225133 a 4.79E-05 -2.6 KLF3 Kruppel-like factor 3 (basic) 4p14 225147 a 1SOE-06 3.7 PSCD3 pleckstrin homology, Sect and 7p22.1 coiled-coil domains 3 (PSCD3), mRNA. 225162 a. 1.4OE-05 -3.8 SH3D19 SH3 domain protein D19 4q31.3 (SH3D19), mRNA. 2251.79 a O.OOO1313 -2.1 HIP2 Huntingtin interacting protein 2 4p14 US 2013/02743 15 A1 Oct. 17, 2013 60
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 225198 a 2.56E-OS -2.7 WAPA VAMP (vesicle-associated 18p11.22 membrane protein)-associated protein A, 33 kDa (VAPA), transcript variant 2, mRNA. 225207 a O.OOO1018 -3.4 PDK4 pyruvate dehydrogenase kinase, 7q21.3-q22.1 isoenzyme 4 (PDK4), mRNA. 225219 a 2.7OE-OS -3.5 SMADS SMAD, mothers against DPP 5q31 homolog 5 (Drosophila) (SMAD5), transcript variant 3, mRNA. 225220 a O.OOO74 -2.2 Homo sapiens, clone 4 MAGE: 4249217, mRNA 225223 a 2.89E-OS -2 SMADS SMAD, mothers against DPP 5q31 homolog 5 (Drosophila) (SMAD5), transcript variant 3, mRNA. 225239 a O.OOO4432 4.3 mmunoglobulin light chain 11 variable region 225243 s at 0.0001.227 -2.6 SLMAP sarcolemma associated protein 3p21.2-p14.3 (SLMAP), mRNA. 225274 a 2.8OE-06 -3 SNRPG Small nuclear ribonucleoprotein 2p13.3 polypeptide G 225310 a O.OOO177 -2.2 RBMX RNA binding motif protein, X- Xq26.3 inked 225326 a 8.88E-OS -2 RBM27 PREDICTED: RNA binding 5 motif protein 27 (RBM27), mRNA. 225.330 a. O.OOO2697 -2.1 IGF1R insulin-like growth factor 1 15q26.3 receptor 225332 a 5.78E-OS -2.1 KRTAP4-7 Keratin associated protein 4-7 17q12-q21 225344 a O.OOO4132 2.8 NCOA7 nuclear receptor coactivator 7 6q22.32 (NCOA7), mRNA. 225352 a O.OOO1828 -2.3 TLOC1 translocation protein 1 3q26.2 (TLOC1), mRNA. 225381 a O.OOO663S -4.3 LOC399959 PREDICTED: hypothetical 11 LOC399959 (LOC399959), mRNA. 225387 a 3.43E-OS -3.2 TM4SF9 Tetraspanin 5 4q23 225416 a O.OOO3496 -2.2 RNF12 Ring finger protein 12 Xq13-q21 225421 a O.OOO9226 -3.1 ACY1L2 aminoacylase 1-like 2 6q15 (ACY1 L2), mRNA. 225.426 a 6.78E-OS -2.5 PPP6C Protein phosphatase 6, catalytic 9q33.3 Subunit 225480 a O.OOO2718 2.2 C1orf122 chromosome 1 open reading 1p34.3 rame 122 (C1orf122), mRNA. 225489 a 3.71E-OS -2 TMEM18 transmembrane protein 18 2p25.3 (TMEM18), mRNA. 225498 a 7.4SE-OS -2.2 CHMP4B chromatin modifying protein 4B 20d 11.22 (CHMP4B), mRNA. 2255.05 s at 1.90E-06 4.1 C20orf&1 chromosome 20 open reading 20p13 frame 81 (C20orf31), mRNA. 225509 a 101E-05 -2.9 SAP3OL Hypothetical protein LOC56757 5q33.2 2255.24 a S.OOE-OS -3.1 ANTXR2 anthrax toxin receptor 2 4q21.21 (ANTXR2), mRNA. 225526 a O.OOO4149 -2 MKLN1. muskelin 1, intracellular 7q32 mediator containing kelch motifs (MKLN1), mRNA. 225546 a 4.45E-05 -2.7 EEF2K Eukaryotic elongation factor-2 16p12.1 kinase 225571 a O.OOO188 -4.7 LIFR leukemia inhibitory factor 5p13-p12 receptor (LIFR), mRNA. 225574 a 8.57E-OS -2.4 MGC101.98 hypothetical protein MGC10198 4q35.1 (MGC10198), mRNA. 225575 a. O.OOO3237 -4.5 LIFR leukemia inhibitory factor 5p13-p12 receptor (LIFR), mRNA. 225611 a O.OOO4119 -2.6 MAST4 Microtubule associated 5q12.3 serine threonine kinase family member 4 2256.26 a O.OOO3153 4.8 PAG1 phosphoprotein associated with 8q21.13 glycosphingolipid microdomains 1 (PAG1), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 61
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 225636 a O.OOO278 2.5 STAT2 signal transducer and activator 12q13.3 of transcription 2, 113 kDa (STAT2), mRNA. 225646 a 1. SOE-OS 4.4 CTSC cathepsin C (CTSC), transcript 11 q14.1-q14.3 variant 2 mRNA. 225647 s at 0.0009077 2.5 CTSC cathepsin C (CTSC), transcript 11 q14.1-q14.3 variant 1 mRNA. 225686 a O.OOO3333 2.2 FAM33A family with sequence similarity 17q23.2 33, member A (FAM33A), mRNA. 225698 a 3.41E-05 -3.2 TIGA1 TIGA1 (TIGA1), mRNA. 5q21-q22 225728 a O.OOO6083 -2.8 ARGBP2 Arg/Abl-interacting protein 4q35.1 ArgBP2 225793 a O.OOO103 -2.1 MGC46719 Lix1 homolog (mouse) like 1q21.1 225799 a 2.29E-OS 5.2 MGC4677 hypothetical protein MGC4677 2p11.2 (MGC4677), mRNA. 225811 a 5.44E-05 -2.1 Transcribed locus, weakly 11 similar to XP 510104.1 PREDICTED: Similar to hypothetical protein FLJ25224 Pan troglodytes 225845 at O.OOO3774 -3.4 BTBD15 BTB (POZ) domain containing 11q24.3 5 (BTBD15), mRNA. 225855 at 7.78E-05 -2.4 EPB41LS erythrocyte membrane protein 214.2 band 4.1 like 5 (EPB41L5), mRNA. 225886 at O.OOOSO86 -2.2 DDX5 RNA-binding protein 45 17q21 (RBP45), putative 225915 at 3. SOE-OS -3.4 CAB39L. Calcium binding protein 39-like 13q14.2 225939 at O.OOO1338 -2.8 EIF4E3 Eukaryotic translation initiation 3p14 actor 4E member 3 225941 at O.OOO2862 -2.2 EIF4E3 Eukaryotic translation initiation 3p14 actor 4E member 3 225946 at O.OOO3S11 -2.3 C12Orf2 Chromosome 12 open reading 12p12.3 rame 2 225947 at 5.75E-05 2.3 MYOHD1 myosin head domain containing 17q12 (MYOHD1), mRNA. 225967 s at 0.0001375 2.5 LOC2841 84 PREDICTED: hypothetical 17 LOC284.184 (LOC284.184), mRNA. 225976 at O.OOO3271 -2 BTF3L4 basic transcription factor 3-like 1p32.3 4 (BTF3L4), mRNA. 225987 at O.OOO3S03 3.3 TNFAIP9 STEAP family member 4 7q21.12 225996 at O.OOO6306 -9.3 MRNA, cDNA 2 DKFZp686N1345 (from clone DKFZp686N1345) 226017 at O.OOO3O1 2.3 CKLFSF7 chemokine-like factor 3p23 superfamily 7 (CKLFSF7), transcript variant 2, mRNA. 226020 s at 7.89E-05 -2.2 OMA1 OMA1 homolog, Zinc 1p32.2-p32.1 metallopeptidase (S. cerevisiae) (OMA1), mRNA. 226038 a O.OOO2389 -3.2 LONRF1 LON peptidase N-terminal 8p23.1 domain and ring finger 1 (LONRF1), mRNA. 226063 a 4.OOE-06 3.3 WAV2 vav 2 oncogene (VAV2), 9q34.1 mRNA. 226066 a O.OOOS179 -3 MITF microphthalmia-associated 3p 14.2-p14.1 transcription factor (MITF), transcript variant 5, mRNA. 226117 a 8.72E-OS -2.7 TIFA TRAF-interacting protein with a 4q25 forkhead-associated domain (TIFA), mRNA. 226120 a O.OOO1436 -2.7 TTC8 tetratricopeptide repeat domain 14q31.3 8 (TTC8), transcript variant 3, mRNA. 226180 a O.OOO9691 -2 WDR36 WD repeat domain 36 5q22.1 (WDR36), mRNA. 226184 a 3.75E-05 -3.6 FMNL2 formin-like 2 (FMNL2), 2d23.3 transcript variant 2, mRNA. US 2013/02743 15 A1 Oct 17, 2013 62
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 226203 a O.OOO3968 -2 CDNA clone IMAGE: 5299888 15 226223 a O.OOO2488 -2.6 PAWR PRKC, apoptosis, WT1, 12q21 regulator 226225 a. O.OOO3786 -3.2 MCC Mutated in colorectal cancers 5q21-q22 226230 a. O.OOO2S89 -2.4 KIAA1387 KIAA1387 protein 2p16.1 226280 a 3.34E-OS -2.8 BNIP2 BCL2 adenovirus E1B 19 kDa 15q22.2 interacting protein 2 226297 a 8.43E-OS -2.1 ESTs 226303 a O.OOO3656 -2.9 PGMS phosphoglucomutase 5 (PGM5), 9q13 mRNA. 226336 a O.OOO1456 -2 PPLA Peptidylprolyl isomerase A 7p13-p11.2 (cyclophilin A) 226344 a O.OOO9627 -2.9 ZMAT1 Zinc finger, matrin type 1 Xq21 (ZMAT1), transcript variant 1, mRNA. 226403 a 9.2OE-06 3.8 TMC4 transmembrane channel-like 4 19q13.42 (TMC4), mRNA. 226472 a O.OOO7652 -2 PPIL4 peptidylprolyl isomerase 6q24-q25 (cyclophilin)-like 4 (PPIL4), mRNA. 226484 a O.OOO9077 2 ZNF651 Zinc finger protein 651 3p22.1 (ZNF651), mRNA. 226499 a O.OOO9453 2.8 MGC61598 Similar to ankyrin-repeat protein 9q34.3 Nrarp 226521 S at 0.0006084 -2.1 FLJ13614 hypothetical protein FLJ13614 4q21.21-q21.23 (FLJ13614), mRNA. 226529 a 4.8SE-OS -2.3 FLJ11273 hypothetical protein FLJ11273 7p21.3 (FLJ11273), mRNA. 226541 a O.OOO1172 -2.3 FBXO3O F-box protein 30 (FBXO30), 6q24 mRNA. 226561 a O.OOO6O2S -2.1 LOC28SO86 Hypothetical protein 2a36.3 LOC28SO86 226599 a O.OOO2151 2.3 KIAA1727 KIAA1727 protein 4q31.3 (KLAA1727), mRNA. 226625 a. 7.3OE-06 -3.8 TGFBR3 Transforming growth factor, 1p33-p32 beta receptor III (betaglycan, 300 kDa) 226663 a 8.6OE-06 2.8 ANKRD10 Ankyrin repeat domain 10 13q34 226668 a 0.0003707 -2.6 WDSUB1 WD repeat, SAM and U-box 2d24.2 domain containing 1 (WDSUB1), mRNA. 226688 a O.OOO2101 -3.3 C3orf23 chromosome 3 open reading 3p21.33-p21.32 frame 23 (C3orf23), transcript variant 1 mRNA. 226695 a O.OOO1301 2.5 PRRX1 paired related homeobox 1 1q24 (PRRX1), transcript variant pmx-1b, mRNA. 226705 a O.OOO2111 -2 FGFR1 Fibroblast growth factor 8p11.2-p11.1 receptor 1 (fms-related tyrosine kinase 2, Pfeiffer syndrome) 226713 a 181E-05 -2.9 C3orf6 Chromosome 3 open reading 3 frame 6 226747 a O.OOO6894 -3.7 KIAA1344 KIAA1344 (KIAA1344), 14q22.1 mRNA. 226751 a 8.3OE-06 -2.4 C2Orf32 chromosome 2 open reading 2p14 frame 32 (C2orf32), mRNA. 226765 a O.OOO2842 2.2 SPTBN1 Spectrin, beta, non-erythrocytic 1 2p21 226777 a 5.39E-OS 4.9 ADAM12 A disintegrin and 10q26.3 metalloproteinase domain 12 (meltrin alpha) 2268.06 s at 1.95E-05 -2.8 MRNA, cDNA 1 DKFZp686.J23256 (from clone DKFZp686.J23256) 226829 at O.OOO6675 2.9 KIAA1914 KIAA1914 (KIAA1914), 10q25.3 transcript variant 2, mRNA. 226867 at O.OOO8554 -2 C9orf SS Chromosome 9 open reading 9p22.1 frame 55 226873 at O.OOO2O67 -2.8 Transcribed locus 16 226899 at 3.1OE-06 3.1 UNCSB unc-5 homolog B (C. elegans) 10q22.1 (UNCSB), mRNA. 226909 at O.OOO4528 -2 KIAA1729 KIAA1729 protein 4p16.1 (KLAA1729), mRNA. US 2013/02743 15 A1 Oct 17, 2013 63
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 226911 at 1.2OE-05 3.7 FLJ39155 hypothetical protein FLJ39155 5p13.2-p13.1 (FLJ39155), transcript variant 4, mRNA. 226933 s at 5.79E-05 3.8 ID4 inhibitor of DNA binding 4, 6p22-p21 dominant negative helix-loop helix protein (ID4), mRNA. 226943 a O.OOO4664 2 MRNA, cDNA DKFZp547P055 12 (from clone DKFZp547P055) 226994 a O.OOO2644 -2.3 DNAJA2 DnaJ (Hsp40) homolog, 16q11.1-q11.2 Subfamily A, member 2 226997 a O.OOO2372 3.7 CDNA FLJ10196 fis, clone 5 HEMBA1004776 227031 a O.OOO1825 -2.4 SNX13 Sorting nexin 13 7p21.1 227070 a O.OOO3008 -2.7 GLT8D2 glycosyltransferase 8 domain 12q containing 2 (GLT8D2), mRNA. 227082 a. O.OOO7234 -2 MRNA, cDNA 3 DKFZp586K1922 (from clone DKFZp586K1922) 227121 a O.OOO6088 -2.1 MRNA, cDNA 3 DKFZp586K1922 (from clone DKFZp586K1922) 227132 a O.OOO2728 -2.4 LOCS1123 HSPCO38 protein (LOC51123), 8q22.3 mRNA. 227138 a O.OOO8198 -2.1 CRTAP cartilage associated protein 3p22.3 (CRTAP), mRNA. 227148 a O.OOO1511 -3.4 PLEKHH2 pleckstrin homology domain 2p21 containing, family H (with MyTH4 domain) member 2 (PLEKHH2), mRNA. 2271 78 a O.OOOS124 -3.9 CUGBP2 CUG triplet repeat, RNA 10p13 binding protein 2 (CUGBP2), transcript variant 2, mRNA. 227197 a O.OOO108 -2.9 SGEF Src homology 3 domain- 3q25.2 containing guanine nucleotide exchange factor 227214 a O.OOO2O58 2.1 GOPC Golgi associated PDZ and 6q21 coiled-coil motif containing 227221 a O.OOO4345 2.1 CDNA FLJ31683 fis, clone 3 NT2RI2005.353 227260 a O.OOO1678 3 Transcribed locus 227 273 a 4.47E-OS -2.6 Transcribed locus 10 227 278 a O.OOOS386 2.3 Transcribed locus, weakly similar to XP 510104.1 PREDICTED: Similar to hypothetical protein FLJ25224 Pan troglodytes 227293 at 8.47E-OS -2.2 LNX Ligand of numb-protein X 4q12 227295 at 2.7OE-06 3.4 IKIP IKK interacting protein (IKIP), 12q23.1 transcript variant 3.1, mRNA. 227317 at O.OOO3721 2.3 LMCD1 LIM and cysteine-rich domains 3p26-p24 1 (LMCD1), mRNA. 227347 x at 2.86E-05 4 HES4 hairy and enhancer of split 4 1p36.33 (Drosophila) (HES4), mRNA. 227372 s at 0.0004102 3.4 BALAP2L1 BAI1-associated protein 2-like 1 7q21.3-q22.1 (BAIAP2L1), mRNA. 227383 at O.OOOS636 2.4 Similar to KIAA0454 protein 1q21.1 227384 S at 0.000128 2.6 Similar to KIAA0454 protein 1q21.1 227396 at O.OOO1476 2.6 Homo sapiens, clone 11 IMAGE: 4454331, mRNA 2274.07 at 9.42E-05 -2.3 FLJ90013 hypothetical protein FLJ90013 4p15.32 (FLJ90013), mRNA. 227529 S. at 0.0001731 -2.9 AKAP12 A kinase (PRKA) anchor protein 6q24-q25 (gravin) 12 227530 at 8.47E-OS -3 AKAP12 A kinase (PRKA) anchor protein 6q24-q25 (gravin) 12 227 636 at O.OOO3391 -2.3 THAP5 THAP domain containing 5 7q31.1 227703 s at 1.29E-OS -5.9 SYTL4 synaptotagmin-like 4 Xq21.33 (granuphilin-a) (SYTL4), mRNA. US 2013/02743 15 A1 Oct 17, 2013 64
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 227708 at O.OOO6098 -2.1 EEF1A1 Eukaryotic translation 6q14.1 elongation factor 1 alpha 1 227719 at 4.OOE-06 -3.6 CDNA FLJ37828 fis, clone 13 BRSSN2OO6575 227728 at 6.11E-OS -2.9 PPM1A Protein phosphatase 1A 14q23.1 (formerly 2C), magnesium dependent, alpha isoform 227827 at O.OOO8248 -4.5 ARGBP2 Arg/Abl-interacting protein 4q35.1 ArgBP2 227850 X. at 0.0001134 2.9 CDC42EP5 CDC42 effector protein (Rho 19q13.42 GTPase binding) 5 (CDC42EP5), mRNA. 227866 a O.OOO7379 -2 RBM16 RNA binding motif protein 16 6q25.1-q25.3 227945 a. O.OOO3107 -2.3 TBC1D1 TBC1 (tre-2/USP6, BUB2, 4p14 codc16) domain family, member 1 (TBC1D1), mRNA. 227952 a O.OOO1513 4.1 Full length insert cDNA clone 4 Y46GO4 227971 a 3.26E-OS -4.1 NRK Nik related kinase (NRK), Xq22.3 mRNA. 228O12 a O.OOO6848 -2 MATR3 Matrin 3 5q31.2 228.027 a 3.77E-05 -4 GPRASP2 G protein-coupled receptor Xq22.1 associated sorting protein 2 (GPRASP2), transcript variant 2, mRNA. 228030 a. O.OOOS637 3 RBM6 RNA binding motif protein 6 3p21.3 228.098 s at 0.0009902 2.2 MYLIP myosin regulatory light chain 6p23-p22.3 interacting protein (MYLIP), mRNA. 228202 a O.OOO1345 -9.3 PLN Phospholamban 6q22.1 228204 a 1.OOE-07 2.9 PSMB4 proteasome (prosome, 1q21 macropain) subunit, beta type, 4 (PSMB4), mRNA. 228253 a 1.1OE-05 3.8 LOXL3 lysyl oxidase-like 3 (LOXL3), 2p13 mRNA. 228297 a O.OOO4293 3.3 CNN3 calponin 3, acidic (CNN3), 1p22-p21 mRNA. 228310 a 8.1OE-06 -3.3 ENAH enabled homolog (Drosophila) 1q42.12 (ENAH), transcript variant 2, mRNA. 228331 a O.OOO2123 3.4 C11orf1 Chromosome 11 open reading 11 q12.1 frame 31 228333 a O.OOOS4O7 -2.2 Full length insert cDNA clone 2 YT94EO2 228335 a. O.OOO878S -3.8 CLDN11 claudin 11 (oligodendrocyte 3q26.2-q26.3 transmembrane protein) (CLDN11), mRNA. 228497 a O.OOO1644 3.2 SLC22A15 Solute carrier family 22 (organic 1p13.1 cation transporter), member 15 (SLC22A15), mRNA. 228523 a O.OOO4047 2 NANOS1 nanos homolog 1 (Drosophila) 10q26.11 (NANOS1), transcript variant 2, mRNA. 228551 a 3.9SE-OS -3.1 MGC24039 Hypothetical protein 12p11.21 MGC24039 228554 a 1.35E-05 -4.7 MRNA, cDNA 11 DKFZp586GO321 (from clone DKFZp586G0321) 228569 a O.OOO681S -2.2 PAPOLA Poly(A) polymerase alpha 14q32.31 228573 a O.OOO1917 -2.5 ANTXR2 Anthrax toxin receptor 2 4q21.21 228577 x at 0.0003076 2.8 ODF2L outer dense fiber of sperm tails 1p22.3 2-like (ODF2L), transcript variant 2 mRNA. 228579 a 7.OOE-07 5.2 KCNQ3 Potassium voltage-gated 8q24 channel, KQT-like subfamily, member 3 228785 a O.OOO6036 2.2 ZNF281 Zinc finger protein 281 1q32.1 228805 a O.OOO3526 -3 FLJ44216 FLJ44216 protein (FLJ44216), 5q.35.2 mRNA. 228841 a O.OOO4789 -2 CDNA FLJ32429 fis, clone 5 SKMUS2OO1014 228.850 S at 0.0005294 2.8 SLIT2 Slithomolog 2 (Drosophila) 4p15.2 US 2013/02743 15 A1 Oct. 17, 2013 65
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 228885 at 182E-05 -4 MAMDC2 MAM domain containing 2 9q21.11 (MAMDC2), mRNA. 228905 at O.OOO1087 -2.4 Transcribed locus, moderately 8 similar to XP 517655.1 PREDICTED: Similar to KIAAO825 protein Pan troglodytes 228961 a O.OOO4391 -2.1 FLJ35954 Hypothetical protein FLJ35954 5q11.2 22.9085 a O.OOO3123 2.5 LRRC3B eucine rich repeat containing 3p24 3B (LRRC3B), mRNA. 229114 a O.OOO2683 -2 GAB1 GRB2-associated binding 4q31.21 protein 1 229119 S at 0.0001491 -2.2 TTC19 Hypothetical protein 17p12 LOC1251SO 229129 a O.OOO6968 -2.2 HNRPD Heterogeneous nuclear 4q21.1-q21.2 ribonucleoprotein D (AU-rich element RNA binding protein 1, 37 kDa) 229130 a. O.OOO2O67 -3 LOC285535 Hypothetical protein 4p16.1 LOC285535 229145 a. 4.45E-05 -4.2 C10orf104 chromosome 10 open reading 10q22.1 rame 104 (C10orf104), mRNA. 229160 a O.OOO3SS2 -6.5 MUM1L1 melanoma associated antigen Xq22.3 (mutated) 1-like 1 (MUM1L1), mRNA. 229200 a O.OOO156 2.3 Hypothetical LOC400813 1q44 229204 a O.OOO3OO7 2.2 HP1-BP74 Heterochromatin protein 1, 1p36.12 binding protein 3 229218 a O.OOO3816 3.3 COL1A2 Collagen, type I, alpha 2 7q22.1 229221 a O.OOO6672 4.1 CD44 CD44 antigen (homing function 11 p.13 and Indian blood group system) 229287 a O.OOO7653 -2.8 Full-length cDNA clone 14 CSODKO1OYA20 of HeLa cells Cot 25-normalized of Homo Sapiens (human) 229299 a O.OOO1103 -2.2 FLJ30596 hypothetical protein FLJ30596 5p13.2 (FLJ30596), mRNA. 229.308 a 2.94E-05 -5.4 Transcribed locus 18 229319 a O.OOO1521 -2.4 Homo sapiens, clone 6 MAGE: 4105966, mRNA 229331 a 0.0007515 2.3 SPATA18 spermatogenesis associated 18 4q11 homolog (rat) (SPATA18), mRNA. 229339 a O.OOO2425 -5 MYOCD Myocardin 7p11.2 229354 a O.OOOS/8 3.3 PDCD6 Aryl-hydrocarbon receptor 5pter-p15.2 reeSSO 2294.31 a O.OOO216 -2.1 RFXAP regulatory factor X-associated 3a14 protein (RFXAP), mRNA. 2294.83 a O.OOO2012 2.5 UBE2H Ubiquitin-conjugating enzyme 7q32 E2H (UBC8 homolog, yeast) 229515 a. O.OOO2226 -2.2 PAWR PRKC, apoptosis, WT1 2d21 regulator 229520 S. at 0.0001174 2.3 C14orf118 Chromosome 14 open reading 4q22.1-q24.3 rame 118 229531 a O.OOO7976 -2.2 Mitochondrial carrier triple Xq22.2 repeat 6 229553 a O.OOO8476 2 PGM2L1 phosphoglucomutase 2-like 1 1a13.4 (PGM2L1), mRNA. 2295.80 a O.OOO8828 -6.5 Transcribed locus 3 229638 a O.OOO8539 2.9 IRX3 iroquois homeobox protein 3 6d12.2 (IRX3), mRNA. 229642 a. O.OOO2914 4.2 ARHGEF7 Rho guanine nucleotide 3d34 exchange factor (GEF) 7 (ARHGEF7), transcript variant 2, mRNA. 229665 a. O.OOO1871 2.2 CSTF3 Hypothetical protein 1p13 LOC283267 229711 S. at 0.0003052 2.1 CDNA FLJ37519 fis, clone 2 BRCAN2OO4699 229748 x at 0.0001962 2.7 LOC285.458 Hypothetical protein 4 LOC285.458 229795 at O.OOO2666 3.4 Transcribed locus 2 US 2013/02743 15 A1 Oct. 17, 2013 66
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 229801 a O.OOO1036 3.4 C10orfaf chromosome 10 open reading 10p14 rame 47 (C10orfA7), mRNA. 2298.30 a. O.OOO4695 3.9 PDGFA Platelet-derived growth factor 7p22 alpha polypeptide 229844 a O.OOO1747 -2.2 Transcribed locus 3 229891 X at 1.34E-05 -2.9 KIAA1704 KIAA1704 13q13-q14 229969 a O.OOO3327 -2.5 Transcribed locus, moderately 6 similar to XP 50823.0.1 PREDICTED: zinc finger protein 195 Pan troglodytes 229994 a O.OOO2738 -3 MRNA, cDNA 1 DKFZp686.J23256 (from clone DKFZp686.J23256) 230000 a O.OOO4792 2.7 C17orf27 Chromosome 17 open reading 17q25.3 rame 27 230030 a. 0.0003377 -5.2 HS6ST2 heparan sulfate 6-O- Xq26.2 Sulfotransferase 2 (HS6ST2), mRNA. 23.0068. S. at 8.34E-05 -3.2 PEG3 paternally expressed 3 (PEG3), 19q13.4 mRNA. 230077 a O.OOO1004 3.9 TFRC Transferrin receptor (p90, 3q29 CD71) 230081 a S.OOE-07 -6.1 PLCXD3 phosphatidylinositol-specific 5p13.1 phospholipase C, X domain containing 3 (PLCXD3), mRNA. 23O130 a. 9.27E-OS 3 SLIT2 Slithomolog 2 (Drosophila) 4p15.2 23O141 a O.OOO2O42 -2 ARID4A AT rich interactive domain 4A 14q23.1 (RBP1-like) 230174 a O.OOO8233 -2.1 LYPLAL1 Lysophospholipase-like 1 1q41 230178 s at 9.98E-OS -2.4 STATIP1 Signal transducer and activator 18q12.2 of transcription 3 interacting protein 1 230270 a 7.57E-05 2.9 ESTs 23O333 a O.OOO8148 2.5 SAT Spermidine?spermine N1- Xp22.1 acetyltransferase 23O336 a O.OOO8645 2.7 Transcribed locus 4 230369 a 0.0005567 -2.1 GPR161 G protein-coupled receptor 161 1q24.2 230387 a O.OOO1569 3.1 ATP2C1 ATPase, Ca++ transporting, type 3q22.1 2C, member 1 230440 a 2.81E-05 3.3 ZNF469 PREDICTED: zinc finger 16 protein 469 (ZNF469), mRNA. 230561 s at 0.0001454 -2.9 FLU23861 hypothetical protein FLJ23861 2a34 (FLJ23861), mRNA. 230574 a O.OOO80S6 2.9 Hypothetical LOC388480 18q21.33 230746 s at 1.5OE-06 13.3 STC1 stanniocalcin 1 (STC1), mRNA. 8p21-p11.2 230758 a O.OOO64 -2.6 Transcribed locus X 230793 a 7.34E-OS -3.1 LRRC16 leucine rich repeat containing 16 6p22.2 (LRRC16), mRNA. 230850 a O.OOO1807 3 FMNL3 Formin-like 3 12q13.12 230885 a O.OOO1271 2.2 SPG7 Spastic paraplegia 7, paraplegin 16q24.3 (pure and complicated autosomal recessive) 230958 s at 4.15E-05 -2.1 MRNA, cDNA 1 DKFZp686.J23256 (from clone DKFZp686.J23256) 231130 at O.OOO9273 -2 FKBP7 FK506 binding protein 7 2a31.2 231183 s at 0.000129 4.3 JAG1 jagged 1 (Alagile syndrome) 20p12.1-p11.23 (JAG1), mRNA. 231202 at O.OOO2962 3.1 FLJ38508 Aldehyde dehydrogenase 1 12q23.3 family, member L2 231411 at O.OOO1224 3.2 LHFP lipoma HMGIC fusion partner 13q12 (LHFP), mRNA. 231597 X at 0.0001876 15.9 ESTs, Weakly similar to T47135 hypothetical protein DKFZp761L0812.1 H. sapiens 231806 s at 0.0009603 2.1 STK36 serine threonine kinase 36 2a35 (fused homolog, Drosophila) (STK36), mRNA. 231825 X at 8.13E-05 3.6 ATF7IP Activating transcription factor 7 12p13.1 interacting protein US 2013/02743 15 A1 Oct 17, 2013 67
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 231882 at O.OOO2463 2.7 CDNA FLJ10674 fis, clone 22 NT2RP2OO6436 231886 at O.OOO3613 3 Similar to hypothetical protein 3q29 LOC284,701 232034 at O.OOO816S 3.9 LOC2O3274 Hypothetical protein 9q21.11 LOC2O3274 232145 at O.OOO2S39 2.1 LOC38.8969 hypothetical LOC38.8969 2p11.2 (LOC38.8969), mRNA. 232150 at 2.29E-OS 3.1 C20orf18 Chromosome 20 open reading 20p13 frame 18 232169 X at 0.0002893 3.1 NDUFS8 NADH dehydrogenase 11 q13 (ubiquinone) Fe—S protein 8, 23 kDa (NADH-coenzyme Q reductase) 232174 a 7.OOE-06 4.1 EXT1 Exostoses (multiple) 1 8q24.11-q24.13 232180 a 8.56E-OS 3.2 UGP2 UDP-glucose 2p14-p13 pyrophosphorylase 2 232215 X at 0.0001855 3.4 FLJ11029 Hypothetical protein FLJ11029 17q23.2 232254 a 8.7SE-OS 2.5 FBXO25 F-box protein 25 8p23.3 232266 x at O.OOO1426 3.9 CDC2LS Cell division cycle 2-like 5 7p13 (cholinesterase-related cell division controller) 232304 a 2.6OE-06 4 PELI1 Pellino homolog 1 (Drosophila) 2p13.3 232347 X at 0.0001636 2.6 CBR4 Carbonic reductase 4 4q32.3 232406 a O.OOO1583 2.9 JAG1 agged 1 (Alagile syndrome) 20p12.1-p11.23 232458 a 1.67E-OS 4.1 COL3A1 Collagen, type III, alpha 1 2a31 (Ehlers-Danlos syndrome type V, autosomal dominant) 232516 X at 7.53E-05 3.3 YAP YY1 associated protein 1 1q22 232530 a. O.OOO6114 2.8 PLD1 Phospholipase D1, 3q26 phophatidylcholine-specific 232538 a O.OOO8.933 2.4 CDNA: FLJ23573 fis, clone 16 LNG12S2O 232541 a O.OOO7593 3.5 EGFR Epidermal growth factor 7p12 receptor (erythroblastic eukemia viral (v-erb-b) oncogene homolog, avian) 232617 a 8.18E-OS 3.2 CTSS cathepsin S (CTSS), mRNA. 1q21 232653 a O.OOO473 3.5 Homo sapiens cDNA FLJ14044 is, clone HEMBA1006124 232702 a O.OOO6941 3 RABGAP1L, RAB GTPase activating protein 1q24 -like 232797 a O.OOO8213 2.3 ITGAV integrin, alpha V (vitronectin 2a31-q32 receptor, alpha polypeptide, antigen CD51) 232814 X at 0.0005021 2.8 C14orf153 Chromosome 14 open reading 14q32.32-q32.33 rame 153 232889 at O.OOO3899 2.6 CDNA clone IMAGE:5576908 5 232952 at O.OOO1405 2.7 DDEF1 HSPCO54 protein 8q24.1-q24.2 233041 x at 0.0004112 3 BTBD9 BTB (POZ) domain containing 9 6p21 233180 at 1.17E-OS 2.8 RNF152 Ring finger protein 152 18q21.33 233274 at 3.OOE-07 3.6 NCK1 NCK adaptor protein 1 3q21 233319 X at 0.0001543 3.1 PHACTR4 Phosphatase and actin regulator 4 1p35.3 233330 s a 4.59E-OS S.6 Similar to Ribosome biogenesis 9q13 protein BMS1 homolog 233406 at O.OOO2846 2.9 KIAAO256 KIAAO256 gene product 15q21.1 233496 s a O.OOO3586 -2 CFL2 Cofilin 2 (muscle) 14q12 233702 X at 0.0007629 3.1 CDNA: FLJ20946 fis, clone 7 ADSEO1819 233849 s a O.OOOS935 -2.3 ARHGAPS Rho GTPase activating protein 5 14q12 (ARHGAPS), transcript variant 2, mRNA. 233912 X at 1.73E-OS 4.4 ELMOD2 ELMO domain containing 2 4q31.21 234.192 s a O.OOO1567 -3.1 GKAP1 G kinase anchoring protein 1 9q21.32 (GKAP1), mRNA. 234.339 s a 6.34E-OS -2.4 GLTSCR2 glioma tumor Suppressor 19q13.3 candidate region gene 2 (GLTSCR2), mRNA. 234464 S a 4.21E-OS 3 EME1 essential meiotic endonuclease 1 17q21.33 homolog 1 (S. pombe) (EME1), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 68
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 234512 x 2a-OE-06 -3.1 LOC4421.59 PREDICTED: similar to Rp17a 6 protein (LOC442159), mRNA. 234562 x 1a80E-06 8.6 CKLFSF8 Chemokine-like factor super 3p23 family 8 234578 at 7.26E-05 4.9 MRNA, cDNA 1 DKFZp434E1812 (from clone DKFZp434E1812) 234675 x 6afs2E-05 4.3 CDNA: FLJ23566 fis, clone 14 LNG1088O 234.723 x 1as(E-06 9.4 CDNA: FLJ21228 fis, clone 7 COLOO739 234753 x (a)002577 5.7 234762 x 8a SE-05 4.1 NLN Neurolysin (metallopeptidase 5q12.3 M3 family) 234788 x (a)001541 3.3 FLJ13611 Hypothetical protein FLJ13611 5q12.3 234873 x (a)00607 -2 234981 X 985E-05 4.4 LOC134147 Similar to mouse 5p15.2 2310016AO9Rik gene 234985 at 9.3OE-06 2.9 LOC143458 Hypothetical protein 11 p.13 LOC143458 234998 at 0.0009467 -2.7 CDNA clone IMAGE: 5313062 15 235.005 at 0.0003344 -2.3 MGC4562 Hypothetical protein MGC4562 15q22.31 235061 at 0.0002578 -2.9 PPM1 K protein phosphatase 1 K (PP2C 4q22.1 domain containing) (PPM1K), mRNA. 235072 S. 666E-05 -3 Transcribed locus 6 235122 at 5.42E-05 3.1 CDNA clone IMAGE: 6254.031 1 235151 at 0.0004926 -2.1 LOC283357 Hypothetical protein 12p13.33 LOC283357 235204 at 1.87E-05 3.1 ENTPD7 Ectonucleoside triphosphate 10 diphosphohydrolase 7 235205 at O.OOO1252 3.5 LOC346887 PREDICTED: Similar to Solute 8 carrier family 16 (monocarboxylic acid transporters), member 14 (LOC346887), mRNA. 235278 at 0.0002619 -3.6 C20orf133 chromosome 20 open reading 20p12.1 rame 133 (C20orf133), transcript variant 2, mRNA. 235309 at p <1e-O7 -4.9 CDNA clone IMAGE: 4140029 16 235327 x (a)002751 2.9 UBXD4 UBX domain containing 4 2p23.3 (UBXD4), mRNA. 235343 at 3.96E-05 4.2 FLJ12505 Hypothetical protein FLJ12505 1q32.3 235374 at 3.04E-05 2.7 MDH1 Malate dehydrogenase 1, NAD 2p13.3 (soluble) 235412 at 0.0002124 3.2 ARHGEF7 Rho guanine nucleotide 13q34 exchange factor (GEF) 7 235433 at 0.0003304 -2.2 SATL1 Spermidine?spermine N1-acetyl Xq21.1 transferase-like 1 235556 at 0.0001833 -2 Transcribed locus, weakly 5 similar to NP 703324.1 glutamic acid-rich protein (garp) Plasmodium falciparum 3D7 2356O1 at 0.0003888 2.7 ESTs 235612 at 2.90E-05 -3.1 Transcribed locus, moderately 1 similar to NP 858931.1 NFS1 nitrogen fixation 1 isoform b precursor; cysteine desulfurase: nitrogen-fixing bacteria S-like protein; nitrogen fixation 1 (S. cerevisiae, homolog) Homo Sapiens 235628 x (a)004.866 2.4 Hypothetical protein 5q23.2 LOC133926 235693 at 0.0003764 2.9 Transcribed locus 5 235725 at O.OOO4132 -2.2 Transcribed locus 14 235927 at 6.41E-05 2.4 XPO1 Exportin 1 (CRM1 homolog, 2p16 yeast) 235944 at 0.0004.031 3.3 HMCN1 hemicentin 1 (HMCN1), 1q25.3-q31.1 mRNA. 236249 at 1.54E-05 2.7 IKIP KK interacting protein (IKIP), 12q23.1 transcript variant 1, mRNA. US 2013/02743 15 A1 Oct 17, 2013 69
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 236251 at 8.16E-OS 2.7 ITGAV Integrin, alpha V (vitronectin 2a31-q32 receptor, alpha polypeptide, antigen CD51) 236678 at O.OOOS987 2.8 JAG1 Jagged 1 (Alagile syndrome) 20p12.1-p11.23 236715 x at 0.0002206 2.5 UACA uveal autoantigen with coiled- 15q22-q24 coil domains and ankyrin repeats (UACA), transcript variant 1, mRNA. 236829 at 6.57E-OS 2.9 TMF1 TATA element modulatory 3p21-p12 factor 1 236883 at O.OOO3O76 2.6 ESTs 236936 at O.OOO7927 -2.4 Transcribed locus 8 236966 at O.OOO47S7 2.6 TXNDC6 thioredoxin domain containing 6 3q22.3 (TXNDC6), mRNA. 236974 at O.OOO3599 2.5 CCNI Cyclin I 4q21.1 237206 at O.OOO3483 -5.9 MYOCD Myocardin 17p11.2 237333 at O.OOO8796 -2 SYNCOILIN Syncoilin, intermediate filament 1 1p34.3-p33 2374.75 X at 0.0001562 4.3 SEPP1 Selenoprotein P. plasma, 1 5q31 237494 at 2.03E-OS 3.2 Transcribed locus 15 237868 x at O.OOO358 3 ESTs, Weakly similar to ALUF HUMAN ALU CLASSF WARNING ENTRY !!! H. sapiens 238.026 at O.OOO790S -2 RPL3SA Ribosomal protein L35a 3q29-qter 238142 at O.OOO678 2.4 LOC2S3982 Hypothetical protein 16p11.2 LOC2S3982 238183 at O.OOO1644 3.7 ESTs 238273 at O.OOO7281 2 Full-length cDNA clone 7 CSODBOOSYG10 of Neuroblastoma Cot 10 normalized of Homo sapiens (human) 238327 a 6.09E-OS 2.6 Similar to MGC52679 protein 22d 13.33 238478 a O.OOO183 -3.7 BNC2 Basonuclin 2 9p22.3-p22.2 238584 a O.OOO21.96 3.3 IQCA IQ motif containing with AAA 2a37.2-q37.3 domain 238613 a 8.97E-OS -2.7 ZAK sterile alpha motif and leucine 2d24.2 Zipper containing kinase AZK (ZAK), transcript variant 2, mRNA. 238642 a. O.OOO3128 3 LOC338692 Ankyrin repeat domain 13 11 q13.2 amily, member D 238673 a 6.02E-OS 4.7 Transcribed locus 8 238714 a O.OOO1825 2.5 RAB12, member RAS oncogene 18p11.22 amily 238719 a O.OOO4981 -2.5 PPP2CA Protein phosphatase 2 (formerly 5q31.1 2A), catalytic Subunit, alpha isoform 238852 a O.OOO3O11 2.3 ESTs 238878 a O.OOO6945 -6.2 ARX Aristaless related homeobox Xp22.1-p21.3 239227 a 9.04E-05 3 EXT1 Exostoses (multiple) 1 8q24.11-q24.13 239246 a O.OOO2103 3 FARP1 FERM, RhoGEF (ARHGEF) 13q32.2 and pleckstrin domain protein 1 (chondrocyte-derived) 239258 a O.OOO9901 3.1 RHOQ Rashomolog gene family, 2p21 member Q 2392.62 a 3.72E-OS -3.8 CDNA FLJ26242 fis, clone 11 DMCOO770 239264 a O.OOO3642 2.2 SEC8L1 SEC8-like 1 (S. cerevisiae) 7q31 2393.67 a O.OOO1649 3.1 BDNF brain-derived neurotrophic 11 p.13 actor (BDNF), transcript variant 6, mRNA. 239516 a O.OOO1834 2.7 LYPLAL1 Lysophospholipase-like 1 1q41 239540 a O.OOO9106 2.6 GTF3C1 General transcription factor IIIC, 16p12 polypeptide 1, alpha 220 kDa 239748 x at O.OOO3286 3.7 OCLA ovarian carcinoma 4p11 immunoreactive antigen 239806 a O.OOO2S2 4.4 Transcribed locus 2 239848 a O.OOOS816 -2.7 GA17 Dendritic cell protein 11 p.13 240216 a O.OOO1639 2.6 ZBTB20 Zinc finger and BTB domain 3q13.2 containing 20 240421 X at 0.0001971 3.7 CDNA clone IMAGE: 5268630 4 US 2013/02743 15 A1 Oct. 17, 2013 70
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 240655 at 3.01E-OS 4.3 ALCAM Activated leukocyte cell 3q13.1 adhesion molecule 240795 at O.OOO1514 2.3 CDNA clone IMAGE: 528.8566 5 241223 X at O.OOO2281 3.2 ESTs, Weakly similar to ALU1 HUMAN ALU SUBFAMILY J SEQUENCE CONTAMINATION WARNING ENTRY H. Sapiens 24.1268 X at 0.0008446 3.5 SAMHD1 SAM domain and HD domain 1 20pter-q12 241.303 X at 0.0007123 2.9 ESTs 241387 at O.OOO812S 2.7 PTK2 PTK2 protein tyrosine kinase 2 8q24-qter 241421 at O.OOOSO12 2.6 Transcribed locus 1 241435 at 1.S2E-OS 4.4 ETS1 V-ets erythroblastosis virus E26 11q23.3 oncogene homolog 1 (avian) 241617 x at O.OOO334 2.6 ESTs, Weakly similar to 81.0024C cytochrome oxidase I H. Sapiens 241627 X at 0.0005548 2.9 FLJ10357 Hypothetical protein FLJ10357 14q11.2 241632 x at 3.7OE-06 3.3 ESTs 241686 X at 3.28E-05 4.4 ESTs, Weakly similar to hypothetical protein FLJ20378 Homo sapiens H. sapiens 24.1718 X at 7.37E-05 3.4 ESTs 241727 X at 0.0003166 2.3 DHFRL1 dihydrofolate reductase-like 1 3q11.2 (DHFRL1), mRNA. 24.1769 at O.OOO4802 2.5 ITGAV integrin, alpha V (vitronectin 2a31-q32 receptor, alpha polypeptide, antigen CD51) 241809 at O.OOO1618 2.5 LOC284.465 Hypothetical protein 1p13.2 LOC284.465 242029 at 9.6OE-06 3.7 FNDC3B Fibronectin type III domain 3q26.31 containing 3B 242051 at 6.2OE-06 -3.7 Transcribed locus X 242100 at 2.4OE-06 4.8 CSS3 chondroitin sulfate synthase 3 5q23.3 (CSS3), mRNA. 242171 at O.OOO7535 3.1 ESTs, Weakly similar to ALU1 HUMAN ALU SUBFAMILY J SEQUENCE CONTAMINATION WARNING ENTRY H. Sapiens 242233 at 7.63E-OS 2.4 KIAA1219 KIAA1219 protein 20d 11.23 242240 at O.OOO1413 3 PTK2 PTK2 protein tyrosine kinase 2 8q24-qter 242329 at 8.94E-OS 3.1 LOC4O1317 PREDICTED: hypothetical 7 LOC4O1317 (LOC4O1317), mRNA. 242363 at 9. OSE-OS -2.7 DNCI2 Dynein, cytoplasmic, 2a31.1 intermediate polypeptide 2 242364 X at 0.0001.237 3.1 EVER1 Epidermodysplasia 17q25.3 verruciformis 1 242369 X at 7.20E-06 4.1 NCOA2 Nuclear receptor coactivator 2 8q13.3 242398 X at 3.00E-06 6.1 MEPSO WD repeat domain 77 1p13.2 242405 at 4.O8E-OS 2 MAML2 Mastermind-like 2 (Drosophila) 11q21 242488 at 2.11E-05 -4.7 CDNA FLJ38396 fis, clone 1 FEBRA2OO7957 242500 at O.OOO7278 2.6 Transcribed locus 6 242546 at 2.7OE-06 5.4 LOC44O156 14q11.1 2425.78 x at 0.0002453 4 SLC22A3 Solute carrier family 22 6q26-q27 (extraneuronal monoamine transporter), member 3 242862 X at 6.29E-05 5.2 ESTs 242999 at 0.0007732 2.3 ARHGEF7 Rho guanine nucleotide 13q34 exchange factor (GEF) 7 (ARHGEF7), transcript variant 2, mRNA. 243 g at 9.74E-OS 2.1 MAP4 microtubule-associated protein 4 3p21 (MAP4), transcript variant 2, mRNA. 243006 at 1.1OE-06 4.7 FYN FYN oncogene related to SRC, 6q21 FGR, YES US 2013/02743 15 A1 Oct. 17, 2013 71
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 243147 X at 7.12E-05 5.7 ESTs, Weakly similar to RMS1 HUMAN REGULATOR OF MITOTICSPINDLE ASSEMBLY 1 H. sapiens 243249 at 7.21E-OS 2.4 ESTs, Weakly similar to hypothetical protein FLJ20378 Homo sapiens H. Sapiens 243305 at O.OOO3329 2.9 KIAA1340 Kelch domain containing 5 12p11.22 243442 X at 0.0001001 3.7 ESTs, Weakly similar to ALU1 HUMAN ALU SUBFAMILY J SEQUENCE CONTAMINATION WARNING ENTRY H. Sapiens 243846 X at 0.0007887 3 FLJ32810 Hypothetical protein FLJ32810 11q22.1 243915 a. O.OOO1482 2.9 ESTs, Weakly similar to 2109260A B cell growth factor H. Sapiens 244007 a O.OOO373 -2 Transcribed locus 9 244022 a. O.OOO7683 2 FNDC3B Fibronectin type III domain 3q26.31 containing 3B 244050 a O.OOO1908 -2.6 LOC4O1494 similar to RIKEN 4933428IO3 9p21.3 (LOC4O1494), mRNA. 244188 a O.OOO4372 3.1 FL21924 Hypothetical protein FLJ21924 1p13 244193 a O.OOO3257 2.1 FLJ13236 Hypothetical protein FLJ13236 2d 13.12 244197 X at 2.3OE-05 3.3 CNOT2 CCR4-NOT transcription 2a15 complex, Subunit 2 244457 a 8.66E-OS 3.1 ITPR2 nositol 14,5-triphosphate 2p11 receptor, type 2 244503 a O.OOO41OS 2.5 ESTs 244633 a O.OOO111 2.5 PIAS2 Protein inhibitor of activated 8q21.1 STAT, 2 244648 a O.OOO3403 3.5 FLJ10996 Hypothetical protein FLJ10996 2d 14.1 244745 a. O.OOO6739 -2.7 RERG RAS-like, estrogen-regulated, 2p12.3 growth inhibitor (RERG), mRNA. 31874 a 8.21E-OS 2.9 GAS2L1 Growth arrest-specific 2 like 1 22q12.2 33323 r at O.OOO1426 S.1 SFN Stratifin p36.11 38069 a O.OOO31.97 2 CLCN7 chloride channel 7 (CLCN7), 6p13 mRNA. 38671 a O.OOO3864 2.4 PLXND1 plexin D1 (PLXND1), mRNA. 3q21.3 39549 a O.OOO8832 2.5 NPAS2 neuronal PAS domain protein 2 2d 11.2 (NPAS2), mRNA. 39891 a O.OOOSS41 2.2 DKFZp547K1113 Hypothetical protein 15q26.1 DKFZp547K1113 40524 a 3.7OE-OS 2.8 PTPN21 protein tyrosine phosphatase, 14q31.3 non-receptor type 21 (PTPN21), mRNA. 41856 a O.OOO8627 2.3 UNCSB Unc-5 homolog B (C. elegans) 10q22.1 44783 s at 6.8SE-OS 3 HEY1 hairy fenhancer-of-split related 8q21 with YRPW motif 1 (HEY1), mRNA. 46665 a S.11E-OS 2.4 SEMA4C Sema domain, immunoglobulin 2d 11.2 domain (Ig), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4C (SEMA4C), mRNA. 47550 at 3.4OE-05 4.4 LZTS1 leucine Zipper, putative tumor 8p22 Suppressor 1 (LZTS1), mRNA. 50376 at O.OOO154 2.1 ZNF444 Zinc finger protein 444 19q13.43 (ZNF444), mRNA. 52255 S at 1.1SE-OS 6.1 COLSA3 collagen, type V, alpha 3 19p 13.2 (COL5A3), mRNA. 55583 at O.OOO1374 3.2 DOCK6 dedicator of cytokinesis 6 19p 13.2 (DOCK6), mRNA. 57539 at O.OOO6736 2.1 ZGPAT Zinc finger, CCCH-type with G 20d 13.3 patch domain (ZGPAT), transcript variant 3, mRNA. 57703 at O.OOO1957 2.4 SENP5 SUMO1, sentrin specific 3q29 peptidase 5 (SENP5), mRNA. 57739 at O.OOO4289 2.2 DND1 Dead end homolog 1 (zebrafish) 5q31.3 US 2013/02743 15 A1 Oct. 17, 2013 72
TABLE 1-continued Gene expression profile. Affymetrix Parametric Fold Change Probe Set P-Value (Tumor? Normal) Gene Symbol Description Map 59433 at 5.44E-05 2.2 Transcribed locus X 61734 at 3.36E-OS 2.7 RCN3 Reticulocal bin 3, EF-hand 19q13.33 calcium binding domain
0299. As indicated in Table 1, 652 genes were up-regu- elevated at least 6-fold in tumor endothelium (Table 2), with lated a 2-fold in ovarian cancer endothelium samples. Of the 7 being elevated more than 10-fold and 2 being elevated more 652 genes that were up-regulated 22-fold, 35 genes were than 28-fold. TABLE 2 Genes up-regulated by e6-fold in the tumor associated endothelium. Entrez Fold chromosomal Gene ID Gene Description difference location Function 25975 EGFL6 EGF-like-domain, 36.8 Xp22 May regulate cell cycle and Multiple 6 oncogenesis 7130 TNFAIP6 Tumor necrosis factor, 29.1 2d23.3 Anti-inflammatory and Alpha-induced protein 6 chondroprotective effect 7291 TWIST1 Basic helix-loop-helix 19.0 7p21.2 inhibits chondrogenesis (bHLH) transcription factor 6781 STC1 Stanniocalcin 1 13.3 8p21-p11.2 Regulates calcium phosphate homeostasis, and cell metabolism 8452S HOP Homeodomain-only 13.1 4q11-q12 Transcriptional repressor. Protein, transcript Modulates serum response variant 2 actor-dependent cardiac specific gene expression and cardiac development 1462 CSPG2 Chondroitin sulfate 10.4 5q14.3 Extracellular matrix proteoglycan 2 component of the vitreous gel. (versican) Anti-cell adhesive. 57125 Plexin domain 10.2 17q21.1 Unknown containing 1 6696 SPP1 Secreted 9.5 4q21-q25 Expressed during phosphoprotein 1 embryogenesis, wound (Osteopontin, bone healing, and tumorigenesis. sialoprotein, Regulates the assembly of Early T-lymphocyte heterotypic fibers composed of activation1) both type 1 and type V collagen. 4318 MMP9 Matrix metallopeptidase 9 9.4 20g 11.2-q13.1 Breakdown of extracellular (gelatinase B, 92 kDa matrix. Plays a role in type IV collagenase) angiogenesis and tumor invasion 3937 Lymphocyte cytosolic 8.7 5q33.1-qter Promotes T-cell protein 2 (SH2 domain development and containing Leukocyte activation protein of 76 kDa) 1521.89 Chemokine-like factor 8.6 3p23 Regulates EGF-induced Superfamily 8 signaling. Regulates cell proliferation S366 PMAIP1 Phorbol-12-myristate 8.5 18q21.32 Unknown 13-acetate-induced protein 1 24147 Four jointed box 1 7.7 11p13 in drosophila, a downstream (drosophila) arget of the Notch signaling pathway, regulates cell growth and differentiation. Not known in human 8038 ADAM12 ADAM metallo 7.6 10q26.3 Critical for tumor peptidase domain 12 development. Involved in cell (meltrin alpha) cell and cell-matrix interactions. 9636 GIP2 Interferon, alpha 6.9 1p36.33 Unclear, may be related to inducible protein (clone regulation of cell proliferation IFI-15K) and differentiation US 2013/02743 15 A1 Oct. 17, 2013 73
TABLE 2-continued Genes up-regulated by 26-fold in the tumor associated endothelium. Entrez Fold chromosomal Gene ID Gene Description difference location Function 25878 MXRAS Matrix-remodeling 6.9 Xp22.33 Matrix remodeling associated 5 1123 CNH1 Chimerin 6.9 2q31-q32.1 Rho GTPase activating protein (chimaerin) 1 3310 HSPA6 Heat shock 70 KDa 6.8 1q23 Involved in protein protein 6 (HSP70B) conformational interactions 11211 FZD10 Frizzled homolog 10 6.7 12q24.33 Receptor for the wingless (Drosophila) Type MMTV integration site family. 10631 POSTN Periostin, osteoblast 6.7 13q13.3 Promotes integrin-dependent specific factor cell adhesion and motility, involved in extracellular matrix deposition 85.477 SCIN Scinderin 6.6 7p21.3 Ca' dependent actin filament Severing protein, regulates cortical actin network dynamics 27242 TNFRSF21 Tumor necrosis factor 6.6 6p21.1-12.2 Unclear; maybe related to Receptor Superfamily, activation of NF-kappaB and Member 21 MAPK8/JNK, induces cell apoptosis, involved in inflammation and immune regulation. 25891 DKFZP586- Regeneration associated 6.2 11p13 Unknown H2123 muscle protease, transcript variant 2 4582 MUC1 Mucin 1, 6.2 1q21 Regulates cell aggregation, transmembrane, adhesion transcript variant 4 79084 MEPSO WD repeat domain 77, 6.1 1p13.2 involved in the methylation WDR77 and assembly of spliceosomal SnRNAs Sm proteins SOSO9 COLSA3 Collagen, type V, alpha 3 6.1 19p 13.2 Extracellular protein, associated with formation of fibrils, and some connective issue pathology Such as inflammation, cancer and atherosclerosis 62OS RPS11 Ribosomal protein S11 6.1 19q13.3 nvolved in the recognition of ermination codons. SS8O3 CENTA2 Centaurin, alpha 2 6.1 17q11.2 A phosphatidylinositide binding protein present in the dense membrane fractions of cell extracts 2295 FOXF2 Forkhead box F2 6.O 6p25.3 Regulates cell proliferation and Survival, associated with BMP and Wnt signaling
TABLE 3 Genes down-regulated in tumor associated endothelium. Entrez Chromosomal Gene ID Gene Description Fold difference location Function 5350 PLN Phospholamban O.108 6q22.1 Inhibits sarcoplasmic reticulum Ca(2+)- ATPase activity SELE Selectin E, O112 1q22-q25 Cell surface lycoprotein. endothelial Inhibits cell adhesion. Adhesion molecule 1 Early marker of inflammation 96.87 GREB1 GREB1 protein, O-116 2p25.1 Transcription factor, transcript variant a inhibits cell proliferation 4969 OGN Osteoglycin O.147 9q22 Regulates collagen osteoinductive fibrillogenesis Factor, mimecan), transcript variant 3 US 2013/02743 15 A1 Oct. 17, 2013 74
TABLE 3-continued Genes down-regulated in tumor associated endothelium. Entrez Chromosomal Gene ID Gene Description Fold difference location Function 2S890 AB13BP ABI gene family, O.152 3q12 May play a role in cell member 3 (NESH) motility by regulating binding protein NESH function 90.161 HS6ST2 Heparn sulfate 6- O.153 Xq26.2 Plays a role in growth O-sulfotransferase 2 factor signaling, cell adhesion, and enzymatic catalysis. Maybe involved in vascularization by mediating FGF signaling 139221 MUM1L1 Melanoma associated 0.155 Xq22.3 Encodes tumor specific antigen (mutated) 1- antigens ike 1 4129 MAOB Monoamine oxidase B O.156 Xp11.23 Regulates neurotransmitters in central nervous system 9452 ITM2A integral membrane O.156 Xq13.3-Xq21.2 Transmembrane protein. protein 2A Marker of early stage of endochondral Ossification 17O3O2 ARX Aristaless related O.162 Xp22.1-p21.3 Organ development. homeobox Bifunctional transcription factor 10659 CUGBP2 CUG triplet repeat, O.163 10p13 Binds and stabilizes RNA binding Protein COX2 mRNA, inhibits 2, transcript variant 2 its translation 5577 PRKAR2B Protein kinase, O.163 7q22 Encodes a regulatory cAMP-dependent subunit RII beta of regulatory, type II, human cAMP-dependent beta protein kinase A 345557 LCXD3 Phosphatidylinositol- O.16S 5p13.1 Quantitatively specific solubilizes phospholipase C, X AChE from purified domain containing 3 synaptic plasma membranes and intact synaptosomes of Torpedo ocellata electric Organ
0300 Multiple genes encoding proteins involved in extra- sion was increased by 3.5-fold in the tumor endothelium. cellular matrix function, such as collagens, TNFAIP6, EGFR is known to be overexpressed in ovarian carcinomas ADAMTS4, MMP9, MMP11, had increased expression in and is predictive of poor outcome (Berchuck et al., Am. J. tumor vasculature compared with normal ovarian Vascula Obstet. Gynecol. 164: 669-674, 1991). Similarly, non-recep ture. The C. integrin (vitronectin receptor) was elevated 2.5- tor kinases such as focal adhesion kinase (FAK or PTK2: fold in tumor endothelium. Several transcription factors were 3.1-fold increase) and Fyn (4.7 fold increase), which are play upregulated in the ovarian cancer Vasculature. For example, functional roles for tumor cells were detected. Genes that are HEYL was increased 3-fold. In addition, several transcription overexpressed on both tumor cells and tumor-associated factors were identified including E2F transcription factor 3 endothelial cells are targets for anti-vascular therapy due to (E2F3; plays a role in cell proliferation) (Black, Proc Natl the ability to target both the epithelial and stromal compart AcadSci U.S.A. 102: 15948-15953, 2005); runt-related tran mentS. Scription factor 1 (RUNX1; plays a role in angiogenesis) 0302) In addition to the 652 genes that were identified as (Iwatsuki et al., Oncogene 24: 1129-1137, 2005), signal being up-regulated in ovarian tumor endothelial cells, 497 transducer and activator of transcription 2 (STAT2; role in genes were down-regulated 22-fold in ovarian cancer endot cellular proliferation) (Gomez and Reich, J. Immunol. 170: helium samples (Table 1). FIG. 1 illustrates the fold changes 5373-5381, 2003), the SNAIL-related zinc-finger transcrip observed in the relative expression levels between microarray tion factor, SLUG (SNAI2) (Perez-Mancera et al., Oncogene data and real-time quantitative RT-PCR data from the pro 24: 3073-3082, 2005), and Twist1 (Mironchik et al. Cancer angiogenic gene signature provided in Table 1. Of the 497 Res. 65: 10801-10809, 2005). These genes were elevated genes that were down-regulated 22-fold, 17 genes were 2-18 fold in the ovarian cancer vasculature relative to normal decreased at least 6-fold (as provide in Table 3). For example, ovarian endothelial cells. monoamine oxidase B (MAOB), a gene responsible for 0301 Additional genes were identified as being overex detoxification and degradation of monoamines was decreased pressed in ovarian tumor endothelial cells that had previously by 6.4-fold in the tumor endothelial cells (Grimsby et al., Nat. been reported to be overexpressed in tumor cells. For Genet. 17: 206-210, 1997). Decorin, a small multi-functional example, epidermal growth factor receptor (EGFR) expres proteoglycan with anti-angiogenic properties, was decreased US 2013/02743 15 A1 Oct. 17, 2013
by 4.8-fold (Sulochana et al., J. Biol. Chem. 280: 27935 thelium (OSE). The expression profile of microdissected pap 27948, 2005). Several other genes with potential anti-angio illary serous ovarian cancers using the same microarray meth genic or anti-proliferative roles such as Fibulin-5 (FBLN-5) ods has previously been reported (Bonome et al. Cancer Res. and checkpoint suppressor 1 (CHES1) were down-regulated 65: 10602-10612, 2005). The current disclosed list of differ by 4.5-fold and 4.3-fold, respectively (Albig and Schiemann, entially expressed genes in tumor-associated endothelial cells DNA Cell Biol. 23: 367-379, 2004; and Scott and Plon, Gene was compared with the gene list identified for laser microdis 359: 119-126, 2005). These findings indicate that tumor sected tumor-epithelial cells. A total of 534 differentially endothelial cell and non-tumor endothelial cell isolates pos regulated genes were uniquely altered (up- or down-regu sess distinct expression profiles. lated) in the endothelial cells. The 28 genes with the greatest level of increase in ovarian tumor endothelial cells are listed Example 4 in Table 4, of which 23 genes had a >6-fold increase in expression. A complete listing of the 534 differentially regu Identification of Tumor Endothelial Markers lated genes is provided in Table 5. Further, FIG. 2 illustrates protein expression levels detected in ovarian endothelial cells 0303. This example provides specific tumor endothelial following staining of samples with immunofluorescently-la cell markers. beled PTK2, Fyn, MMP-9, 32-arrestin, Jagged 1 and 0304 Differentially regulated genes expressed in tumor PLXDC1, respectively. associated endothelium were identified by comparing tumor 0305 These findings identify tumor endothelial cell spe associated endothelium versus normal endothelium with cific genes that can be used as biomarkers and potential tar tumor-associated epithelial cells versus ovarian Surface epi gets for treatment of ovarian cancer. TABLE 4 Genes specifically regulated in tumor-endothelium Entrez Fold Chromosomal Gene ID Gene Description difference location Function 25975 EGFL6 EGF-like-domain, multiple 6 36.848 Xp22 May regulate cell cycle (EGFL6) and oncogenesis 7130 TNFAIP6 Tumor necrosis factor, alpha- 29.062 2d23.3 Anti-inflammatory and induced protein 6 (TNFAIP6) chondroprotective effect 7291 TWIST1 Twist homolog 1 18.969 7p21.2 inhibits chondrogenesis (acrocephalosyndactyly 3: Saethre-Chotzen syndrome) (Drosophila) (TWIST1) 6781 STC1 Stanniocalcin 1 (STC1) 13.326 8p21-p11.2 Regulates calcium phosphate homeostasis, and cell metabolism 8452S HOP Homeodomain-only protein 13.144 4q11-q12 Transcriptional (HOP), transcript variant 2 repressor. Modulates serum response actor-dependent cardiac-specific gene expression and cardiac development 1462 CSPG2 Chondroitin Sulfate 10.355 5q14.3 Extracellular matrix proteoglycan 2 (versican) component of the (CSPG2) vitreous gel. Anti-cell adhesive. 57125 PLXDC1 Plexin domain containing 1 10.215 17q21.1 Unknown (PLXDC1) 4318 MMP9 Matrix metallopeptidase 9 9.389 20q11.2-q13.1 Breakdown of (gelatinase B, 92 kDa extracellular gelatinase, 92 kDa type IV matrix. Plays a role in collagenase) (MMP9) angiogenesis and tumor invasion 3937 LCP2 Lymphocyte cytosolic protein 8.744 5q33.1-qter Promotes T-cell 2 (SH2 domain containing development and leukocyte protein of 76 kDa) activation (LCP2) 5366 PMAIP1 Phorbol-12-myristate-13- 8.543 18q21.32 Unknown acetate-induced protein 1 (PMAIP1) 8O38 ADAM12 ADAM metallopeptidase 7.605 10q26.3 Involved in cell-cell and domain 12 (meltrin alpha) cell-matrix interactions (ADAM12), transcript variant 1 25878 MXRAS Matrix-remodeling associated 6.865 Xp22.33 Matrix remodeling 5 (MXRA5) 1123 CHN1 Chimerin (chimaerin) 1 6.857 2a31-q32.1 Rho GTPase activating (CHN1) protein 3310 HSPA6 Heat shock 70 kDa protein 6 6.76 1923 Involved in protein conformational interactions US 2013/02743 15 A1 Oct. 17, 2013 76
TABLE 4-continued Genes specifically regulated in tumor-endothelium
Entrez Fold Chromosomal Gene ID Gene Description difference location Function 10631 POSTN Periostin, osteoblast specific 6.732 13q13.3 Promotes integrin factor (POSTN) dependent cell adhesion and motility, involved in extracellular matrix deposition 11211 FZD10 Frizzled homolog 10 6.701 12q24.33 Receptor for the (Drosophila) (FZD10) wingless type MMTV integration site family 27242 TNFRSF21 Tumor necrosis factor 6.649 6p21.1-12.2 Activates NF-kappaB receptor Superfamily, member and MAPK8/JNK, 21 (TNFRSF21) induces cell apoptosis, involved in inflammation and immune regulation. 25891 DKFZP586H2123 Regeneration associated 6.199 11p13 Unknown muscle protease, transcript variant 2 79084 MEPSO WD repeat domain 77 6.144. 1 p13.2 Involved in the methylation and assembly of spliceosomal SnRNAs Sm proteins SOSO9 COLSA3 Collagen, type V, alpha 3 6.118 19p 13.2 Extracellular protein, (COL5A3) associated with ormation of fibrils, and Some connective tissue pathology Such as inflammation, cancer and atherosclerosis 62OS RPS11 Ribosomal protein S11 6.095 19q13.3 nvolved in the (RPS11) recognition of ermination codons SS8O3 CENTA2 Centaurin, alpha 2 (CENTA2) 6.09 17q11.2 A. phosphatidylinositide binding protein present in the dense membrane ractions of cell extracts 9.0161 HS6ST2 Heparan sulfate 6-O- 0.153 Xq26.2 Plays a role in growth sulfotransferase 2 (HS6ST2) actor signaling, cell adhesion, and enzymatic catalysis. Maybe involved in vascularization by mediating FGF signaling 4969 OGN Osteoglycin (osteoinductive 0.147 9q22 Regulates collagen factor, mimecan) (OGN), fibrillogenesis transcript variant 3 96.87 GREB1 GREB1 protein, transcript 0.116 2p25.1 Transcription factor; variant a inhibits cell proliferation 6401 SELE Selectin E (endothelial 0.112 1d22-q25 Cell surface lycoprotein. adhesion molecule 1) (SELE) Inhibits cell adhesion. Early marker of inflammation 5350 PLN Phospholamban 0.108 6q22.1 Inhibits sarcoplasmic reticulum Ca(2+)- ATPase activity
TABLE 5 Genes specifically regulated in tumor endothelium. Fold difference Parametric of geom means Gene p-value (Tumor? Normal) Probe set Description UG cluster symbol Map p < 1e–07 29.062 206026 s at tumor necrosis factor, HS.437322 TNFAIP6 2d23.3 alpha-induced protein 6 (TNFAIP6), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 77
TABLE 5-continued Genes specifically regulated in tumor endothelium. Fold difference Parametric of geom means Gene (Tumor? Normal) Probe set Description OG cluster symbol Map O.274 213803 at Karyopherin (importin) S.S32793 17q21.32 beta 1 235309 at CDNA clone S.526499 MAGE: 4140029 2.85 228204 at proteasome (prosome, S.895.45 PSMB4 macropain) subunit, beta ype, 4 (PSMB4), mRNA. 13.144 211597 s at homeodomain-only S.121443 HOP protein (HOP), transcript variant 2, mRNA. 3.116 213848 at Dual specificity HS.3843 DUSP7 phosphatase 7 3.599 233274 at NCK adaptor protein 1 S.477693 NCK1 O.266 212653 s at EH domain binding S.271667 EHBP1 2p15 protein 1 (EHBP1), mRNA. 4e-007 10.215 214081 at plexin domain containing S.125036 17q21.1 (PLXDC1), mRNA. 4e-007 0.27 213364 S at sorting nexin 1 (SNX1), S.188634 SNX1 15q22.31 ranscript variant 2, mRNA. 4e-007 36.848 219454 at EGF-like-domain, HS12844 EGFL6 Xp22 multiple 6 (EGFL6), mRNA. 4e-007 18.969 213943 at wist homolog 1 HS.66744 TWIST1 7p21.2 (acrocephalosyndactyly 3: Saethre-Chotzen syndrome) (Drosophila) (TWIST1), mRNA. 4e-007 0.327 225123 at Sestrin 3 HS12O633 SESN3 11q21 Se-OO7 O.16S 230081 at phosphatidylinositol HS1454.04 PLCXD3 5p13.1 specific phospholipase C, X domain containing 3 (PLCXD3), mRNA. 5.249 228579 at Potassium voltage-gated HS.374023 channel, KQT-like subfamily, member 3 8e-007 6.199 213661 at regeneration associated HSSSO44 11 p.13 muscle protease (DKFZP586H2123), transcript variant 2, mRNA. 6.701 219764 at frizzled homolog 10 S.31664 12q24.33 (Drosophila) (FZD10), mRNA. 3.38 212044 S at ribosomal protein L27a S.523463 11 p.15 (RPL27A), mRNA. 213574 s at Karyopherin (importin) S.S32793 17q21.32 beta 1 7.684 219700 at plexin domain containing S.125036 17q21.1 1 (PLXDC1), mRNA. .2e-006 O.294 222791 at round spermatid basic S.486.285 RSBN1 1p13.2 protein 1 (RSBN1), mRNA. .2e-006 O.232 221589 s at Aldehyde dehydrogenase S.293970 14q24.3 6 family, member A1 .2e-006 7.724. 204285 s at phorbol-12-myristate-13 HS.96 PMAIP1 18q21.32 acetate-induced protein 1 (PMAIP1), mRNA. 4e-006 10.546 204595 S at stanniocalcin 1 (STC1), S.25590 STC1 8p21-p11.2 mRNA. Se-OO6 3.695 225147 at pleckstrin homology, S.487479 PSCD3 7p22.1 Sect and coiled-coil domains 3 (PSCD3), mRNA. Se-OO6 9.368 234723 x at CDNA: FLJ21228 fis, S.30671 6 7 clone COLOO739 Se-OO6 O.374 212498 at Membrane-associated S.432862 MARCH 5p15.2 ring finger (C3HC4) 6 VI Se-OO6 13.326 230746 s at stanniocalcin 1 (STC1), S.25590 STC1 mRNA. US 2013/02743 15 A1 Oct. 17, 2013 78
TABLE 5-continued Genes specifically regulated in tumor endothelium. Fold difference Parametric of geom means Gene p-value (Tumor? Normal) Probe set Description UG cluster symbol 6.09 21.9358 s at centaurin, alpha 2 HS.514063 CENTA2 17q11.2 (CENTA2), mRNA. 18e-006 O346 201425 at aldehyde dehydrogenase HS.436437 ALDH2 12q24.2 2 family (mitochondrial) (ALDH2), nuclear gene encoding mitochondrial protein, mRNA. 4.094 2255.05 s at chromosome 20 open HS.29341 20p13 reading frame 81 (C20orf31), mRNA. 2.1e-006 O.284 21993.9 s at cold shock domain HS.6985S CSDE1 containing E1, RNA binding (CSDE1), transcript variant 2, mRNA. 4.136 22105.9 s at coactosin-like 1 S.289092 COTL1 16q24.1 (Dictyostelium) (COTL1), mRNA. 6.649 21.8856 at umor necrosis factor S.443577 TNFRSF21 6p21.1-12.2 receptor Superfamily, member 21 (TNFRSF21), mRNA. 2.4e-006 3.826 205068 s at Rho GTPase activating S.293593 ARHGAP26 protein 26 (ARHGAP26), mRNA. 2.4e-006 4.807 242100 at chondroitin sulfate S.213137 CSS3 synthase 3 (CSS3), mRNA. 2.4e-006 S.981 206377 at orkhead box F2 S.484423 FOXF2 6p25.3 (FOXF2), mRNA. 2.4e-006 0.327 234512 X at PREDICTED: Similar to S.S35174 LOC4421.59 Rpl7a protein (LOC442159), mRNA. 4.037 232304 at Pellino homolog 1 HS7886 PELI1 2p13.3 (Drosophila) 5.391 242546 at LOC44O156 14q11.1 3.362 227295 at KK interacting protein IKIP 12q23.1 (IKIP), transcript variant 3.1, mRNA. 2.8e-006 4.563 1553575 at Unknown 2.9e-OO6 3.761 214924 S at OGT(O-Glc-NAc S.S35711 OIP106 3p25.3-p24.1 ransferase)-interacting protein 106 KDa (OIP106), mRNA. 6.144 242398 x at WD repeat domain 77 S.204773 MEP50 1p13.2 8.543 204286 s at phorbol-12-myristate-13 HS.96 PMAIP1 18q21.32 acetate-induced protein 1 (PMAIP1), mRNA. 3.271 241632 x at ESTs O.258 209512 at hydroxysteroid S594.86 HSDL2 dehydrogenase like 2 (HSDL2), mRNA. 4e-006 4.384 219359 at hypothetical protein S.353181 11 p15.5 FLJ22635 (FLT22635), mRNA. 4e-006 O.279 227719 at CDNA FLJ37828 fis, S.123119 13 clone BRSSN2OO6575 4e-006 3.268 226063 at vav 2 oncogene (VAV2), S.36992.1 WAV2 9q34.1 mRNA. 4e-006 8.744 205269 at lymphocyte cytosolic LCP2 protein 2 (SH2 domain containing leukocyte protein of 76 kDa) (LCP2), mRNA. 4.1e-006 3.906 215599 at SMA4 HS.482411 SMA4 5q13 4.2e-006 O434 214527 s at polyglutamine binding protein 1 PQBP1 Xp11.23 (PQBP1), transcript variant 5, mRNA. 4.068 220817 at transient receptor HS.262960 TRPC4 13q13.1-q13.2 potential cation channel, subfamily C, member 4 (TRPC4), mRNA. US 2013/02743 15 A1 Oct. 17, 2013 79
TABLE 5-continued Genes specifically regulated in tumor endothelium. Fold difference Parametric of geom means Gene p-value (Tumor? Normal) Probe set Description OG cluster symbol Map 4.6e-006 0.237 202920 at ankyrin 2, neuronal S.137367 ANK2 (ANK2), transcript variant 2, mRNA. 4.8e-006 6.86S 209596 at matrix-remodelling S.369422 MXRAS Xp22.33 associated 5 (MXRA5), mRNA. O.324 201737 s at membrane-associated S.432862 38417 5p15.2 ring finger (C3HC4) 6 (MARCH6), mRNA. O.256 224763 at ribosomal protein L37 S.805.45 RPL37 (RPL37), mRNA. 6.857 212624 S at chimerin (chimaerin) 1 S.38O138 CHN1 (CHN1), transcript variant 2, mRNA. 5.8e-006 222486 s at ADAM metallopeptidase S.5341.15 ADAMTS1 21q21.2 with thrombospondin type 1 motif, 1 (ADAMTS1), mRNA. O.294 202908 at Wolfram syndrome 1 S.5186O2 (wolframin) (WFS1), mRNA. O.267 242051 at Transcribed locus S.130260 X O.376 205412 at acetyl-Coenzyme A S.232.375 ACAT1 11q22.3-q23.1 acetyltransferase 1 (acetoacetyl Coenzyme A thiolase) (ACAT1), nuclear gene encoding mitochondrial protein, mRNA. 7.988 224.549 X at O.253 213272 s at promethin (LOC57146), S.258212 LOCS7146 16p12 mRNA. 4.12 242369 x at Nuclear receptor S.446678 NCOA2 8q13.3 coactivator 2 O.26 226625 at Transforming growth S.482390 1p33-p32 factor, beta receptor III (betaglycan, 300 kDa) O.245 21951.1 s at Synuclein, alpha S.426463 SNCAIP interacting protein (synphilin) (SNCAIP), mRNA. 7.8e-006 4.059 222449 at transmembrane, prostate S.517155 TMEPAI 20d 13.31-q13.33 androgen induced RNA (TMEPAI), transcript variant 3, mRNA. 8e-006 2.237 212351 at eukaryotic translation initiation factor 2B, subunit 5 epsilon, 82 kDa (EIF2B5), mRNA. 3.836 219634 at carbohydrate (chondroitin S.17569 CHST11 4) Sulfotransferase 11 (CHST11), mRNA. O418 226751 at chromosome 2 open S.212885 2p14 reading frame 32 (C2orf32), mRNA. 5.037 209081 s at collagen, type XVIII, S.S17356 COL18A1 21q22.3 alpha 1 (COL18A1), transcript variant 2, mRNA. 2.362 205812 s at transmembrane emp24 S.279929 TMED9 protein transport domain containing 9 (TMED9), mRNA. 2.862 234985 at Hypothetical protein S.20586S LOC143458 11 p.13 LOC143458 3.652 242029 at Fibronectin type III S.15943O 3q26.31 domain containing 3B O.386 211666 x at ribosomal protein L3 S.119598 RPL3 22d 13 (RPL3), mRNA.