US 20090297536A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0297536A1 Chin et al. (43) Pub. Date: Dec. 3, 2009

(54) COMPOSITIONS, KITS, AND METHODS FOR Related U.S. Application Data IDENTIFICATION, ASSESSMENT, (60) Provisional application No. 60/575,795, filed on May PREVENTION AND THERAPY OF CANCER 28, 2004, provisional application No. 60/580,337, filed on Jun. 15, 2004. (75) Inventors: Lynda Chin, Brookline, MA (US); Publication Classification Cameron W. Brennan, New York, NY (US); Ronald A. DePinho, (51) Int. Cl. Brookline, MA (US); Andrew J. A 6LX 39/395 (2006.01) Aguirre, Boston, MA (US) CI2O I/68 (2006.01) C40B 30/00 (2006.01) AOIK 67/00 (2006.01) Correspondence Address: A 6LX 3L/7052 (2006.01) FOLEY HOAG, LLP A638/02 (2006.01) PATENT GROUP, WORLD TRADE CENTER A638/16 (2006.01) WEST C07K I4/00 (2006.01) 155 SEAPORT BLVD C7H 2L/00 (2006.01) BOSTON, MA 02110 (US) C07K 6/00 (2006.01) A6IP35/00 (2006.01) (73) Assignee: Dana-Farber Cancer Institute, (52) U.S. Cl...... 424/172.1; 435/6:506/7; 800/10: Inc., Boston, MA (US) 424/183.1; 424/178.1: 514/44 A: 514/2: 514/12: 530/350:536/23.1; 530/389.1 (21) Appl. No.: 11/597,825 (57) ABSTRACT The invention relates to compositions, kits, and methods for (22) PCT Filed: May 27, 2005 detecting, characterizing, preventing, and treating human cancer. A variety of chromosomal regions (MCRs) and mark (86). PCT No.: PCT/US05/18850 ers corresponding thereto, are provided, wherein alterations in the copy number of one or more of the MCRs and/or S371 (c)(1), alterations in the amount, structure, and/or activity of one or (2), (4) Date: Jun. 16, 2009 more of the markers is correlated with the presence of cancer. Patent Application Publication Dec. 3, 2009 Sheet 1 of 6 US 2009/0297536A1

Patent Application Publication Dec. 3, 2009 Sheet 2 of 6 US 2009/0297536A1

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Patent Application Publication Dec. 3, 2009 Sheet 3 of 6 US 2009/0297536A1

Patent Application Publication Dec. 3, 2009 Sheet 4 of 6 US 2009/0297536A1

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Patent Application Publication Dec. 3, 2009 Sheet 6 of 6 US 2009/0297536A1

US 2009/0297536A1 Dec. 3, 2009

COMPOSITIONS, KITS, AND METHODS FOR the majority of amplified and deleted loci and resident , IDENTIFICATION, ASSESSMENT, the presumed cancer-relevant targets remain to be discovered. PREVENTION AND THERAPY OF CANCER SUMMARY OF THE INVENTION CROSS REFERENCE TO RELATED 0005. The present invention is based, at least in part, on the APPLICATIONS identification of specific regions of the genome (referred to 0001. This application claims priority to U.S. Provisional herein as minimal common regions (MCRS)), of recurrent Application No. 60/575,795, which was filed on May 28, copy number change which are contained within certain chro 2004 and U.S. Provisional Application No. 60/580,337, mosomal regions (loci) and are associated with cancer. These which was filed on Jun. 15, 2004, both of which are hereby MCRs were identified using a novel cDNA or oligomer-based incorporated by refemce in there entirety. platform and bioinformatics tools which allowed for the high resolution characterization of copy-number alterations in the pancreatic adenocarcinoma genome (see Example 1). The GOVERNMENT FUNDING present invention is based, also in part, on the identification of markers residing within the MCRs of the invention, which are 0002 Work described herein was supported, at least in also associated with cancer. part, by National Institutes of Health (NIH) under grant 0006. Accordingly, in one aspect, the present invention RO1CA99041, RO1CA86379, RO1CA84628, and T32 provides methods of assessing whether a subject is afflicted CA09382. The government may therefore have certain rights with cancer or at risk for developing cancer, comprising com to this invention. paring the copy number of an MCR in a subject sample to the normal copy number of the MCR, wherein the MCR is BACKGROUND OF THE INVENTION selected from the group consisting of the MCRs listed in Table 1, and wherein an altered copy number of the MCR in 0003 Cancer represents the phenotypic end-point of mul the sample indicates that the subject is afflicted with cancer or tiple genetic lesions that endow cells with a full range of at risk for developing cancer. In one embodiment, the copy biological properties required for tumorigenesis. Indeed, a number is assessed by fluorescent in situ hybridization hallmark genomic feature of many cancers, including, for (FISH). In another embodiment, the copy number is assessed example, pancreatic cancer, breast cancer, ovarian cancer, by quantitative PCR (qPCR). Instillanother embodiment, the and colon cancer, is the presence of numerous complex chro normal copy number is obtained from a control sample. In yet mosome structural aberrations—including non-reciprocal another embodiment, the sample is selected from the group translocations, amplifications and deletions. consisting of tissue, whole blood, serum, plasma, buccal 0004 Karyotype analyses (Johansson, B., et al. (1992) scrape, saliva, cerebrospinal fluid, urine, stool, bile, pancre Cancer 69, 1674-81; Bardi, G., et al. (1993) BrJ Cancer 67, atic juice, and pancreatic tissue. 1106-12; Griffin, C. A., et al. (1994) Genes 0007. In another aspect, the invention provides methods of Cancer 9, 93-100: Griffin, C.A., et al. (1995) Cancer Res 55, assessing whether a Subject is afflicted with cancer or at risk 2394-9; Gorunova, L., et al. (1995) Genes Chromosomes for developing cancer comprising comparing the amount, Cancer 14, 259-66; Gorunova, L., et al. (1998) Genes Chro structure, and/or activity of a marker in a subject sample, mosomes Cancer 23, 81-99), chromosomal CGH and array wherein the marker is a marker which resides in an MCR CGH (Wolf Metal. (2004) Neoplasia 6 (3)240; Kimura Y, et listed in Table 1, and the normal amount, structure, and/or al. (2004) Mod. Pathol. 21 May (epub): Pinkel, et al. (1998) activity of the marker, wherein a significant difference Nature Genetics 20:211; Solinas-Toldo, S., et al. (1996) Can between the amount, structure, and/or activity of the marker cer Res 56, 3803-7: Mahlamaki, E. H., et al. (1997) Genes in the sample and the normal amount, structure, and/or activ Chromosomes Cancer 20, 383-91; Mahlamaki, E. H., et al. ity is an indication that the subject is afflicted with cancer or (2002) Genes Chromosomes Cancer 35, 353-8: Fukushige, at risk for developing cancer. In one embodiment, the marker S., et al. (1997) Genes Chromosomes Cancer 19:161-9; Cur is selected from the group consisting of the markers listed in tis, L.J., et al. (1998) Genomics 53,42-55; Ghadimi, B.M., et Table 4 or Table 5. In another embodiment, the amount of the al. (1999) Am J Pathol 154, 525-36; Armengol, G., et al. marker is determined by determining the level of expression (2000) Cancer Genet Cytogenet 116, 133-41), fluorescence of the marker. In yet another embodiment, the level of expres in situ hybridization (FISH) analysis (Nilsson Metal. (2004) sion of the marker in the sample is assessed by detecting the Int J Cancer 109(3):363-9: Kawasaki Ket al. (2003) Int J presence in the sample of a corresponding to the Mol. Med 12(5):727-31) and loss of heterozygosity (LOH) marker. The presence of the protein may be detected using a mapping (Wang Z C et al. (2004) Cancer Res 64(1):64-71; reagent which specifically binds with the protein. In one Seymour, A. B., et al. (1994) Cancer Res 54,2761-4; Hahn, S. embodiment, the reagent is selected from the group consist A., et al. (1995) Cancer Res 55, 4670-5; Kimura, M., et al. ing of an antibody, an antibody derivative, and an antibody (1996) Genes Chromosomes Cancer 17, 88-93) have identi fragment. In another embodiment, the level of expression of fied recurrent regions of copy number change orallelic loss in the marker in the sample is assessed by detecting the presence various cancers. For example, in pancreatic cancer, frequent in the sample of a transcribed polynucleotide or portion gains have been mapped to 3q, 5p. 7p. 8q, 11q, 12p. 17q and thereof, wherein the transcribed polynucleotide comprises 20d and losses to 3p, 4q, 6q 8p, 9p, 10q, 12q, 13q, 17p. 18q. the marker. In one embodiment, the transcribed polynucle and 21q and 22d. In some instances, validated oncogenes and otide is an mRNA or cDNA. The level of expression of the tumor Suppressor genes residing within these loci have been marker in the sample may also be assessed by detecting the identified, including MYC (8q24), p'Y' (9p21), p53 presence in the sample of a transcribed polynucleotide which (17p13), SMAD4 (18q21) and AKT2 (19q13). However, for anneals with the marker or anneals with a portion of a poly US 2009/0297536A1 Dec. 3, 2009 nucleotide wherein the polynucleotide comprises the marker, indication that the test compound is efficacious for inhibiting under stringent hybridization conditions. cancer and wherein a significantly lower amount and/or activ 0008. In another embodiment, the amount of the marker is ity of a marker in the first sample which is amplified in cancer, determined by determining copy number of the marker. The relative to the second sample, is an indication that the therapy copy number of the MCRs or markers may be assessed by is efficacious for inhibiting cancer in the Subject. In one comparative genomic hybridization (CGH), e.g., array CGH. embodiment, the marker is selected from the group consisting In still another embodiment, the normal amount, structure, of the markers listed in Table 4 or Table 5. and/or activity is obtained from a control sample. In yet 0012 Another aspect of the invention provides methods of another embodiment, the sample is selected from the group selecting a composition capable of modulating cancer com consisting of tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, bile, pancre prising obtaining a sample comprising cancer cells; contact atic juice, and pancreatic tissue. ing said cells with a test compound; and determining the 0009. In another aspect, the invention provides methods ability of the test compound to modulate the amount and/or for monitoring the progression of cancer in a subject com activity of a marker, wherein the marker is a marker which prising a) detecting in a Subject sample at a first point in time, resides in an MCR listed in Table 1, thereby identifying a the amount and/or activity of a marker, wherein the marker is modulator of cancer. In one embodiment, the marker is a marker which resides in an MCR listed in Table 1; b) selected from the group consisting of the markers listed in repeating step a) at a Subsequent point in time; and c) com Table 4 or Table 5. The cells may be isolated from, e.g., an paring the amount and/or activity detected in steps a) and b). animal model of cancer, a cancer cell line, e.g., a pancreatic and therefrom monitoring the progression of cancer in the cancer cell line originating from a pancreatic tumor, or from subject. In one embodiment, the marker is selected from the a Subject Suffering from cancer. group consisting of the markers listed in Table 4 or Table 5. In 0013 Yet another aspect of the invention provides meth another embodiment, the sample is selected from the group ods of selecting a composition capable of modulating cancer consisting of tissue, whole blood, serum, plasma, buccal comprising contacting a marker with a test compound; and scrape, saliva, cerebrospinal fluid, urine, stool, bile, pancre determining the ability of the test compound to modulate the atic juice, and pancreatic tissue. In still another embodiment, amount and/or activity of a marker, wherein the marker is a the sample comprises cells obtained from the Subject. In yet marker which resides in an MCR listed in Table 1, thereby another embodiment, between the first point in time and the identifying a composition capable of modulating cancer. In Subsequent point in time, the Subject has undergone treatment one embodiment, the marker is selected from the group con for cancer, has completed treatment for cancer, and/or is in sisting of the markers listed in Table 4 or Table 5. In another remission. embodiment, the method further comprises administering the 0010. In still another aspect, the invention provides meth test compound to an animal model of cancer. In still another ods of assessing the efficacy of a test compound for inhibiting embodiment, the modulator inhibits the amount and/or activ cancer in a Subject comprising comparing the amount and/or ity of a or protein corresponding to a marker set forth in activity of a marker in a first sample obtained from the subject Table 1 which is amplified, e.g., a marker selected from the and maintained in the presence of the test compound, wherein markers listed in Table 5. In yet another embodiment, the the marker is a marker which resides in an MCR listed in modulator increases the amount and/or activity of a gene or Table 1, and the amount and/or activity of the marker in a protein corresponding to a marker set forth in Table 1 which second sample obtained from the Subject and maintained in is deleted, e.g., a marker selected from the markers listed in the absence of the test compound, wherein a significantly Table 4. higher amount and/or activity of a marker in the first sample 0014. In another aspect, the invention provides kits for which is deleted in cancer, relative to the second sample, is an assessing the ability of a compound to inhibit cancer com indication that the test compound is efficacious for inhibiting prising a reagent for assessing the amount, structure, and/or cancer, and wherein a significantly lower amount and/or activity of a marker, wherein the marker is a marker which activity of the marker in the first sample which is amplified in resides in an MCR listed in Table 1. In one embodiment, the cancer, relative to the second sample, is an indication that the marker selected from the group consisting of the markers test compound is efficacious for inhibiting cancer in the Sub listed in Table 4 or Table 5. ject. In one embodiment, the first and second samples are 0015 The invention also provides kits for assessing portions of a single sample obtained from the Subject. In whether a subject is afflicted with cancer comprising a another embodiment, the first and second samples are por reagent for assessing the copy number of an MCR selected tions of pooled samples obtained from the Subject. In one from the group consisting of the MCRs listed in Table 1, as embodiment, the marker is selected from the group consisting well as kits for assessing whether a subject is afflicted with of the markers listed in Table 4 or Table 5. cancer, the kit comprising a reagent for assessing the amount, 0011. In yet another aspect, the invention provides meth structure, and/or activity of a marker. In one embodiment, the ods of assessing the efficacy of a therapy for inhibiting cancer marker selected from the group consisting of the markers in a subject comprising comparing the amount and/or activity listed in Table 4 or Table 5. of a marker in the first sample obtained from the subject prior 0016. In another aspect, the invention provides kits for to providing at least a portion of the therapy to the Subject, assessing the presence of human cancer cells comprising an wherein the marker is a marker which resides in an MCR antibody or fragment thereof, wherein the antibody or frag listed in Table 1, and the amount and/or activity of the marker ment thereof specifically binds with a protein corresponding in a second sample obtained from the Subject following pro to a marker, wherein the marker is a marker which resides in vision of the portion of the therapy, wherein a significantly an MCR listed in Table 1. In one embodiment, the marker higher amount and/or activity of a marker in the first sample selected from the group consisting of the markers listed in which is deleted in cancer, relative to the second sample, is an Table 4 or Table 5. US 2009/0297536A1 Dec. 3, 2009

0017. In still another aspect, the invention provides kits for bind to a protein corresponding to a marker selected from the assessing the presence of cancer cells comprising a nucleic markers listed in Table 4 or Table 5. acid probe wherein the probe specifically binds with a tran 0025. In yet another aspect, the invention provides an iso scribed polynucleotide corresponding to a marker, wherein lated nucleic acid molecule, or fragment thereof, contained the marker is a marker which resides in an MCR listed in within an MCR selected from the MCRs listed in Table 1, Table 1. In one embodiment, the marker selected from the wherein said nucleic acid molecule has an altered amount, group consisting of the markers listed in Table 4 or Table 5. structure, and/or activity in cancer. The invention also pro 0018. In yet another aspect, the invention provides meth vides an isolated polypeptide encoded by the nucleic acid ods of treating a subject afflicted with cancer comprising molecules. administering to the Subject a modulator of the amount and/or activity of a gene or protein corresponding to a marker, BRIEF DESCRIPTION OF THE DRAWINGS wherein the marker is a marker which resides in an MCR 0026 FIGS. 1A-1C depict the genomic profiles from pan listed in Table 1. In one embodiment, the marker selected creatic adenocarcinoma samples. Array-CGH profiles with from the group consisting of the markers listed in Table 4 or x-axis coordinates representing cDNA probes ordered by Table 5. genomic map positions. Segmented data is displayed in red, 0019. The invention also provides methods of treating a median filtered (3 nearest neighbors) in blue and raw data in Subject afflicted with cancer comprising administering to the black. (1A) Whole-genome profiles of primary tumor speci Subject a compound which inhibits the amount and/or activity men PA.T.7692 (top) and cell line Panc 10.05 (bottom). Note of a gene or protein corresponding to a marker which resides the presence of focal high-level amplifications and deletions in an MCR listed in Table 1 which is amplified in cancer, e.g., as well as large regional gains and losses in both samples. a marker selected from the markers listed in Table 5, thereby (1B) Recurrence of chromosomal alterations. Top: Integer treating a Subject afflicted with cancer. In one embodiment, value recurrence of CNAs in segmented data (Y-axis) plotted the compound is administered in a pharmaceutically accept for each cDNA probe evenly aligned along the x-axis in able formulation. In another embodiment, the compound is an genome order. Dark red or green bars denote gain or loss of antibody or an antigen binding fragment thereof, which spe material. Bright red or green bars represent cifically binds to a protein corresponding to the marker. For probes within regions of amplification or deletion. Bottom: example, the antibody may be conjugated to a toxin or a TreeView showing discrete CNAs within all samples. Red chemotherapeutic agent. In still another embodiment, the represents chromosomal gain and green denotes chromo compound is an RNA interfering agent, e.g., an siRNA mol somal loss. (1C) CGH profiles of 12p12.3-q13.3 (Locus ecule oran shRNA molecule, which inhibits expression of a #15 of Table 1) in three samples illustrating the definition of gene corresponding to the marker. In yet another embodi the physical extent, peak profile and MCRs for that locus. ment, the compound is an antisense oligonucleotide comple Note that the left MCR is defined by the overlap between mentary to a gene corresponding to the marker. In still another samples on top and bottom, while the right MCR is defined by embodiment, the compound is a peptide or peptidomimetic, a the overlap between the two samples on top. Since data points Small molecule which inhibits activity of the marker, e.g., a are plotted on the X-axis by genomic map positions, gaps in small molecule which inhibits a protein-protein interaction the profiles encompass regions of copy number transition for between a marker and a target protein, or an aptamer which which there is no data point. inhibits expression or activity of the marker. (0027 FIGS. 2A-2B depict that QPCR verifies complexity 0020. In another aspect, the invention provides methods of within CNAs. (2A) CGH profiles (Left panel) treating a Subject afflicted with cancer comprising adminis showing amplification of a discrete region of 7q22 in both the tering to the Subject a compound which increases expression AsPC-1 cell line and PA.T. 14172 (Locus # 9, Table 1), with or activity of a gene or protein corresponding to a marker MCR defined by both samples (outlined by dashed lines). which resides in an MCR listed in Table 1 which is deleted in Letters A-D indicate the relative positions of QPCR assays cancer, e.g., a marker selected from the markers listed in Table (Right panel), which confirm the gene copy alterations in 4, thereby treating a subject afflicted with cancer. In one AsPC-1 (dark gray bars) and PA.T. 14172 (light gray bars). embodiment, the compound is a Small molecule. (2B) Chromosome 9 array-CGH profile (Left panel) for a 0021. The invention also includes methods of treating a complex CNA in the HUP-T3 cell line. Homozygous deletion Subject afflicted with cancer comprising administering to the of the known target p16' is confirmed by QPCR (Right Subject a protein corresponding to a marker, e.g., a marker panel), which also verifies the existence of two discrete, focal selected from the markers listed in Table 4, thereby treating a amplicons and a narrow region of one-copy loss revealed by subject afflicted with cancer. In one embodiment, the protein array-CGH. Note that CNAs covered by only one or two is provided to the cells of the Subject, by a vector comprising probes are not identified by the segmentation algorithm. a polynucleotide encoding the protein. In still another (0028 FIGS. 3A-3C depict that combined array-CGH and embodiment, the compound is administered in a pharmaceu expression analysis facilitates identification of candidate tically acceptable formulation. genes. (3A) Analysis of 17q23.2-25.3 locus (Locus #21, 0022. The present invention also provides isolated pro Table 1) in cell line Hup T3. Top panel: array-CGH profile of teins, or fragments thereof, corresponding to a marker HUP-T3. Bottom panel: Expression profile of genes on selected from the markers listed in Table 4 or Table 5. Affymetrix U133A array within the specified locus for the 0023. In another aspect, the invention provides isolated HUP-T3 cell line. The subset of genes exhibiting prominent nucleic acid molecules, or fragments thereof, corresponding gene-dosage correlated expression fall within the peak of the to a marker selected from the markers listed in Table 4 or locus (arrows). (3B) Analysis of 9p24.3-21.2 locus (Locus Table 5. #41, Table 1) in the cell line BXPC-3. Top panel: array-CGH 0024. In still another aspect, the invention provides iso profile of 9p region. Bottom panel: AffymetrixTM expression lated antibodies, or fragments thereof, which specifically profile of genes mapping to the same region. Note the dra US 2009/0297536A1 Dec. 3, 2009 matically reduced expression of the p16' gene (arrows) defining discrete locus boundaries. Longer regions were not within the MCR. (3C) Correlation of p16'Y' expression and discarded, but were not included in defining locus bound copy number in 24 cell lines was analyzed. Note the bimodal aries; 4) informative to spans were compared across Samples distribution of both expression values and copy number val to identify overlapping groups of positive-value or negative ues for this gene across all samples (green lines). The box value segments; each group defines a locus; and 5) MCRS outlined in dots defines those samples (BXPC-3, MiaPaca, were defined as contiguous spans having at least 75% of the Capan 1, Hup-T3 and Dan-G) in which p16' is homozy peak recurrence as calculated by counting the occurrence of gously deleted and not expressed. The box outlined in solid highly altered segments. If two MCRs were separated by a lines encloses samples (Panc-1, Panc 03.27, SW 1990, Panc 08.13, Hup-T4 and Panc 02.13) in which p16' is present gap of only one probe position, they were joined. If there were but with absent or reduced expression. more than 3 MCRs in a locus, the whole region was reported 0029 FIG. 4 depicts a histogram of segmented profiles as a single complex MCR. showing the peak at a Log ratio of 0 as a result of mode 0036) A locus-identification algorithm was used that centering. Outer lines mark 3% (del) and 97% (amp) quan defines informative CNAS on the basis of size and achieve tiles; inner lines mark+/-4 standard deviation of middle 50% ment of a high significance threshold for the amplitude of of data. change. Overlapping CNAs from multiple profiles were then 0030 FIG. 5 depicts a comparison of array-CGH profiles merged in an automated fashion to define a discrete "locus of of pancreatic adenocarcinoma primary tumors and cell lines. regional copy number change, the bounds of which represent A pseudo-karyotype presentation of an integer-value recur the combined physical extend to these overlapping CNAs rence plot by chromosome is shown here for both primary (FIG.1C). Each locus was characterized by a peak profile, the tumors (PT) and cell lines (CL). Gain is shown on the right of width and amplitude of which reflect the contour of the most the chromosome and loss on the left of the chromosome. prominent amplification or deletion for that locus. Further Important regions of similarity between PT and CL are high more, within each locus, one or more minimal common lighted with boxes and chromosomes with prominent dis regions (MCRs) were identified across multiple tumor crepancies between PT and CL are circled. samples (FIG. 1C), with each MCR potentially harboring a 0031 FIG. 6 depicts a whole-genome correlation of copy distinct cancer-relevant gene targeted for copy number alter number and expression in the cell line Capan-1. Median fil ation across the sample set. tered (width=31 probes) array-CGH and expression data are 0037. The locus-identification algorithm defined discrete indicated in light blue and gold, respectively. Note that fluc MCRs within the dataset which were annotated in terms of tuations in average gene expression correlate with changes in recurrence, amplitude of change and representation in both chromosome copy number (R=0.66). cell lines and primary tumors. These discrete MCRs were prioritized based on four criteria that emphasize recurrent DETAILED DESCRIPTION OF THE INVENTION high-threshold changes in both primary tumors and cell lines 0032. The present invention is based, at least in part, on the (see Example 1). Implementation of this prioritization identification of specific regions of the genome (referred to scheme yielded 64 MCRs of the present invention within 54 herein as minimal common regions (MCRS)), of recurrent independent loci, that satisfied at least three of the four crite copy number change which are contained within certain chro ria (see Table 1). mosomal regions (loci) and are associated with cancer. These 0038. The confidence-level ascribed to these prioritized MCRs were identified using a novel clNA or oligomer-based loci was further validated by real-time quantitative PCR platform and bioinformatics tools which allowed for the high (QPCR), which demonstrated 100% concordance with 16 resolution characterization of copy-number alterations in the selected MCRs defined by array-CGH. When the MCRs in pancreatic adenocarcinoma genome (see Example 1). Table 1 were combined with an additional 81 MCRs (within 0033. To arrive at the identified loci and MCRs, array 66 distinct loci) satisfying 2 out of 4 criteria, this genomic comparative genomic hybridization (array-CGH) was ulti characterization has produced a set of 145 MCRs within 121 lized to define copy number aberrations (CNAs) (gains and independent loci (Table 3). losses of chromosomal regions) in pancreatic adenocarci 0039. The MCRs identified herein possess a median size noma cell lines and tumor specimens. of 2.7 Mb, with 21 (33%) MCRs spanning 1 Mb or less 0034 Segmentation analysis of the raw profiles to filter (median of 0.33 Mb) and possess an average of 15 annotated noise from the dataset (as described by Olshen and Venkatra genes. Table 1 lists the cytogenetic bands for each of the 54 man, Olshen, A. B., and Venkatraman, E. S. (2002) ASA independent loci as well as the locus boundary (Mb) and locus Proceedings of the Joint Statistical Meetings 2530-2535: peak profile. The positions of each of the identified MCRs are Ginzinger, D. G. (2002) Exp Hematol 30, 503-12: Golub, T. also listed in Table 1, as well as the size and recurrence for R., et al. (1999) Science 286,531-7: Hyman, E., et al. (2002) each. For example, locus #3 represents a chromosomal region Cancer Res 62, 6240-5; Lucito, R., et al. (2003) Genome Res of 5q31.1-q21.1 and has a locus boundary of 133.51-134.33. 13, 2291-305) was performed and used to identify statisti This locus contains an MCR at position 133.53-133.56. cally significant changepoints in the data. 0040 Also in Table 1, the loci and MCRs are indicated as 0035) Identification of loci was based on an automated having either “gain and amplification” or “loss and deletion.” computer algorithm that utilized several basic criteria as fol indicating that each locus and MCR has either (1) increased lows: 1) segments above or below certain percentiles were copy number and/or expression or (2) decreased copy number identified as altered; 2) if two or more altered segments were and/or expression, or deletion, in cancer. Furthermore, genes adjacent in a single profile separated by less than 500KB, the known to play important roles in the pathogenesis of pancre entire region spanned by the segments was considered to be atic adenocarcinoma (the p16'' and TP53 tumor suppres an altered span; 3) highly altered segments or spans that were sors and the MYC, KRAS2 and AKT2 oncogenes) are present shorter than 20 MB were retained as “informative spans' for within the loci and are also set forth in Table 1. US 2009/0297536A1 Dec. 3, 2009

0041 Complementary expression profile analysis of a sig object of the article. By way of example, “an element’ means nificant fraction of the genes residing within the MCRs of the one element or more than one element. present invention provided a subset of markers with statisti cally significant association between gene dosage and mRNA 0050. The terms “tumor' or “cancer” refer to the presence expression. Table 4 lists the markers of the invention which of cells possessing characteristics typical of cancer-causing reside in MCRs of deletion and which consequently display cells, such as uncontrolled proliferation, immortality, meta decreased expression by comparison across pancreatic cancer static potential, rapid growth and proliferation rate, and cer cell lines. Table 5 lists the markers of the invention which tain characteristic morphological features. Cancer cells are reside in MCRs of amplification that are overexpressed by often in the form of a tumor, but such cells may exist alone comparison, across pancreatic cancer cell lines. Additional within an animal, or may be a non-tumorigenic cancer cell, markers within the MCRs that have not yet been annotated such as a leukemia cell. As used herein, the term "cancer' may also be used as markers for cancer as described herein, includes premalignant as well as malignant cancers. Cancers and are included in the invention. include, but are not limited to, pancreatic cancer, e.g., pan 0042. The novel methods for identifying chromosomal regions of altered copy number, as described herein, may be creatic adenocarcinoma, melanomas, breast cancer, lung can applied to various data sets for various diseases, including, cer, bronchus cancer, colorectal cancer, prostate cancer, pan but not limited to, cancer. Other methods may be used to creas cancer, Stomach cancer, ovarian cancer, urinary bladder determine copy number aberrations as are known in the art, cancer, brain or central nervous system cancer, peripheral including, but not limited to oligonucleotide-based microar nervous system cancer, esophageal cancer, cervical cancer, rays (Brennan, et al. (2004) In Press, Lucito, et al. (2003) uterine or endometrial cancer, cancer of the oral cavity or Genome Res. 13:2291-2305; Bignell et al. (2004) Genome pharynx, liver cancer, kidney cancer, testicular cancer, biliary Res. 14:287-295; Zhao, et al (2004) Cancer Research, In tract cancer, Small bowel or appendix cancer, salivary gland Press), and other methods as described herein including, for example, hybridization methods (FISH). cancer, thyroid gland cancer, adrenal gland cancer, osteosar 0043. The amplification or deletion of the MCRs identi coma, chondrosarcoma, cancer of hematological tissues, and fied herein correlate with the presence of cancer, e.g., pancre the like. atic cancer and other epithelial cancers. Furthermore, analy 0051. The term “pancreatic cancer or “neoplasia” as used sis of copy number and/or expression levels of the genes herein, includes Panns, adenomas, adenocarcinomas, gastri residing within each MCR has led to the identification of nomas, somatostatinomas, insulinomas and glucagonomas of individual markers and combinations of markers described the pancreas. herein, the increased and decreased expression and/or increased and decreased copy number of which correlate with 0052. As used herein, the term “adenocarcinoma’ is car the presence of cancer, e.g., pancreatic cancer, e.g., in a Sub cinoma that develops in the lining or inner Surface of an organ ject. and is derived from glandular tissue or in which the tumor 0044 Accordingly, methods are provided herein for cells form recognizable glandular structures. detecting the presence of cancer in a sample, the absence of 0053 As used interchangeably herein, the terms, “pancre cancer in a sample, and other characteristics of cancer that are atic adenocarcinoma, or “pancreatic ductal adenocarci relevant to prevention, diagnosis, characterization, and noma’ is an adenocarcinoma of the pancreas. In one embodi therapy of cancer in a subject by evaluating alterations in the ment, pancreatic adenocarcinomas arise from the progression amount, structure, and/or activity of a marker. For example, of premalignant lesions that occur in the pancreatic ducts evaluation of the presence, absence or copy number of the (pancreatic intraepithelial neoplasia, referred to herein as MCRs identified herein, or by evaluating the copy number, “Pan IN’). The methods described herein may be used to expression level, protein level, protein activity, presence of detect premalignant cancers, e.g., Panns, as well as malig mutations (e.g., Substitution, deletion, or addition mutations) nant cancerS. which affect activity of the marker, or methylation status of any one or more of the markers within the MCRs (e.g., the 0054. A "minimal common region (MCR), as used markers set forth in Tables 4 and 5), is within the scope of the herein, refers to a contiguous chromosomal region which invention. displays either gain and amplification (increased copy num 0045 Methods are also provided herein for the identifica ber) or loss and deletion (decreased copy number) in the tion of compounds which are capable of inhibiting cancer, in genome of a cancer. An MCR includes at least one nucleic a Subject, and for the treatment, prevention, and/or inhibition acid sequence which has increased or decreased copy number of cancer using a modulator, e.g., an agonist or antagonist, of and which is associated with a cancer. The MCRs of the a gene or protein marker of the invention. instant invention include, but are not limited to, those set forth 0046 Although the MCRs and markers described herein in Table 1. were identified in pancreatic cancer samples, the methods of 0055. A “marker' is a gene or protein which may be the invention are in no way limited to use for the prevention, altered, wherein said alteration is associated with cancer. The diagnosis, characterization, therapy and prevention of pan alteration may be in amount, structure, and/or activity in a creatic cancer, e.g., the methods of the invention may be cancertissue or cancer cell, as compared to its amount, struc applied to any cancer, as described herein. ture, and/or activity, in a normal or healthy tissue or cell (e.g., 0047 Various aspects of the invention are described in a control), and is associated with a disease state. Such as further detail in the following subsections: cancer. For example, a marker of the invention which is asso ciated with cancer may have altered copy number, expression 1. DEFINITIONS level, protein level, protein activity, or methylation status, in 0048. As used herein, each of the following terms has the a cancertissue or cancer cell as compared to a normal, healthy meaning associated with it in this section. tissue or cell. Furthermore, a “marker” includes a molecule 0049. The articles “a” and “an are used herein to refer to whose structure is altered, e.g., mutated (contains an allelic one or to more than one (i.e. to at least one) of the grammatical variant), e.g., differs from the wildtype sequence at the nucle US 2009/0297536A1 Dec. 3, 2009

otide or amino acid level, e.g., by Substitution, deletion, or an expression level or copy number in a test sample that is addition, when present in a tissue or cell associated with a greater than the standard error of the assay employed to assess disease state. Such as cancer. expression or copy number, and is preferably at least twice, 0056. The term “altered amount of a marker or “altered and more preferably three, four, five or ten or more times the level of a marker refers to increased or decreased copy expression level or copy number of the marker or MCR in a number of a marker or chromosomal region, e.g., MCR, and/ control sample (e.g., sample from a healthy Subject not or increased or decreased expression level of a particular afflicted with cancer) and preferably, the average expression marker gene or genes in a cancer sample, as compared to the level or copy number of the marker or MCR in several control expression level or copy number of the marker in a control samples. sample. The term “altered amount of a marker also includes an increased or decreased protein level of a marker in a 0062 “Methylation status of a marker refers to the sample, e.g., a cancer sample, as compared to the protein level methylation pattern, e.g., methylation of the promoter of the of the marker in a normal, control sample. Furthermore, an marker, and/or methylation levels of the marker. DNA methy altered amount of a marker may be determined by detecting lation is a heritable, reversible and epigenetic change. Yet, the methylation status of a marker, as described herein, which DNA methylation has the potential to alter gene expression, may affect the expression or activity of a marker. which has developmental and genetic consequences. DNA 0057 The amount of a marker, e.g., expression or copy methylation has been linked to cancer, as described in, for number of a marker or MCR, or protein level of a marker, in example, Laird, et al. (1994) Human Molecular Genetics a subject is “significantly higher or lower than the normal 3:1487-1495 and Laird, P. (2003) Nature 3:253-266, the con amount of a marker or MCR, if the amount of the marker is tents of which are incorporated herein by reference. For greater or less, respectively, than the normal level by an example, methylation of CpG oligonucleotides in the promot amount greater than the standard error of the assay employed ers of tumor Suppressor genes can lead to their inactivation. In to assess amount, and preferably at least twice, and more addition, alterations in the normal methylation process are preferably three, four, five, ten or more times that amount. associated with genomic instability (Lengauer et al. Proc. Alternately, the amount of the marker or MCR in the subject Natl. Acad. Sci. USA94:2545-2550, 1997). Such abnormal can be considered “significantly higher or lower than the epigenetic changes may be found in many types of cancer and normal amount if the amount is at least about two, and pref can, therefore, serve as potential markers for oncogenic trans erably at least about three, four, or five times, higher or lower, formation. respectively, than the normal amount of the marker or MCR. 0063 Methods for determining methylation include 0058. The "copy number of a gene' or the "copy number restriction landmark genomic scanning (Kawai et al., Mol. of a marker” refers to the number of DNA sequences in a cell Cell. Biol. 14:7421-7427, 1994), methylation-sensitive arbi encoding a particular gene product. Generally, for a given trarily primed PCR (Gonzalgo et al., Cancer Res. 57:594-599, gene, a mammal has two copies of each gene. The copy 1997); digestion of genomic DNA with methylation-sensitive number can be increased, however, by gene amplification or restriction enzymes followed by Southern analysis of the duplication, or reduced by deletion. regions of interest (digestion-Southern method); PCR-based 0059. The “normal” copy number of a marker or MCR or process that involves digestion of genomic DNA with methy “normal level of expression of a marker is the level of lation-sensitive restriction enzymes prior to PCR amplifica expression, copy number of the marker, or copy number of the tion (Singer-Sam et al., Nucl. Acids Res. 18.687, 1990); MCR, in a biological sample, e.g., a sample containing tissue, genomic sequencing using bisulfite treatment (Frommer et whole blood, serum, plasma, buccal scrape, saliva, cere al., Proc. Natl. Acad. Sci. USA89:1827-1831, 1992); methy broSpinal fluid, urine, stool, bile, pancreatic juice, and pan lation-specific PCR (MSP) (Herman et al. Proc. Natl. Acad. creatic tissue, from a subject, e.g. a human, not afflicted with Sci. USA 93:9821-9826, 1992); and restriction enzyme CaCC. digestion of PCR products amplified from bisulfite-converted 0060. The term “altered level of expression” of a markeror DNA (Sadri and Hornsby, Nucl. Acids Res. 24:5058-5059, MCR refers to an expression level or copy number of a marker 1996; and Xiong and Laird, Nucl. Acids. Res. 25:2532-2534, in a test sample e.g., a sample derived from a patient Suffering 1997): PCR techniques for detection of gene mutations (Kup from cancer, that is greater or less than the standard error of puswamyet et al., Proc. Natl. Acad. Sci. USA 88: 1143-1147, the assay employed to assess expression or copy number, and 1991) and quantitation of allelic-specific expression (Szabo is preferably at least twice, and more preferably three, four, and Mann, Genes Dev. 9:3097-3108, 1995; and Singer-Sam five or ten or more times the expression level or copy number et al., PCR Methods Appl. 1:160-163, 1992); and methods of the marker or MCR in a control sample (e.g., sample from described in U.S. Pat. No. 6,251,594, the contents of which a healthy Subjects not having the associated disease) and are incorporated herein by reference. An integrated genomic preferably, the average expression level or copy number of the and epigenomic analysis as described in Zardo, et al. (2000) marker or MCR in several control samples. The altered level Nature Genetics 32:453-458, may also be used. of expression is greater or less than the standard error of the 0064. The term “altered activity” of a marker refers to an assay employed to assess expression or copy number, and is activity of a marker which is increased or decreased in a preferably at least twice, and more preferably three, four, five disease state, e.g., in a cancer Sample, as compared to the or ten or more times the expression level or copy number of activity of the marker in a normal, control sample. Altered the marker or MCR in a control sample (e.g., sample from a activity of a marker may be the result of for example, altered healthy Subjects not having the associated disease) and pref expression of the marker, altered protein level of the marker, erably, the average expression level or copy number of the altered structure of the marker, or, e.g., an altered interaction marker or MCR in several control samples. with other involved in the same or different pathway 0061 An "overexpression' or “significantly higher level as the marker or altered interaction with transcriptional acti of expression or copy number of a marker or MCR refers to vators or inhibitors, or altered methylation status. US 2009/0297536A1 Dec. 3, 2009

0065. The term “altered structure' of a marker refers to the recurrence or resistance of tumors before, during or after presence of mutations or allelic variants within the marker therapy. The predictive functions of the marker may be con gene or maker protein, e.g., mutations which affect expres firmed by, e.g., (1) increased or decreased copy number (e.g., sion or activity of the marker, as compared to the normal or by FISH or qPCR), overexpression or underexpression (e.g., wild-type gene or protein. For example, mutations include, by ISH, Northern Blot, or qPCR), increased or decreased but are not limited to substitutions, deletions, or addition protein level (e.g., by IHC), or increased or decreased activity mutations. Mutations may be present in the coding or non (determined by, for example, modulation of a pathway in coding region of the marker. which the marker is involved), e.g., in more than about 5%, 0066. A “marker nucleic acid' is a nucleic acid (e.g., 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, DNA, mRNA, cDNA) encoded by or corresponding to a 25%, or more of human cancers; (2) its presence or absence in marker of the invention. For example, Such marker nucleic a biological sample, e.g., a sample containing tissue, whole acid molecules include DNA (e.g., cDNA) comprising the blood, serum, plasma, buccal scrape, Saliva, cerebrospinal entire or a partial sequence of any of the nucleic acid fluid, urine, stool, bile, pancreatic juice, and pancreatic tissue sequences set forth in Tables 4 or 5 or the complement or from a Subject, e.g. a human, afflicted with cancer; (3) its hybridizing fragment of such a sequence. The marker nucleic presence or absence in clinical Subset of patients with cancer acid molecules also include RNA comprising the entire or a (e.g., those responding to a particular therapy or those devel partial sequence of any of the nucleic acid sequences set forth oping resistance). Diagnostic markers also include 'surrogate in Tables 4 or 5 or the complement of Such a sequence, markers, e.g., markers which are indirect markers of disease wherein all thymidine residues are replaced with uridine resi progression. dues. A “marker protein’ is a protein encoded by or corre (0071. The term “probe' refers to any molecule which is sponding to a marker of the invention. A marker protein capable of selectively binding to a specifically intended target comprises the entire or apartial sequence of a protein encoded molecule, for example a marker of the invention. Probes can by any of the sequences set forth in Tables 4 or 5 or a fragment be either synthesized by one skilled in the art, or derived from thereof. The terms “protein’ and “polypeptide' are used appropriate biological preparations. For purposes of detec interchangeably herein. tion of the target molecule, probes may be specifically 0067. A “marker,” as used herein, includes any nucleic designed to be labeled, as described herein. Examples of acid sequence present in an MCR as set forth in Table 1, or a molecules that can be utilized as probes include, but are not protein encoded by Such a sequence. limited to, RNA, DNA, proteins, antibodies, and organic 0068 Markers identified herein include diagnostic and OOCS. therapeutic markers. A single marker may be a diagnostic 0072. As used herein, the term “promoter/regulatory marker, a therapeutic marker, or both a diagnostic and thera sequence” means a nucleic acid sequence which is required peutic marker. for expression of a gene product operably linked to the pro 0069. As used herein, the term “therapeutic marker' moter/regulatory sequence. In some instances, this sequence includes markers, e.g., markers set forth in Tables 4 and 5. may be the core promoter sequence and in other instances, which are believed to be involved in the development (includ this sequence may also include an enhancer sequence and ing maintenance, progression, angiogenesis, and/or metasta other regulatory elements which are required for expression sis) of cancer. The cancer-related functions of a therapeutic of the gene product. The promoter/regulatory sequence may, marker may be confirmed by, e.g., (1) increased or decreased for example, be one which expresses the gene product in a copy number (by, e.g., fluorescence in situ hybridization tissue-specific manner. (FISH) or quantitative PCR (qPCR)) or mutation (e.g., by 0073. An "RNA interfering agent” as used herein, is sequencing), overexpression or underexpression (e.g., by in defined as any agent which interferes with or inhibits expres situ hybridization (ISH), Northern Blot, or qPCR), increased sion of a target gene, e.g., a marker of the invention, by RNA or decreased protein levels (e.g., by immunohistochemistry interference (RNAi). Such RNA interfering agents include, (IHC)), or increased or decreased protein activity (deter but are not limited to, nucleic acid molecules including RNA mined by, for example, modulation of a pathway in which the molecules which are homologous to the target gene, e.g., a marker is involved), e.g., in more than about 5%, 6%, 7%, 8%, marker of the invention, or a fragment thereof, short interfer 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, or more of ing RNA (siRNA), and small molecules which interfere with human cancers; (2) the inhibition of cancer cell proliferation or inhibit expression of a target gene by RNA interference and growth, e.g., in soft agar, by, e.g., RNA interference (RNAi). (“RNAi) of the marker; (3) the ability of the marker to (0074) “RNA interference (RNAi) is an evolutionally con enhance transformation of mouse embryo fibroblasts (MEFs) served process whereby the expression or introduction of by oncogenes, e.g., Myc and RAS, or by RAS alone; (4) the RNA of a sequence that is identical or highly similar to a ability of the marker to enhance or decrease the growth of target gene results in the sequence specific degradation or tumor cell lines, e.g., in Softagar; (5) the ability of the marker specific post-transcriptional gene silencing (PTGS) of mes to transform primary mouse cells in SCID explant; and/or; (6) senger RNA (mRNA) transcribed from that targeted gene (see the prevention of maintenance or formation of tumors, e.g., Coburn, G. and Cullen, B. (2002).J of Virology 76(18): 9225), tumors arising de novo in an animal or tumors derived from thereby inhibiting expression of the target gene. In one human cancer cell lines, by inhibiting or activating the embodiment, the RNA is double stranded RNA (dsRNA). marker. In one embodiment, a therapeutic marker may be This process has been described in plants, invertebrates, and used as a diagnostic marker. mammalian cells. In nature, RNAi is initiated by the dsRNA 0070. As used herein, the term “diagnostic marker' specific endonuclease Dicer, which promotes processive includes markers, e.g., markers set forth in Tables 4 and 5. cleavage of long dsRNA into double-stranded fragments which are useful in the diagnosis of cancer, e.g., over- or termed siRNAs. siRNAs are incorporated into a protein com under-activity emergence, expression, growth, remission, plex that recognizes and cleaves target mRNAs. RNAi can US 2009/0297536A1 Dec. 3, 2009 also be initiated by introducing nucleic acid molecules, e.g., I0081. A “transcribed polynucleotide' is a polynucleotide synthetic siRNAs or RNA interfering agents, to inhibit or (e.g. an RNA, a cDNA, or an analog of one of an RNA or silence the expression of target genes. As used herein, “inhi cDNA) which is complementary to or homologous with all or bition of target gene expression” or “inhibition of marker a portion of a mature RNA made by transcription of a marker gene expression' includes any decrease in expression or pro of the invention and normal post-transcriptional processing tein activity or level of the target gene (e.g., a marker gene of (e.g. splicing), if any, of the transcript, and reverse transcrip the invention) or protein encoded by the target gene, e.g., a tion of the transcript. marker protein of the invention. The decrease may be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or I0082 “Complementary” refers to the broad concept of more as compared to the expression of a target gene or the sequence complementarity between regions of two nucleic activity or level of the protein encoded by a target gene which acid strands or between two regions of the same nucleic acid has not been targeted by an RNA interfering agent. Strand. It is known that an adenine residue of a first nucleic 0075 "Short interfering RNA' (siRNA), also referred to acid region is capable of forming specific hydrogen bonds herein as “small interfering RNA is defined as an agent (“base pairing) with a residue of a second nucleic acid region which functions to inhibit expression of a target gene, e.g., by which is antiparallel to the first region if the residue is thym RNAi. An siRNA may be chemically synthesized, may be ine or uracil. Similarly, it is known that a cytosine residue of produced by in vitro transcription, or may be produced within a first nucleic acid strand is capable of base pairing with a a host cell. In one embodiment, siRNA is a double stranded residue of a second nucleic acid strand which is antiparallel to RNA (dsRNA) molecule of about 15 to about 40 nucleotides the first Strand if the residue is guanine. A first region of a in length, preferably about 15 to about 28 nucleotides, more nucleic acid is complementary to a second region of the same preferably about 19 to about 25 nucleotides in length, and or a different nucleic acid if, when the two regions are more preferably about 19, 20, 21, or 22 nucleotides in length, arranged in an antiparallel fashion, at least one nucleotide and may contain a 3' and/or 5' overhang on each strand having residue of the first region is capable of base pairing with a a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of residue of the second region. Preferably, the first region com the overhang is independent between the two strands, i.e., the prises a first portion and the second region comprises a second length of the over hang on one strand is not dependent on the portion, whereby, when the first and second portions are length of the overhang on the second strand. Preferably the arranged in an antiparallel fashion, at least about 50%, and siRNA is capable of promoting RNA interference through preferably at least about 75%, at least about 90%, or at least degradation or specific post-transcriptional gene silencing about 95% of the nucleotide residues of the first portion are (PTGS) of the target messenger RNA (mRNA). capable of base pairing with nucleotide residues in the second 0076. In another embodiment, an siRNA is a small hairpin (also called stem loop) RNA (shRNA). In one embodiment, portion. More preferably, all nucleotide residues of the first these shRNAs are composed of a short (e.g., 19-25 nucle portion are capable of base pairing with nucleotide residues in otide) antisense strand, followed by a 5-9 nucleotide loop, and the second portion. the analogous sense Strand. Alternatively, the sense strand I0083. The terms “homology' or “identity, as used inter may precede the nucleotide loop structure and the antisense changeably herein, refer to sequence similarity between two strand may follow. These shRNAs may be contained in plas polynucleotide sequences or between two polypeptide mids, retroviruses, and lentiviruses and expressed from, for sequences, with identity being a more strict comparison. The example, the pol III U6 promoter, or another promoter (see, phrases “percent identity or homology' and “% identity or e.g., Stewart, et al. (2003) RNAApril: 9(4):493-501 incorpo homology” refer to the percentage of sequence similarity rated be reference herein). found in a comparison of two or more polynucleotide 0077 RNA interfering agents, e.g., siRNA molecules, sequences or two or more polypeptide sequences. “Sequence may be administered to a patient having or at risk for having similarity refers to the percent similarity in cancer, to inhibit expression of a marker gene of the invention, sequence (as determined by any suitable method) between e.g., a marker gene which is overexpressed in cancer (such as two or more polynucleotide sequences. Two or more the markers listed in Table 5) and thereby treat, prevent, or sequences can be anywhere from 0-100% similar, or any inhibit cancer in the subject. integer value there between. Identity or similarity can be 0078. A "constitutive' promoter is a nucleotide sequence determined by comparing a position in each sequence that which, when operably linked with a polynucleotide which may be aligned for purposes of comparison. When a position encodes or specifies a gene product, causes the gene product in the compared sequence is occupied by the same nucleotide to be produced in a living human cell under most or all base or amino acid, then the molecules are identical at that physiological conditions of the cell. position. A degree of similarity or identity between poly 0079 An “inducible' promoter is a nucleotide sequence nucleotide sequences is a function of the number of identical which, when operably linked with a polynucleotide which or matching nucleotides at positions shared by the polynucle encodes or specifies a gene product, causes the gene product otide sequences. A degree of identity of polypeptide to be produced in a living human cell Substantially only when sequences is a function of the number of identical amino acids an inducer which corresponds to the promoteris present in the at positions shared by the polypeptide sequences. A degree of cell. homology or similarity of polypeptide sequences is a function 0080. A “tissue-specific' promoter is a nucleotide of the number of amino acids at positions shared by the sequence which, when operably linked with a polynucleotide polypeptide sequences. The term 'substantial homology, as which encodes or specifies a gene product, causes the gene used herein, refers to homology of at least 50%, more pref product to be produced in a living human cell Substantially erably, 60%, 70%, 80%, 90%, 95% or more. only if the cell is a cell of the tissue type corresponding to the I0084. A marker is “fixed' to a substrate if it is covalently or promoter. non-covalently associated with the substrate such the sub US 2009/0297536A1 Dec. 3, 2009

strate can be rinsed with a fluid (e.g. standard Saline citrate, 0097. 7) making antibodies, antibody fragments or anti pH 7.4) without a substantial fraction of the marker dissoci body derivatives that are useful for treating cancer and/ ating from the Substrate. or assessing whether a Subject is afflicted with cancer, 0085. As used herein, a “naturally-occurring nucleic acid 0.098 8) assessing the presence of cancer cells; molecule refers to an RNA or DNA molecule having a nucle 0099 9) assessing the efficacy of one or more test com otide sequence that occurs in nature (e.g. encodes a natural pounds for inhibiting cancer in a Subject; protein). 0.100 10) assessing the efficacy of a therapy for inhib I0086 Cancer is “inhibited if at least one symptom of the iting cancer in a subject; cancer is alleviated, terminated, slowed, or prevented. As 0101 11) monitoring the progression of cancer in a used herein, cancer is also “inhibited' if recurrence or Subject; metastasis of the cancer is reduced, slowed, delayed, or pre 0102 12) selecting a composition or therapy for inhib vented. iting cancer, e.g., in a Subject: 0087. A kit is any manufacture (e.g. a package or con 0.103 13) treating a subject afflicted with cancer; tainer) comprising at least one reagent, e.g. a probe, for spe 0.104 14) inhibiting cancer in a subject; cifically detecting a marker of the invention, the manufacture 0105 15) assessing the carcinogenic potential of a test being promoted, distributed, or sold as a unit for performing compound; and the methods of the present invention. 010.6 16) preventing the onset of cancer in a subject at risk for developing cancer. II. Uses of the Invention 0107 The invention thus includes a method of assessing whether a subject is afflicted with cancer or is at risk for 0088. The present invention is based, in part, on the iden developing cancer. This method comprises comparing the tification of chromosomal regions (MCRs) which are struc amount, structure, and/or activity, e.g., the presence, absence, turally altered leading to a different copy number in cancer copy number, expression level, protein level, protein activity, cells as compared to normal (i.e. non-cancerous) cells. Fur presence of mutations, e.g., mutations which affect activity of thermore, the present invention is based, in part, on the iden the marker (e.g., Substitution, deletion, or addition muta tification of markers, e.g., markers which reside in the MCRs tions), and/or methylation status, of a marker in a subject of the invention, which have an altered amount, structure, sample with the normal level. A significant difference and/or activity in cancer cells as compared to normal (i.e., between the amount, structure, or activity of the marker in the non-cancerous) cells. The markers of the invention corre subject sample and the normal level is an indication that the spond to DNA, cDNA, RNA, and polypeptide molecules subject is afflicted with cancer. The invention also provides a which can be detected in one or both of normal and cancerous method for assessing whether a subject is afflicted with can cells. cer or is at risk for developing cancer by comparing the level 0089. The amount, structure, and/or activity, e.g., the pres of expression of marker(s) within an MCR or copy number of ence, absence, copy number, expression level, protein level. an MCR in a cancer sample with the level of expression of protein activity, presence of mutations, e.g., mutations which marker(s) within an MCR or copy number of an MCR in a affect activity of the marker (e.g., Substitution, deletion, or normal, control sample. A significant difference between the addition mutations), and/or methylation status, of one or level of expression of marker(s) within an MCR or copy more of these markers in a sample, e.g., a sample containing number of the MCR in the subject sample and the normal tissue, whole blood, serum, plasma, buccal scrape, Saliva, level is an indication that the subject is afflicted with cancer. cerebrospinal fluid, urine, stool, bile, pancreatic juice, and The MCR is selected from the group consisting of those listed pancreatic tissue, is herein correlated with the cancerous state in Table 1. of the tissue. In addition, the presence, absence, and/or copy 0108. The marker is selected from the group consisting of number of one or more of the MCRs of the invention in a the markers listed in Tables 4 and 5. Table 4 lists markers sample is also correlated with the cancerous state of the which have a highly significant correlation between gene tissue. The invention thus provides compositions, kits, and expression and gene dosage (p, 0.05). The level of expression methods for assessing the cancerous state of cells (e.g. cells or copy number of these markers is decreased in Samples obtained from a non-human, cultured non-human cells, and in histologically identified as pancreatic cancer, e.g., pancreatic Vivo cells) as well as methods for treatment, prevention, and/ adenocarcinoma. Table 4 also lists the chromosome, physical or inhibition of cancer using a modulator, e.g., an agonist or position in Mb, Gene Weight, p-value, AffymetrixTM probe(s) antagonist, of a marker of the invention. number corresponding to each UniGene ID, Genebank 0090 The compositions, kits, and methods of the inven Accession No. (i.e., “GI” number), and SEQID NO. for each tion have the following uses, among others: of the markers. Although one or more molecules correspond 0091. 1) assessing whether a subject is afflicted with ing to the markers listed in Table 4 may have been described Cancer, by others, the significance of these markers with regard to the 0092. 2) assessing the stage of cancer in a human Sub cancerous state of cells, has not previously been identified. ject; 0109 Table 5 also lists markers which have a highly sig nificant correlation between gene expression and gene dosage 0093. 3) assessing the grade of cancer in a subject; (p, 0.05). The level of expression or copy number of these 0094 4) assessing the benign or malignant nature of markers is increased in Samples histologically identified as cancer in a subject; pancreatic cancer, e.g., pancreatic adenocarcinoma. Table 5 0.095 5) assessing the metastatic potential of cancerina also lists the chromosome, physical position in Mb, Gene Subject; Weight, p-value. AffymetrixTM probe(s) number correspond 0096 6) assessing the histological type of neoplasm ing to each UniGene ID, Genebank Accession No. (i.e., “GI’ associated with cancer in a subject; number), and SEQ ID NO. for each of these markers. US 2009/0297536A1 Dec. 3, 2009

Although one or more molecules corresponding to the mark MCR or panel of markers or MCRs of the invention be ers listed in Table 5 may have been described by others, the selected such that a positive result is obtained in at least about significance of these markers with regard to the cancerous 20%, and preferably at least about 40%, 60%, or 80%, and state of cells, has not previously been identified. more preferably, in substantially all, subjects afflicted with 0110. Any marker or combination of markers listed in cancer, of the corresponding stage, grade, histological type, Tables 4 or 5 or any MCR or combination of MCRs listed in or benign/premalignant/malignant nature. Preferably, the Table 1, may be used in the compositions, kits, and methods marker or panel of markers of the invention is selected Such of the present invention. In general, it is preferable to use that a PPV (positive predictive value) of greater than about markers for which the difference between the amount, e.g., 10% is obtained for the general population (more preferably level of expression or copy number, and/or activity of the coupled with an assay specificity greater than 99.5%). marker or MCR in cancer cells and the amount, e.g., level of (0.115. When a plurality of markers or MCRs of the inven expression or copy number, and/or activity of the same tion are used in the compositions, kits, and methods of the marker in normal cells, is as great as possible. Although this invention, the amount, structure, and/or activity of each difference can be as small as the limit of detection of the marker or level of expression or copy number can be com method for assessing amount and/or activity of the marker, it pared with the normal amount, structure, and/or activity of is preferred that the difference be at least greater than the each of the plurality of markers or level of expression or copy standard error of the assessment method, and preferably a number, in non-cancerous samples of the same type, either in difference of at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-15-, 20 a single reaction mixture (i.e. using reagents, such as different 25-, 100-, 500-, 1000-fold or greater than the amount, e.g., fluorescent probes, for each marker) or in individual reaction level of expression or copy number, and/or activity of the mixtures corresponding to one or more of the markers or same biomarker in normal tissue. MCRS. 0111. It is understood that by routine screening of addi 0116. In one embodiment, a significantly altered amount, tional Subject samples using one or more of the markers of the structure, and/or activity of more than one of the plurality of invention, it will be realized that certain of the markers have markers, or significantly altered copy number of one or more altered amount, structure, and/or activity in cancers of various of the MCRs in the sample, relative to the corresponding types, including specific pancreatic cancers, as well as other normal levels, is an indication that the subject is afflicted with cancers, e.g., carcinoma, Sarcoma, lymphoma or leukemia, cancer. For example, a significantly lower copy number in the examples of which include, but are not limited to, melanomas, sample of each of the plurality of markers or MCRs, relative breast cancer, lung cancer, bronchus cancer, colorectal can to the corresponding normal levels or copy number, is an cer, prostate cancer, pancreas cancer, stomach cancer, ovarian indication that the subject is afflicted with cancer. In yet cancer, urinary bladder cancer, brain or central nervous sys another embodiment, a significantly enhanced copy number tem cancer, peripheral nervous system cancer, esophageal of one or more markers or MCRs and a significantly lower cancer, cervical cancer, uterine or endometrial cancer, cancer level of expression or copy number of one or more markers or of the oral cavity or pharynx, liver cancer, kidney cancer, MCRs in a sample relative to the corresponding normal lev testicular cancer, biliary tract cancer, Small bowel or appendix els, is an indication that the subject is afflicted with cancer. cancer, salivary gland cancer, thyroid gland cancer, adrenal Also, for example, a significantly enhanced copy number in gland cancer, osteosarcoma, chondrosarcoma, cancer of the sample of each of the plurality of markers or MCRs, hematological tissues, and the like. relative to the corresponding normal copy number, is an indi 0112 For example, it will be confirmed that some of the cation that the subject is afflicted with cancer. In yet another markers of the invention have altered amount, structure, and/ embodiment, a significantly enhanced copy number of one or or activity in some, i.e., 10%, 20%, 30%, or 40%, or most (i.e. more markers or MCRS and a significantly lower copy num 50% or more) or substantially all (i.e. 80% or more) of cancer, ber of one or more markers or MCRs in a sample relative to e.g., pancreatic cancer. Furthermore, it will be confirmed that the corresponding normal levels, is an indication that the certain of the markers of the invention are associated with subject is afflicted with cancer. cancer of various histologic Subtypes. 0117. When a plurality of markers or MCRs are used, it is 0113. In addition, as a greater number of subject samples preferred that 2, 3, 4, 5, 8, 10, 12, 15, 20, 30, or 50 or more are assessed for altered amount, structure, and/or activity of individual markers or MCRs be used or identified, wherein the markers or altered expression or copy number MCRs of fewer markers or MCRs are preferred. the invention and the outcomes of the individual subjects 0118. Only a small number of markers are known to be from whom the samples were obtained are correlated, it will associated with, for example, pancreatic cancer (e.g., AKT2, also be confirmed that markers have altered amount, struc p16'Y', c-MYC, SMAD4, and TP53; Lynch, supra). These ture, and/or activity of certain of the markers or altered markers or other markers which are known to be associated expression or copy number of MCRs of the invention are with other types of cancer may be used together with one or strongly correlated with malignant cancers and that altered more markers of the invention in, for example, a panel of expression of other markers of the invention are strongly markers. In addition, frequent gains have been mapped to 3q, correlated with benign tumors or premalignant states. The 5p, 7p. 8q, 11q, 12p. 17q and 20d and losses to 3p, 4q, 6q 8p. compositions, kits, and methods of the invention are thus 9p, 10q, 12q, 13q, 17p. 18q and 21q and 22d. in pancreatic useful for characterizing one or more of the stage, grade, cancer. In some instances, validated oncogenes and tumor histological type, and benign/premalignant/malignant nature Suppressor genes residing within these loci have been identi of cancer in Subjects. fied, including MYC (8q24), p16'Y''' (9p21), p53 (17p13), 0114. When the compositions, kits, and methods of the SMAD4 (18q21) and AKT2 (19q13). It is well known that invention are used for characterizing one or more of the stage, certain types of genes, such as oncogenes, tumor suppressor grade, histological type, and benign/premalignant/malignant genes, growth factor-like genes, protease-like genes, and pro nature of cancer, in a Subject, it is preferred that the marker or tein kinase-like genes are often involved with development of US 2009/0297536A1 Dec. 3, 2009

cancers of various types. Thus, among the markers of the nucleic acid (e.g. a genomic DNA, an mRNA, a spliced invention, use of those which correspond to proteins which mRNA, a cDNA, or the like) include complementary nucleic resemble known proteins encoded by known oncogenes and acids. For example, the nucleic acid reagents may include tumor Suppressor genes, and those which correspond to pro oligonucleotides (labeled or non-labeled) fixed to a substrate, teins which resemble growth factors, proteases, and protein labeled oligonucleotides not bound with a substrate, pairs of kinases, are preferred. PCR primers, molecular beacon probes, and the like. 0119. It is recognized that the compositions, kits, and I0123. The kit of the invention may optionally comprise methods of the invention will be of particular utility to sub additional components useful for performing the methods of jects having an enhanced risk of developing cancer, and their the invention. By way of example, the kit may comprise fluids medical advisors. Subjects recognized as having an enhanced (e.g., SSC buffer) suitable for annealing complementary risk of developing cancer, include, for example, Subjects hav nucleic acids or for binding an antibody with a protein with ing a familial history of cancer, Subjects identified as having which it specifically binds, one or more sample compart a mutant oncogene (i.e. at least one allele), and Subjects of ments, an instructional material which describes performance advancing age. of a method of the invention, a sample of normal cells, a 0120 An alteration, e.g. copy number, amount, structure, sample of cancer cells, and the like. and/or activity of a marker in normal (i.e. non-cancerous) 0.124 Akit of the invention may comprise a reagent useful human tissue can be assessed in a variety of ways. In one for determining protein level or protein activity of a marker. In embodiment, the normal level of expression or copy number another embodiment, a kit of the invention may comprise a is assessed by assessing the level of expression and/or copy reagent for determining methylation status of a marker, or number of the marker or MCR in a portion of cells which may comprise a reagent for determining alteration of struc appear to be non-cancerous and by comparing this normal ture of a marker, e.g., the presence of a mutation. level of expression or copy number with the level of expres 0.125. The invention also includes a method of making an sion or copy number in a portion of the cells which are isolated hybridoma which produces an antibody useful in Suspected of being cancerous. For example, when laparos methods and kits of the present invention. A protein corre copy or other medical procedure, reveals the presence of a sponding to a marker of the invention may be isolated (e.g. by tumor on one portion of an organ, the normal level of expres purification from a cell in which it is expressed or by tran sion or copy number of a marker or MCR may be assessed Scription and translation of a nucleic acid encoding the pro using the non-affected portion of the organ, and this normal tein in Vivo or in vitrousing known methods) and a vertebrate, level of expression or copy number may be compared with the preferably a mammal such as a mouse, rat, rabbit, or sheep, is level of expression or copy number of the same marker in an immunized using the isolated protein. The vertebrate may affected portion (i.e., the tumor) of the organ. Alternately, and optionally (and preferably) be immunized at least one addi particularly as further information becomes available as a tional time with the isolated protein, so that the vertebrate result of routine performance of the methods described exhibits a robust immune response to the protein. Splenocytes herein, population-average values for “normal” copy number, are isolated from the immunized vertebrate and fused with an amount, structure, and/or activity of the markers or MCRs of immortalized cell line to form hybridomas, using any of a the invention may be used. In other embodiments, the “nor variety of methods well known in the art. Hybridomas formed mal’ copy number, amount, structure, and/or activity of a in this manner are then screened using standard methods to marker or MCR may be determined by assessing copy num identify one or more hybridomas which produce an antibody ber, amount, structure, and/or activity of the marker or MCR which specifically binds with the protein. The invention also in a subject sample obtained from a non-cancer-afflicted Sub includes hybridomas made by this method and antibodies ject, from a subject sample obtained from a subject before the made using Such hybridomas. Suspected onset of cancer in the Subject, from archived Sub 0.126 The invention also includes a method of assessing ject samples, and the like. the efficacy of a test compound for inhibiting cancer cells. As 0121 The invention includes compositions, kits, and described above, differences in the amount, structure, and/or methods for assessing the presence of cancer cells in a sample activity of the markers of the invention, or level of expression (e.g. an archived tissue sample or a sample obtained from a or copy number of the MCRs of the invention, correlate with Subject). These compositions, kits, and methods are Substan the cancerous state of cells. Although it is recognized that tially the same as those described above, except that, where changes in the levels of amount, e.g., expression or copy necessary, the compositions, kits, and methods are adapted number, structure, and/or activity of certain of the markers or for use with certain types of samples. For example, when the expression or copy number of the MCRs of the invention sample is a parafinized, archived human tissue sample, it may likely result from the cancerous state of cells, it is likewise be necessary to adjust the ratio of compounds in the compo recognized that changes in the amount may induce, maintain, sitions of the invention, in the kits of the invention, or the and promote the cancerous state. Thus, compounds which methods used. Such methods are well known in the art and inhibit cancer, in a subject may cause a change, e.g., a change within the skill of the ordinary artisan. in expression and/or activity of one or more of the markers of 0122) The invention thus includes a kit for assessing the the invention to a level nearer the normal level for that marker presence of cancer cells (e.g. in a sample Such as a subject (e.g., the amount, e.g., expression, and/or activity for the sample). The kit may comprise one or more reagents capable marker in non-cancerous cells). of identifying a marker or MCR of the invention, e.g., binding 0127. This method thus comprises comparing amount, specifically with a nucleic acid or polypeptide corresponding e.g., expression, and/or activity of a marker in a first cell to a marker or MCR of the invention. Suitable reagents for sample and maintained in the presence of the test compound binding with a polypeptide corresponding to a marker of the and amount, e.g., expression, and/or activity of the marker in invention include antibodies, antibody derivatives, antibody a second cell sample and maintained in the absence of the test fragments, and the like. Suitable reagents for binding with a compound. A significant increase in the amount, e.g., expres US 2009/0297536A1 Dec. 3, 2009

Sion, and/or activity of a marker listed in Table 4 (e.g., a changes induce, maintain, and promote the cancerous state of marker that was shown to be decreased in cancer), a signifi cancer cells. Thus, cancer characterized by an increase in the cant decrease in the amount, e.g., expression, and/or activity amount, e.g., expression, and/or activity of one or more mark of a marker listed in Table 5 (e.g., a marker that was shown to ers listed in Table 5 (e.g., a marker that was shown to be be increased in cancer), is an indication that the test com increased in cancer), can be inhibited by inhibiting amount, pound inhibits cancer. The cell samples may, for example, be e.g., expression, and/or activity of those markers. Likewise, aliquots of a single sample of normal cells obtained from a cancer characterized by a decrease in the amount, e.g., Subject, pooled samples of normal cells obtained from a Sub expression, and/or activity of one or more markers listed in ject, cells of a normal cell lines, aliquots of a single sample of Table 4 (e.g., a marker that was shown to be decreased in cancer, cells obtained from a Subject, pooled samples of can cancer), can be inhibited by enhancing amount, e.g., expres cer, cells obtained from a subject, cells of a cancer cell line, Sion, and/or activity of those markers. cells from an animal model of cancer, or the like. In one I0131. Amount and/or activity of a marker listed in Table 5 embodiment, the samples are cancer cells obtained from a (e.g., a marker that was shown to be increased in cancer), can subject and a plurality of compounds known to be effective be inhibited in a number of ways generally known in the art. for inhibiting various cancers, are tested in order to identify For example, an antisense oligonucleotide can be provided to the compound which is likely to best inhibit the cancer in the the cancer cells in order to inhibit transcription, translation, or Subject. both, of the marker(s). An RNA interfering agent, e.g., an 0128. This method may likewise be used to assess the siRNA molecule, which is targeted to a marker listed in Table efficacy of a therapy, e.g., chermotherapy, radiation therapy, 5, can be provided to the cancer cells in order to inhibit Surgery, or any other therapeutic approach useful for inhibit expression of the target marker, e.g., through degradation or ing cancer in a Subject. In this method, the amount, e.g., specific post-transcriptional gene silencing (PTGS) of the expression, and/or activity of one or more markers of the messenger RNA (mRNA) of the target marker. Alternately, a invention in a pair of samples (one Subjected to the therapy, polynucleotide encoding an antibody, an antibody derivative, the other not subjected to the therapy) is assessed. As with the or an antibody fragment, e.g., a fragment capable of binding method of assessing the efficacy of test compounds, if the an antigen, and operably linked with an appropriate promoter therapy induces a significant decrease in the amount, e.g., or regulator region, can be provided to the cell in order to expression, and/or activity of a marker listed in Table 5 (e.g., generate intracellular antibodies which will inhibit the func a marker that was shown to be increased in cancer), blocks tion, amount, and/or activity of the protein corresponding to induction of a marker listed in Table 5 (e.g., a marker that was the marker(s). Conjugated antibodies or fragments thereof, shown to be increased in cancer), or if the therapy induces a e.g., chemolabeled antibodies, radiolabeled antibodies, or significant enhancement of the amount, e.g., expression, and/ immunotoxins targeting a marker of the invention may also be or activity of a marker listed in Table 4 (e.g., a marker that was administered to treat, prevent or inhibit cancer. shown to be decreased in cancer), then the therapy is effica 0.132. A small molecule may also be used to modulate, cious for inhibiting cancer. As above, if samples from a e.g., inhibit, expression and/or activity of a marker listed in selected subject are used in this method, then alternative Table 5. In one embodiment, a small molecule functions to therapies can be assessed in vitro in order to select a therapy disrupt a protein-protein interaction between a marker of the most likely to be efficacious for inhibiting cancer in the sub invention and a target molecule or ligand, thereby modulat ject. ing, e.g., increasing or decreasing the activity of the marker. 0129. This method may likewise be used to monitor the I0133. Using the methods described herein, a variety of progression of cancer in a Subject, wherein if a sample in a molecules, particularly including molecules Sufficiently Subject has a significant decrease in the amount, e.g., expres small that they are able to cross the cell membrane, can be Sion, and/or activity of a marker listed in Table 5 (e.g., a screened in order to identify molecules which inhibit amount marker that was shown to be increased in cancer, or blocks and/or activity of the marker(s). The compound so identified induction of a marker listed in Table 5 (e.g., a marker that was can be provided to the subject in order to inhibit amount shown to be increased in cancer), or a significant enhance and/or activity of the marker(s) in the cancer cells of the ment of the amount, e.g., expression, and/or activity of a Subject. marker listed in Table 4 (e.g., a marker that was shown to be I0134. Amount and/or activity of a marker listed in Table 4 decreased in cancer), during the progression of cancer, e.g., at (e.g., a marker that was shown to be decreased in cancer), can a first point in time and a Subsequent point in time, then the be enhanced in a number of ways generally known in the art. cancer has improved. In yet another embodiment, between For example, a polynucleotide encoding the marker and oper the first point in time and a Subsequent point in time, the ably linked with an appropriate promoter/regulator region can Subject has undergone treatment, e.g., chermotherapy, radia be provided to cells of the subject in order to induce enhanced tion therapy, Surgery, or any other therapeutic approach useful expression and/or activity of the protein (and mRNA) corre for inhibiting cancer, has completed treatment, or is in remis sponding to the marker therein. Alternatively, if the protein is S1O. capable of crossing the cell membrane, inserting itself in the 0130. As described herein, cancer in subjects is associated cell membrane, or is normally a secreted protein, then amount with an increase in amount, e.g., expression, and/or activity of and/or activity of the protein can be enhanced by providing one or more markers listed in Table 5 (e.g., a marker that was the protein (e.g. directly or by way of the bloodstream) to shown to be increased in cancer), and/or a decrease in cancer cells in the Subject. A Small molecule may also be used amount, e.g., expression, and/or activity of one or more mark to modulate, e.g., increase, expression or activity of a marker ers listed in Table 4 (e.g., a marker that was shown to be listed in Table 4. Furthermore, in another embodiment, a decreased in cancer). While, as discussed above, some of modulator of a marker of the invention, e.g., a small molecule, these changes in amount, e.g., expression, and/or activity may be used, for example, to re-express a silenced gene, e.g., number result from occurrence of the cancer, others of these a tumor Suppressor, in order to treat or prevent cancer. For US 2009/0297536A1 Dec. 3, 2009

example, such a modulator may interfere with a DNA binding 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which natu element or a methyltransferase. rally flank the nucleic acid molecule in genomic DNA of the 0135. As described above, the cancerous state of human cell from which the nucleic acid is derived. Moreover, an cells is correlated with changes in the amount and/or activity "isolated nucleic acid molecule, such as a cDNA molecule, of the markers of the invention. Thus, compounds which can be substantially free of other cellular material or culture induce increased expression or activity of one or more of the medium when produced by recombinant techniques, or Sub markers listed in Table 5 (e.g., a marker that was shown to be stantially free of chemical precursors or other chemicals increased in cancer), decreased amount and/or activity of one when chemically synthesized. or more of the markers listed in Table 4 (e.g., a marker that was shown to be decreased in cancer), can induce cell car 0.139. A nucleic acid molecule of the present invention, cinogenesis. The invention also includes a method for assess e.g., a nucleic acid molecules encoding a protein correspond ing the human cell carcinogenic potential of a test compound. ing to a marker listed in Tables 4 or 5, can be isolated using This method comprises maintaining separate aliquots of standard molecular biology techniques and the sequence human cells in the presence and absence of the test com information in the database records described herein. Using pound. Expression or activity of a marker of the invention in all or a portion of Such nucleic acid sequences, nucleic acid each of the aliquots is compared. A significant increase in the molecules of the invention can be isolated using standard amount and/or activity of a marker listed in Table 5 (e.g., a hybridization and cloning techniques (e.g., as described in marker that was shown to be increased in cancer), or a sig Sambrook et al., ed., Molecular Cloning. A Laboratory nificant decrease in the amount and/or activity of a marker Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold listed in Table 4 (e.g., a marker that was shown to be Spring Harbor, N.Y., 1989). decreased in cancer), in the aliquot maintained in the presence 0140. A nucleic acid molecule of the invention can be of the test compound (relative to the aliquot maintained in the amplified using cDNA, mRNA, or genomic DNA as a tem absence of the test compound) is an indication that the test compound possesses human cell carcinogenic potential. The plate and appropriate oligonucleotide primers according to relative carcinogenic potentials of various test compounds standard PCR amplification techniques. The nucleic acid can be assessed by comparing the degree of enhancement or molecules so amplified can be cloned into an appropriate inhibition of the amount and/or activity of the relevant mark vector and characterized by DNA sequence analysis. Further ers, by comparing the number of markers for which the more, oligonucleotides corresponding to all or a portion of a amount and/or activity is enhanced or inhibited, or by com nucleic acid molecule of the invention can be prepared by paring both. standard synthetic techniques, e.g., using an automated DNA 0.136 Various aspects of the invention are described in synthesizer. further detail in the following subsections. 0.141. In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic III. Isolated Nucleic Acid Molecules acid molecule which has a nucleotide sequence complemen 0.137. One aspect of the invention pertains to isolated tary to the nucleotide sequence of a nucleic acid correspond nucleic acid molecules that correspond to a marker of the ing to a marker of the invention or to the nucleotide sequence invention, including nucleic acids which encode a polypep of a nucleic acid encoding a protein which corresponds to a tide corresponding to a marker of the invention or a portion of marker of the invention. A nucleic acid molecule which is Such a polypeptide. The nucleic acid molecules of the inven complementary to a given nucleotide sequence is one which tion include those nucleic acid molecules which reside in the is Sufficiently complementary to the given nucleotide MCRs identified herein. Isolated nucleic acid molecules of sequence that it can hybridize to the given nucleotide the invention also include nucleic acid molecules Sufficient sequence thereby forming a stable duplex. for use as hybridization probes to identify nucleic acid mol 0.142 Moreover, a nucleic acid molecule of the invention ecules that correspond to a marker of the invention, including can comprise only a portion of a nucleic acid sequence, nucleic acid molecules which encode a polypeptide corre wherein the full length nucleic acid sequence comprises a sponding to a marker of the invention, and fragments of Such marker of the invention or which encodes a polypeptide cor nucleic acid molecules, e.g., those suitable for use as PCR responding to a marker of the invention. Such nucleic acid primers for the amplification or mutation of nucleic acid molecules can be used, for example, as a probe or primer. The molecules. As used herein, the term “nucleic acid molecule' probe?primer typically is used as one or more substantially is intended to include DNA molecules (e.g., cDNA or purified oligonucleotides. The oligonucleotide typically genomic DNA) and RNA molecules (e.g., mRNA) and ana comprises a region of nucleotide sequence that hybridizes logs of the DNA or RNA generated using nucleotide analogs. understringent conditions to at least about 7, preferably about The nucleic acid molecule can be single-stranded or double 15, more preferably about 25, 50, 75, 100,125, 150, 175,200, stranded, but preferably is double-stranded DNA. 250, 300, 350, or 400 or more consecutive nucleotides of a 0138 An "isolated nucleic acid molecule is one which is nucleic acid of the invention. separated from other nucleic acid molecules which are 0.143 Probes based on the sequence of a nucleic acid mol present in the natural source of the nucleic acid molecule. ecule of the invention can be used to detect transcripts or Preferably, an "isolated nucleic acid molecule is free of genomic sequences corresponding to one or more markers of sequences (preferably protein-encoding sequences) which the invention. The probe comprises a label group attached naturally flank the nucleic acid (i.e., sequences located at the thereto, e.g., a radioisotope, a fluorescent compound, an 5' and 3' ends of the nucleic acid) in the genomic DNA of the enzyme, or an enzyme co-factor. Such probes can be used as organism from which the nucleic acid is derived. For part of a diagnostic test kit for identifying cells or tissues example, in various embodiments, the isolated nucleic acid which mis-express the protein, such as by measuring levels of molecule can contain less than about 5 kB, 4 kB, 3 kB, 2kB, a nucleic acid molecule encoding the protein in a sample of US 2009/0297536A1 Dec. 3, 2009

cells from a subject, e.g., detecting mRNA levels or deter tial amino acid residues. A “non-essential amino acid resi mining whether agene encoding the protein has been mutated due is a residue that can be altered from the wild-type or deleted. sequence without altering the biological activity, whereas an 0144. The invention further encompasses nucleic acid “essential amino acid residue is required for biological activ molecules that differ, due to degeneracy of the genetic code, ity. For example, amino acid residues that are not conserved from the nucleotide sequence of nucleic acid molecules or only semi-conserved among homologs of various species encoding a protein which corresponds to a marker of the may be non-essential for activity and thus would be likely invention, and thus encode the same protein. targets for alteration. Alternatively, amino acid residues that 0145. In addition to the nucleotide sequences described in are conserved among the homologs of various species (e.g., Tables 4 or 5, it will be appreciated by those skilled in the art murine and human) may be essential for activity and thus that DNA sequence polymorphisms that lead to changes in would not be likely targets for alteration. the amino acid sequence can exist within a population (e.g., 0150. Accordingly, another aspect of the invention per the human population). Such genetic polymorphisms can tains to nucleic acid molecules encoding a polypeptide of the exist among individuals within a population due to natural invention that contain changes in amino acid residues that are allelic variation. An allele is one of a group of genes which not essential for activity. Such polypeptides differ in amino occur alternatively at a given genetic locus. In addition, it will acid sequence from the naturally-occurring proteins which be appreciated that DNA polymorphisms that affect RNA correspond to the markers of the invention, yet retain biologi expression levels can also exist that may affect the overall cal activity. In one embodiment, Such a protein has an amino expression level of that gene (e.g., by affecting regulation or acid sequence that is at least about 40% identical, 50%, 60%, degradation). 70%, 80%, 90%, 95%, or 98% identical to the amino acid 0146. As used herein, the phrase “allelic variant” refers to sequence of one of the proteins which correspond to the a nucleotide sequence which occurs at a given locus or to a markers of the invention. polypeptide encoded by the nucleotide sequence. 0151. An isolated nucleic acid molecule encoding a vari 0147 As used herein, the terms “gene' and “recombinant ant protein can be created by introducing one or more nucle gene' refer to nucleic acid molecules comprising an open otide substitutions, additions or deletions into the nucleotide reading frame encoding a polypeptide corresponding to a sequence of nucleic acids of the invention, such that one or marker of the invention. Such natural allelic variations can more amino acid residue substitutions, additions, or deletions typically result in 1-5% Variance in the nucleotide sequence are introduced into the encoded protein. Mutations can be of a given gene. Alternative alleles can be identified by introduced by standard techniques, such as site-directed sequencing the gene of interest in a number of different indi mutagenesis and PCR-mediated mutagenesis. Preferably, viduals. This can be readily carried out by using hybridization conservative amino acid substitutions are made at one or more probes to identify the same genetic locus in a variety of predicted non-essential amino acid residues. A "conservative individuals. Any and all Such nucleotide variations and result amino acid substitution' is one in which the amino acid ingamino acid polymorphisms or variations that are the result residue is replaced with an amino acid residue having a simi of natural allelic variation and that do not alter the functional lar side chain. Families of amino acid residues having similar activity are intended to be within the scope of the invention. side chains have been defined in the art. These families 0148. In another embodiment, an isolated nucleic acid include amino acids with basic side chains (e.g., lysine, argi molecule of the invention is at least 7, 15, 20, 25, 30, 40, 60, nine, histidine), acidic side chains (e.g., aspartic acid, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, glutamic acid), uncharged polar side chains (e.g., glycine, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, asparagine, glutamine, serine, threonine, tyrosine, cysteine), 2600, 2800, 3000, 3500, 4000, 4500, or more nucleotides in non-polar side chains (e.g., alanine, Valine, leucine, isoleu length and hybridizes under Stringent conditions to a nucleic cine, proline, phenylalanine, methionine, tryptophan), beta acid molecule corresponding to a marker of the invention or to branched side chains (e.g., threonine, Valine, isoleucine) and a nucleic acid molecule encoding a protein corresponding to aromatic side chains (e.g., tyrosine, phenylalanine, tryp a marker of the invention. As used herein, the term “hybrid tophan, histidine). Alternatively, mutations can be introduced izes under stringent conditions is intended to describe con randomly along all or part of the coding sequence. Such as by ditions for hybridization and washing under which nucleotide saturation mutagenesis, and the resultant mutants can be sequences at least 60% (65%, 70%, preferably 75%) identical screened for biological activity to identify mutants that retain to each other typically remain hybridized to each other. Such activity. Following mutagenesis, the encoded protein can be stringent conditions are known to those skilled in the art and expressed recombinantly and the activity of the protein can be can be found in sections 6.3.1-6.3.6 of Current Protocols in determined. Molecular Biology, John Wiley & Sons, N.Y. (1989). A pre 0152 The present invention encompasses antisense ferred, non-limiting example of Stringent hybridization con nucleic acid molecules, i.e., molecules which are comple ditions are hybridization in 6x sodium chloride/sodium cit mentary to a sense nucleic acid of the invention, e.g., comple rate (SSC) at about 45° C., followed by one or more washes in mentary to the coding strand of a double-stranded cDNA 0.2xSSC, 0.1% SDS at 50-65° C. molecule corresponding to a marker of the invention or 0149. In addition to naturally-occurring allelic variants of complementary to an mRNA sequence corresponding to a a nucleic acid molecule of the invention that can exist in the marker of the invention. Accordingly, an antisense nucleic population, the skilled artisan will further appreciate that acid molecule of the invention can hydrogen bond to (i.e. sequence changes can be introduced by mutation thereby anneal with) a sense nucleic acid of the invention. The anti leading to changes in the amino acid sequence of the encoded sense nucleic acid can be complementary to an entire coding protein, without altering the biological activity of the protein Strand, or to only a portion thereof, e.g., all or part of the encoded thereby. For example, one can make nucleotide Sub protein coding region (or open reading frame). An antisense stitutions leading to amino acid substitutions at “non-essen nucleic acid molecule can also be antisense to all or part of a US 2009/0297536A1 Dec. 3, 2009

non-coding region of the coding strand of a nucleotide achieve Sufficient intracellular concentrations of the antisense sequence encoding a polypeptide of the invention. The non molecules, vector constructs in which the antisense nucleic coding regions (“5' and 3“untranslated regions') are the 5' acid molecule is placed under the control of a strong pol II or and 3' sequences which flank the coding region and are not pol III promoter are preferred. translated into amino acids. 0.155. An antisense nucleic acid molecule of the invention 0153. An antisense oligonucleotide can be, for example, can be an O-anomeric nucleic acid molecule. An O-anomeric about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucle nucleic acid molecule forms specific double-stranded hybrids otides in length. Anantisense nucleic acid of the invention can with complementary RNA in which, contrary to the usual be constructed using chemical synthesis and enzymatic liga O-units, the strands run parallel to each other (Gaultier et al., tion reactions using procedures known in the art. For 1987, Nucleic Acids Res. 15:6625-6641). The antisense example, an antisense nucleic acid (e.g., an antisense oligo nucleic acid molecule can also comprise a 2'-O-methylribo nucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides nucleotide (Inoue et al., 1987, Nucleic Acids Res. 15:6131 designed to increase the biological stability of the molecules 6148) or a chimeric RNA-DNA analogue (Inoue et al., 1987, or to increase the physical stability of the duplex formed FEBS Lett. 215:327-330). between the antisense and sense nucleic acids, e.g., phospho 0156 The invention also encompasses ribozymes. rothioate derivatives and acridine substituted nucleotides can Ribozymes are catalytic RNA molecules with ribonuclease be used. Examples of modified nucleotides which can be used activity which are capable of cleaving a single-stranded to generate the antisense nucleic acid include 5-fluorouracil, nucleic acid. Such as an mRNA, to which they have a comple 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, mentary region. Thus, ribozymes (e.g., hammerhead Xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) ribozymes as described in Haselhoff and Gerlach, 1988, uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-car Nature 334:585-591) can be used to catalytically cleave boxymethylaminomethyluracil, dihydrouracil, beta-D-galac mRNA transcripts to thereby inhibit translation of the protein tosylqueosine, inosine, N6-isopentenyladenine, 1-meth encoded by the mRNA. A ribozyme having specificity for a ylguanine, 1-methylinosine, 2,2-dimethylguanine, nucleic acid molecule encoding a polypeptide corresponding 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-me to a marker of the invention can be designed based upon the thylcytosine, N6-adenine, 7-methylguanine, 5-methylami nucleotide sequence of a cDNA corresponding to the marker. nomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta For example, a derivative of a Tetrahymena L-19 IVS RNA D-mannosylcueosine, 5'-methoxycarboxymethyluracil, can be constructed in which the nucleotide sequence of the 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, active site is complementary to the nucleotide sequence to be uracil-5-oxyacetic acid (v), Wybutoxosine, pseudouracil, cleaved (see Cechet al. U.S. Pat. No. 4,987,071; and Cechet queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, al. U.S. Pat. No. 5,116,742). Alternatively, an mRNA encod 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ing a polypeptide of the invention can be used to select a ester, uracil-5-oxyacetic acid (V), 5-methyl-2-thiouracil, catalytic RNA having a specific ribonuclease activity from a 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2.6- pool of RNA molecules (see, e.g., Bartel and Szostak, 1993, diaminopurine. Alternatively, the antisense nucleic acid can Science 261:14-11-1418). be produced biologically using an expression vector into 0157. The invention also encompasses nucleic acid mol which a nucleic acid has been Sub-cloned in an antisense ecules which form triple helical structures. For example, orientation (i.e., RNA transcribed from the inserted nucleic expression of a polypeptide of the invention can be inhibited acid will be of an antisense orientation to a target nucleic acid by targeting nucleotide sequences complementary to the of interest, described further in the following subsection). regulatory region of the gene encoding the polypeptide (e.g., 0154 The antisense nucleic acid molecules of the inven the promoter and/or enhancer) to form triple helical structures tion are typically administered to a subject or generated in situ that prevent transcription of the gene in target cells. See such that they hybridize with or bind to cellular mRNA and/or generally Helene (1991) Anticancer Drug Des. 6(6):569-84; genomic DNA encoding a polypeptide corresponding to a Helene (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher selected marker of the invention to thereby inhibit expression (1992) Bioassays 14(12):807-15. of the marker, e.g., by inhibiting transcription and/or transla 0158. In various embodiments, the nucleic acid molecules tion. The hybridization can be by conventional nucleotide of the invention can be modified at the base moiety, Sugar complementarity to form a stable duplex, or, for example, in moiety or phosphate backbone to improve, e.g., the stability, the case of an antisense nucleic acid molecule which binds to hybridization, or solubility of the molecule. For example, the DNA duplexes, through specific interactions in the major deoxyribose phosphate backbone of the nucleic acid mol groove of the double helix. Examples of a route of adminis ecules can be modified to generate peptide nucleic acid mol tration of antisense nucleic acid molecules of the invention ecules (see Hyrup et al., 1996, Bioorganic & Medicinal includes direct injection at a tissue site or infusion of the Chemistry 4(1): 5-23). As used herein, the terms “peptide antisense nucleic acid into an ovary-associated body fluid. nucleic acids” or “PNAs refer to nucleic acid mimics, e.g., Alternatively, antisense nucleic acid molecules can be modi DNA mimics, in which the deoxyribose phosphate backbone fied to target selected cells and then administered systemi is replaced by a pseudopeptide backbone and only the four cally. For example, for systemic administration, antisense natural nucleobases are retained. The neutral backbone of molecules can be modified such that they specifically bind to PNAS has been shown to allow for specific hybridization to receptors or antigens expressed on a selected cell Surface, DNA and RNA under conditions of low ionic strength. The e.g., by linking the antisense nucleic acid molecules to pep synthesis of PNA oligomers can be performed using standard tides or antibodies which bind to cell surface receptors or Solid phase peptide synthesis protocols as described in Hyrup antigens. The antisense nucleic acid molecules can also be et al. (1996), supra; Perry-O'Keefeet al. (1996) Proc. Natl. delivered to cells using the vectors described herein. To Acad. Sci. USA 93:14670-675. US 2009/0297536A1 Dec. 3, 2009

0159 PNAs can be used in therapeutic and diagnostic different portions of the nucleic acid in Such an orientation applications. For example, PNAS can be used as antisense or that when the complementary regions are annealed with one antigene agents for sequence-specific modulation of gene another, fluorescence of the fluorophore is quenched by the expression by, e.g., inducing transcription or translation quencher. When the complementary regions of the nucleic arrestor inhibiting replication. PNAS can also be used, e.g., in acid molecules are not annealed with one another, fluores the analysis of single base pair mutations in a gene by, e.g., cence of the fluorophore is quenched to a lesser degree. PNA directed PCR clamping; as artificial restriction enzymes Molecular beacon nucleic acid molecules are described, for when used in combination with other enzymes, e.g., S1 example, in U.S. Pat. No. 5,876,930. nucleases (Hyrup (1996), supra; or as probes or primers for DNA sequence and hybridization (Hyrup, 1996, supra; Perry IV. Isolated Proteins and Antibodies O'Keefe et al., 1996, Proc. Natl. Acad. Sci. USA 93:14670 675). 0163. One aspect of the invention pertains to isolated pro 0160. In another embodiment, PNAS can be modified, e.g., teins which correspond to individual markers of the inven to enhance their stability or cellular uptake, by attaching tion, and biologically active portions thereof, as well as lipophilic or other helper groups to PNA, by the formation of polypeptide fragments suitable for use as immunogens to PNA-DNA chimeras, or by the use of liposomes or other raise antibodies directed against a polypeptide corresponding techniques of drug delivery known in the art. For example, to a marker of the invention. In one embodiment, the native PNA-DNA chimeras can be generated which can combine the polypeptide corresponding to a marker can be isolated from advantageous properties of PNA and DNA. Such chimeras cells or tissue sources by an appropriate purification scheme allow DNA recognition enzymes, e.g., RNASE H and DNA using standard protein purification techniques. In another polymerases, to interact with the DNA portion while the PNA embodiment, polypeptides corresponding to a marker of the portion would provide high binding affinity and specificity. invention are produced by recombinant DNA techniques. PNA-DNA chimeras can be linked using linkers of appropri Alternative to recombinant expression, a polypeptide corre ate lengths selected in terms of base Stacking, number of sponding to a marker of the invention can be synthesized bonds between the nucleobases, and orientation (Hyrup, chemically using standard peptide synthesis techniques. 1996, supra). The synthesis of PNA-DNA chimeras can be 0164. An "isolated” or “purified protein or biologically performed as described in Hyrup (1996), supra, and Finnet al. active portion thereof is substantially free of cellular material (1996) Nucleic Acids Res. 24(17):3357-63. For example, a or other contaminating proteins from the cell or tissue source DNA chain can be synthesized on a solid support using stan from which the protein is derived, or substantially free of dard phosphoramidite coupling chemistry and modified chemical precursors or other chemicals when chemically Syn nucleoside analogs. Compounds such as 5'-(4-methoxytrityl) thesized. The language “substantially free of cellular mate amino-5'-deoxy-thymidine phosphoramidite can be used as a rial' includes preparations of protein in which the protein is link between the PNA and the 5' end of DNA (Maget al., separated from cellular components of the cells from which it 1989, Nucleic Acids Res. 17:5973-88). PNA monomers are is isolated or recombinantly produced. Thus, protein that is then coupled in a step-wise manner to produce a chimeric substantially free of cellular material includes preparations of molecule with a 5' PNA segment and a 3' DNA segment (Finn protein having less than about 30%. 20%, 10%, or 5% (by dry et al., 1996, Nucleic Acids Res. 24(17):3357-63). Alterna weight) of heterologous protein (also referred to herein as a tively, chimeric molecules can be synthesized with a 5' DNA “contaminating protein'). When the protein or biologically segment and a 3' PNA segment (Peterser et al., 1975, Bioor active portion thereof is recombinantly produced, it is also ganic Med. Chem. Lett. 5:1119-11124). preferably substantially free of culture medium, i.e., culture 0161 In other embodiments, the oligonucleotide can medium represents less than about 20%, 10%, or 5% of the include other appended groups such as peptides (e.g., for volume of the protein preparation. When the protein is pro targeting host cell receptors in vivo), or agents facilitating duced by chemical synthesis, it is preferably substantially transport across the cell membrane (see, e.g., Letsinger et al., free of chemical precursors or other chemicals, i.e., it is 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et separated from chemical precursors or other chemicals which al., 1987, Proc. Natl. Acad. Sci. USA 84:648-652; PCT Pub are involved in the synthesis of the protein. Accordingly such lication No. WO 88/09810) or the blood-brain barrier (see, preparations of the protein have less than about 30%, 20%, e.g., PCT Publication No. WO 89/10134). In addition, oligo 10%, 5% (by dry weight) of chemical precursors or com nucleotides can be modified with hybridization-triggered pounds other than the polypeptide of interest. cleavage agents (see, e.g., Krol et al., 1988, Bio/Techniques 0.165 Biologically active portions of a polypeptide corre 6:958-976) or intercalating agents (see, e.g., Zon, 1988, sponding to a marker of the invention include polypeptides Pharm. Res. 5:539-549). To this end, the oligonucleotide can comprising amino acid sequences Sufficiently identical to or be conjugated to another molecule, e.g., a peptide, hybridiza derived from the amino acid sequence of the protein corre tion triggered cross-linking agent, transportagent, hybridiza sponding to the marker (e.g., the protein encoded by the tion-triggered cleavage agent, etc. nucleic acid molecules listed in Tables 4 or 5), which include 0162 The invention also includes molecular beacon fewer amino acids than the full length protein, and exhibit at nucleic acid molecules having at least one region which is least one activity of the corresponding full-length protein. complementary to a nucleic acid molecule of the invention, Typically, biologically active portions comprise a domain or Such that the molecular beacon is useful for quantitating the motif with at least one activity of the corresponding protein. presence of the nucleic acid molecule of the invention in a A biologically active portion of a protein of the invention can sample. A "molecular beacon’ nucleic acid is a nucleic acid be a polypeptide which is, for example, 10, 25, 50, 100 or molecule comprising a pair of complementary regions and more amino acids in length. Moreover, other biologically having a fluorophore and a fluorescent quencher associated active portions, in which other regions of the protein are therewith. The fluorophore and quencher are associated with deleted, can be prepared by recombinant techniques and US 2009/0297536A1 Dec. 3, 2009

evaluated for one or more of the functional activities of the rithm for comparing nucleotide or amino acid sequences, a native form of a polypeptide of the invention. PAM120 weight residue table can, for example, be used with 0166 Preferred polypeptides have an amino acid a k-tuple value of 2. sequence of a protein encoded by a nucleic acid molecule 0169. The percent identity between two sequences can be listed in Tables 4 or 5. Other useful proteins are substantially determined using techniques similar to those described identical (e.g., at least about 40%, preferably 50%, 60%, above, with or without allowing gaps. In calculating percent 70%, 80%, 90%, 95%, or 99%) to one of these sequences and identity, only exact matches are counted. retain the functional activity of the protein of the correspond 0170 The invention also provides chimeric or fusion pro ing naturally-occurring protein yet differ in amino acid teins corresponding to a marker of the invention. As used herein, a “chimeric protein’ or “fusion protein comprises all sequence due to natural allelic variation or mutagenesis. or part (preferably a biologically active part) of a polypeptide 0167 To determine the percent identity of two amino acid corresponding to a marker of the invention operably linked to sequences or of two nucleic acids, the sequences are aligned a heterologous polypeptide (i.e., a polypeptide other than the for optimal comparison purposes (e.g., gaps can be intro polypeptide corresponding to the marker). Within the fusion duced in the sequence of a first amino acid or nucleic acid protein, the term “operably linked' is intended to indicate that sequence for optimal alignment with a second amino or the polypeptide of the invention and the heterologous nucleic acid sequence). The amino acid residues or nucle polypeptide are fused in-frame to each other. The heterolo otides at corresponding amino acid positions or nucleotide gous polypeptide can be fused to the amino-terminus or the positions are then compared. When a position in the first carboxyl-terminus of the polypeptide of the invention. sequence is occupied by the same amino acid residue or 0171 One useful fusion protein is a GST fusion protein in nucleotide as the corresponding position in the second which a polypeptide corresponding to a marker of the inven sequence, then the molecules are identical at that position. tion is fused to the carboxyl terminus of GST sequences. Such The percent identity between the two sequences is a function fusion proteins can facilitate the purification of a recombinant of the number of identical positions shared by the sequences polypeptide of the invention. (i.e., 96 identity of identical positions/total # of positions (e.g., 0172. In another embodiment, the fusion protein contains overlapping positions)x100). In one embodiment the two a heterologous signal sequence at its amino terminus. For sequences are the same length. example, the native signal sequence of a polypeptide corre sponding to a marker of the invention can be removed and 0168 The determination of percent identity between two replaced with a signal sequence from another protein. For sequences can be accomplished using a mathematical algo example, the gp67 secretory sequence of the baculovirus rithm. A preferred, non-limiting example of a mathematical envelope protein can be used as a heterologous signal algorithm utilized for the comparison of two sequences is the sequence (Ausubel et al.,ed., Current Protocols in Molecular algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. Biology, John Wiley & Sons, NY, 1992). Other examples of USA 87:2264-2268, modified as in Karlin and Altschul eukaryotic heterologous signal sequences include the secre (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an tory sequences of melittin and human placental alkaline phos algorithm is incorporated into the NBLAST and XBLAST phatase (Stratagene; LaJolla, Calif.). In yet another example, programs of Altschul, et al. (1990).J. Mol. Biol. 215:403-410. useful prokaryotic heterologous signal sequences include the BLAST nucleotide searches can be performed with the phoA secretory signal (Sambrook et al., Supra) and the pro NBLAST program, score=100, wordlength=12 to obtain tein A secretory signal (Pharmacia Biotech; Piscataway, nucleotide sequences homologous to a nucleic acid mol N.J.). ecules of the invention. BLAST protein searches can be per 0173. In yet another embodiment, the fusion protein is an formed with the XBLAST program, score=50, wordlength=3 immunoglobulin fusion protein in which all or part of a to obtain amino acid sequences homologous to a protein polypeptide corresponding to a marker of the invention is molecules of the invention. To obtain gapped alignments for fused to sequences derived from a member of the immuno comparison purposes, Gapped BLAST can be utilized as globulin protein family. The immunoglobulin fusion proteins described in Altschul et al. (1997) Nucleic Acids Res. of the invention can be incorporated into pharmaceutical 25:3389-3402. Alternatively, PSI-Blast can be used to per compositions and administered to a subject to inhibit an inter form an iterated search which detects distant relationships action between a ligand (soluble or membrane-bound) and a between molecules. When utilizing BLAST, Gapped protein on the Surface of a cell (receptor), to thereby suppress BLAST, and PSI-Blast programs, the default parameters of signal transduction in vivo. The immunoglobulin fusion pro the respective programs (e.g., XBLAST and NBLAST) can tein can be used to affect the bioavailability of a cognate be used. See http://www.ncbi.nlm.nih.gov. Another pre ligand of a polypeptide of the invention. Inhibition of ligand/ ferred, non-limiting example of a mathematical algorithm receptor interaction can be useful therapeutically, both for utilized for the comparison of sequences is the algorithm of treating proliferative and differentiative disorders and for Myers and Miller, (1988) Comput Appl Biosci, 4:11-7. Such modulating (e.g. promoting or inhibiting) cell Survival. More an algorithm is incorporated into the ALIGN program (ver over, the immunoglobulin fusion proteins of the invention can sion 2.0) which is part of the GCG sequence alignment soft be used as immunogens to produce antibodies directed ware package. When utilizing the ALIGN program for com against a polypeptide of the invention in a Subject, to purify paring amino acid sequences, a PAM120 weight residue ligands and in Screening assays to identify molecules which table, a gap length penalty of 12, and a gap penalty of 4 can be inhibit the interaction of receptors with ligands. used. Yet another useful algorithm for identifying regions of 0.174 Chimeric and fusion proteins of the invention can be local sequence similarity and alignment is the FASTA algo produced by standard recombinant DNA techniques. In rithm as described in Pearson and Lipman (1988) Proc. Natl. another embodiment, the fusion gene can be synthesized by Acad. Sci. USA 85:2444-2448. When using the FASTA algo conventional techniques including automated DNA synthe US 2009/0297536A1 Dec. 3, 2009 sizers. Alternatively, PCR amplification of gene fragments (e.g., for phage display). There are a variety of methods which can be carried out using anchor primers which give rise to can be used to produce libraries of potential variants of the complementary overhangs between two consecutive gene polypeptides of the invention from a degenerate oligonucle fragments which can Subsequently be annealed and re-ampli otide sequence. Methods for synthesizing degenerate oligo fied to generate a chimeric gene sequence (see, e.g., Ausubel nucleotides are known in the art (see, e.g., Narang, 1983, et al., Supra). Moreover, many expression vectors are com Tetrahedron 39:3; Itakura et al., 1984, Annu. Rev. Biochem. mercially available that already encode a fusion moiety (e.g., 53:323: Itakura et al., 1984, Science 198:1056; Ike et al., 1983 a GST polypeptide). A nucleic acid encoding a polypeptide of Nucleic Acid Res. 11:477). the invention can be cloned into Such an expression vector 0178. In addition, libraries of fragments of the coding Such that the fusion moiety is linked in-frame to the polypep sequence of a polypeptide corresponding to a marker of the tide of the invention. invention can be used to generate a variegated population of 0.175. A signal sequence can be used to facilitate secretion polypeptides for Screening and Subsequent selection of vari and isolation of the secreted protein or other proteins of ants. For example, a library of coding sequence fragments can interest. Signal sequences are typically characterized by a be generated by treating a double stranded PCR fragment of core of hydrophobic amino acids which are generally cleaved the coding sequence of interest with a nuclease under condi from the mature protein during secretion in one or more tions wherein nicking occurs only about once per molecule, cleavage events. Such signal peptides contain processing sites denaturing the double stranded DNA, renaturing the DNA to that allow cleavage of the signal sequence from the mature form double stranded DNA which can include sense/anti proteins as they pass through the Secretory pathway. Thus, the sense pairs from different nicked products, removing single invention pertains to the described polypeptides having a stranded portions from reformed duplexes by treatment with signal sequence, as well as to polypeptides from which the S1 nuclease, and ligating the resulting fragment library into signal sequence has been proteolytically cleaved (i.e., the an expression vector. By this method, an expression library cleavage products). In one embodiment, a nucleic acid can be derived which encodes amino terminal and internal sequence encoding a signal sequence can be operably linked fragments of various sizes of the protein of interest. in an expression vector to a protein of interest, such as a 0179. Several techniques are known in the art for screen protein which is ordinarily not secreted or is otherwise diffi ing gene products of combinatorial libraries made by point cult to isolate. The signal sequence directs secretion of the mutations or truncation, and for screening cDNA libraries for protein, such as from a eukaryotic host into which the expres gene products having a selected property. The most widely sion vector is transformed, and the signal sequence is subse used techniques, which are amenable to high throughput quently or concurrently cleaved. The protein can then be analysis, for screening large gene libraries typically include readily purified from the extracellular medium by art recog cloning the gene library into replicable expression vectors, nized methods. Alternatively, the signal sequence can be transforming appropriate cells with the resulting library of linked to the protein of interest using a sequence which facili vectors, and expressing the combinatorial genes under con tates purification, such as with a GST domain. ditions in which detection of a desired activity facilitates 0176 The present invention also pertains to variants of the isolation of the vector encoding the gene whose product was polypeptides corresponding to individual markers of the detected. Recursive ensemble mutagenesis (REM), a tech invention. Such variants have an altered amino acid sequence nique which enhances the frequency of functional mutants in which can function as either agonists (mimetics) orasantago the libraries, can be used in combination with the screening nists. Variants can be generated by mutagenesis, e.g., discrete assays to identify variants of a protein of the invention (Arkin point mutation or truncation. An agonist can retain Substan and Yourvan, 1992, Proc. Natl. Acad. Sci. USA 89:7811 tially the same, or a subset, of the biological activities of the 7815; Delgrave et al., 1993, Protein Engineering 6(3):327 naturally occurring form of the protein. An antagonist of a 331). protein can inhibit one or more of the activities of the natu 0180. An isolated polypeptide corresponding to a marker rally occurring form of the protein by, for example, competi of the invention, or a fragment thereof, can be used as an tively binding to a downstream or upstream member of a immunogen to generate antibodies using standard techniques cellular signaling cascade which includes the protein of inter for polyclonal and monoclonal antibody preparation. The est. Thus, specific biological effects can be elicited by treat full-length polypeptide or protein can be used or, alterna ment with a variant of limited function. Treatment of a subject tively, the invention provides antigenic peptide fragments for with a variant having a subset of the biological activities of the use as immunogens. The antigenic peptide of a protein of the naturally occurring form of the protein can have fewer side invention comprises at least 8 (preferably 10, 15, 20, or 30 or effects in a subject relative to treatment with the naturally more) amino acid residues of the amino acid sequence of one occurring form of the protein. of the polypeptides of the invention, and encompasses an 0177 Variants of a protein of the invention which function epitope of the protein such that an antibody raised against the as either agonists (mimetics) or as antagonists can be identi peptide forms a specific immune complex with a marker of fied by Screening combinatorial libraries of mutants, e.g., the invention to which the protein corresponds. Preferred truncation mutants, of the protein of the invention foragonist epitopes encompassed by the antigenic peptide are regions or antagonist activity. In one embodiment, a variegated that are located on the Surface of the protein, e.g., hydrophilic library of variants is generated by combinatorial mutagenesis regions. Hydrophobicity sequence analysis, hydrophilicity at the nucleic acid level and is encoded by a variegated gene sequence analysis, or similar analyses can be used to identify library. A variegated library of variants can be produced by, hydrophilic regions. for example, enzymatically ligating a mixture of synthetic 0181 An immunogen typically is used to prepare antibod oligonucleotides into gene sequences such that a degenerate ies by immunizing a Suitable (i.e. immunocompetent) subject set of potential protein sequences is expressible as individual Such as a rabbit, goat, mouse, or other mammal or vertebrate. polypeptides, or alternatively, as a set of larger fusion proteins An appropriate immunogenic preparation can contain, for US 2009/0297536A1 Dec. 3, 2009

example, recombinantly-expressed or chemically-synthe cation No. WO 92/20791; PCT Publication No. WO sized polypeptide. The preparation can further include an 92/15679; PCT Publication No. WO 93/01288: PCT Publi adjuvant, such as Freund's complete or incomplete adjuvant, cation No. WO 92/01047: PCT Publication No. WO or a similar immunostimulatory agent. 92/09690; PCT Publication No. WO 90/02809; Fuchs et al. 0182. Accordingly, another aspect of the invention per (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum. tains to antibodies directed against a polypeptide of the inven Antibod. Hybridomas 3:81–85; Huse et al. (1989) Science tion. The terms “antibody” and “antibody substance' as used 246:1275-1281; Griffiths et al. (1993) EMBO.J. 12:725-734. interchangeably herein refer to immunoglobulin molecules 0185. Additionally, recombinant antibodies, such as chi and immunologically active portions of immunoglobulin meric and humanized monoclonal antibodies, comprising molecules, i.e., molecules that contain an antigenbinding site both human and non-human portions, which can be made which specifically binds an antigen, such as a polypeptide of using standard recombinant DNA techniques, are within the the invention. A molecule which specifically binds to a given Scope of the invention. Such chimeric and humanized mono polypeptide of the invention is a molecule which binds the clonal antibodies can be produced by recombinant DNA tech polypeptide, but does not substantially bind other molecules niques known in the art, for example using methods described in a sample, e.g., a biological sample, which naturally con in PCT Publication No. WO 87/02671; European Patent tains the polypeptide. Examples of immunologically active Application 184, 187: European Patent Application 171.496; portions of immunoglobulin molecules include F(ab) and European Patent Application 173,494; PCT Publication No. F(ab')2 fragments which can be generated by treating the WO 86/01533; U.S. Pat. No. 4,816,567; European Patent antibody with an enzyme such as pepsin. The invention pro Application 125,023: Better et al. (1988) Science 240: 1041 vides polyclonal and monoclonal antibodies. The term 1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439 "monoclonal antibody” or "monoclonal antibody composi 3443; Liu et al. (1987).J. Immunol. 139:3521-3526; Sun et al. tion', as used herein, refers to a population of antibody mol (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et ecules that contain only one species of an antigenbinding site al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) capable of immunoreacting with a particular epitope. Nature 314:446-449; and Shaw et al. (1988).J. Natl. Cancer 0183 Polyclonal antibodies can be prepared as described Inst. 80: 1553-1559); Morrison (1985) Science 229:1202 above by immunizing a suitable subject with a polypeptide of 1207: Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No. the invention as an immunogen. The antibody titer in the 5.225,539; Jones et al. (1986) Nature 321:552-525; Verho immunized subject can be monitored over time by standard eyan et al. (1988) Science 239:1534; and Beidler et al. (1988) techniques, such as with an enzyme linked immunosorbent J. Immunol. 141:4053-4060. assay (ELISA) using immobilized polypeptide. If desired, the 0186 Completely human antibodies are particularly desir antibody molecules can be harvested or isolated from the able for therapeutic treatment of human subjects. Such anti subject (e.g., from the blood or serum of the subject) and bodies can be produced using transgenic mice which are further purified by well-known techniques, such as protein A incapable of expressing endogenous immunoglobulin heavy chromatography to obtain the IgG fraction. At an appropriate and light chains genes, but which can express human heavy time after immunization, e.g., when the specific antibody and light chain genes. The transgenic mice are immunized in titers are highest, antibody-producing cells can be obtained the normal fashion with a selected antigen, e.g., all or a from the subject and used to prepare monoclonal antibodies portion of a polypeptide corresponding to a marker of the by standard techniques, such as the hybridoma technique invention. Monoclonal antibodies directed against the anti originally described by Kohler and Milstein (1975) Nature gen can be obtained using conventional hybridoma technol 256:495-497, the human B cell hybridoma technique (see ogy. The human immunoglobulin transgenes harbored by the Kozbor et al., 1983, Immunol. Today 4:72), the EBV-hybri transgenic mice rearrange during B cell differentiation, and doma technique (see Cole et al., pp. 77-96 In Monoclonal Subsequently undergo class Switching and somatic mutation. Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985) or Thus, using such a technique, it is possible to produce thera trioma techniques. The technology for producing hybridomas peutically useful IgG, IgA and IgE antibodies. For an over is well known (see generally Current Protocols in Immunol View of this technology for producing human antibodies, see ogy, Coligan et al. ed., John Wiley & Sons, New York, 1994). Lonberg and Huszar (1995) Int. Rev. Immunol. 13:65-93). For Hybridoma cells producing a monoclonal antibody of the a detailed discussion of this technology for producing human invention are detected by screening the hybridoma culture antibodies and human monoclonal antibodies and protocols Supernatants for antibodies that bind the polypeptide of inter for producing such antibodies, see, e.g., U.S. Pat. No. 5,625, est, e.g., using a standard ELISA assay. 126; U.S. Pat. No. 5,633,425; U.S. Pat. No. 5,569,825: U.S. 0184 Alternative to preparing monoclonal antibody-se Pat. No. 5,661.016; and U.S. Pat. No. 5,545,806. In addition, creting hybridomas, a monoclonal antibody directed againsta companies such as Abgenix, Inc. (Freemont, Calif.), can be polypeptide of the invention can be identified and isolated by engaged to provide human antibodies directed against a Screening a recombinant combinatorial immunoglobulin Selected antigen using technology similar to that described library (e.g., an antibody phage display library) with the above. polypeptide of interest. Kits for generating and screening 0187 Completely human antibodies which recognize a phage display libraries are commercially available (e.g., the Selected epitope can be generated using a technique referred Pharmacia Recombinant Phage Antibody System, Catalog to as 'guided selection. In this approach a selected non No. 27-94.00-01; and the Stratagene SurfzAP Phage Display human monoclonal antibody, e.g., a murine antibody, is used Kit, Catalog No. 240612). Additionally, examples of methods to guide the selection of a completely human antibody rec and reagents particularly amenable for use in generating and ognizing the same epitope (Jespers et al., 1994, Bio/technol screening antibody display library can be found in, for ogy 12:899-903). example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 0188 An antibody, antibody derivative, or fragment 92/18619; PCT Publication No. WO 91/1.7271; PCT Publi thereof, which specifically binds a marker of the invention US 2009/0297536A1 Dec. 3, 2009 20 which is overexpressed in cancer (e.g., a marker set forth in of transporting another nucleic acid to which it has been Table 5), may be used to inhibit activity of a marker, e.g., a linked. One type of vector is a “plasmid', which refers to a marker set forth in Table 5, and therefore may be administered circular double stranded DNA loop into which additional to a Subject to treat, inhibit, or prevent cancer in the Subject. DNA segments can be ligated. Another type of vector is a viral Furthermore, conjugated antibodies may also be used to treat, vector, wherein additional DNA segments can be ligated into inhibit, or prevent cancer in a Subject. Conjugated antibodies, the viral genome. Certain vectors are capable of autonomous preferably monoclonal antibodies, or fragments thereof, are replication in a host cell into which they are introduced (e.g., antibodies which are joined to drugs, toxins, or radioactive bacterial vectors having a bacterial origin of replication and atoms, and used as delivery vehicles to deliver those sub episomal mammalian vectors). Other vectors (e.g., non-epi stances directly to cancer cells. The antibody, e.g., an anti Somal mammalian vectors) are integrated into the genome of body which specifically binds a marker of the invention (e.g., a host cell upon introduction into the host cell, and thereby are a marker listed in Table 5), is administered to a subject and replicated along with the host genome. Moreover, certain binds the marker, thereby delivering the toxic substance to the vectors, namely expression vectors, are capable of directing cancer cell, minimizing damage to normal cells in other parts the expression of genes to which they are operably linked. In of the body. general, expression vectors of utility in recombinant DNA 0189 Conjugated antibodies are also referred to as techniques are often in the form of plasmids (vectors). How "tagged,” “labeled, or “loaded.” Antibodies with chemo ever, the invention is intended to include such other forms of therapeutic agents attached are generally referred to as expression vectors, such as viral vectors (e.g., replication chemolabeled. Antibodies with radioactive particles attached defective retroviruses, adenoviruses and adeno-associated are referred to as radiolabeled, and this type of therapy is viruses), which serve equivalent functions. known as radioimmunotherapy (RIT). Aside from being used 0193 The recombinant expression vectors of the inven to treat cancer, radiolabeled antibodies can also be used to tion comprise a nucleic acid of the invention in a form Suitable detect areas of cancer spread in the body. Antibodies attached for expression of the nucleic acid in a host cell. This means to toxins are called immunotoxins. that the recombinant expression vectors include one or more 0190. Immunotoxins are made by attaching toxins (e.g., regulatory sequences, selected on the basis of the host cells to poisonous Substances from plants or bacteria) to monoclonal be used for expression, which is operably linked to the nucleic antibodies. Immunotoxins may be produced by attaching acid sequence to be expressed. Within a recombinant expres monoclonal antibodies to bacterial toxins such as diphtherial sion vector, “operably linked' is intended to mean that the toxin (DT) or pseudomonal exotoxin (PE40), or to plant tox nucleotide sequence of interest is linked to the regulatory ins such as ricin A or saporin. sequence(s) in a manner which allows for expression of the 0191 An antibody directed against a polypeptide corre nucleotide sequence (e.g., in an in vitro transcription/transla sponding to a marker of the invention (e.g., a monoclonal tion system or in a host cell when the vector is introduced into antibody) can be used to isolate the polypeptide by standard the host cell). The term “regulatory sequence' is intended to techniques, such as affinity chromatography or immunopre include promoters, enhancers and other expression control cipitation. Moreover, such an antibody can be used to detect elements (e.g., polyadenylation signals). Such regulatory the marker (e.g., in a cellular lysate or cell Supernatant) in sequences are described, for example, in Goeddel, Methods in order to evaluate the level and pattern of expression of the Enzymology. Gene Expression Technology Vol. 185, Aca marker. The antibodies can also be used diagnostically to demic Press, San Diego, Calif. (1991). Regulatory sequences monitor protein levels in tissues or body fluids (e.g. in an include those which direct constitutive expression of a nucle ovary-associated body fluid) as part of a clinical testing pro otide sequence in many types of host cell and those which cedure, e.g., to, for example, determine the efficacy of a given direct expression of the nucleotide sequence only in certain treatment regimen. Detection can be facilitated by coupling host cells (e.g., tissue-specific regulatory sequences). It will the antibody to a detectable substance. Examples of detect be appreciated by those skilled in the art that the design of the able Substances include various enzymes, prosthetic groups, expression vector can depend on Such factors as the choice of fluorescent materials, luminescent materials, bioluminescent the host cell to be transformed, the level of expression of materials, and radioactive materials. Examples of Suitable protein desired, and the like. The expression vectors of the enzymes include horseradish peroxidase, alkaline phos invention can be introduced into host cells to thereby produce phatase, B-galactosidase, or acetylcholinesterase; examples proteins or peptides, including fusion proteins or peptides, of Suitable prosthetic group complexes include streptavidin/ encoded by nucleic acids as described herein. biotin and avidin/biotin; examples of suitable fluorescent 0194 The recombinant expression vectors of the inven materials include umbelliferone, fluorescein, fluorescein tion can be designed for expression of a polypeptide corre isothiocyanate, rhodamine, dichlorotriazinylamine fluores sponding to a marker of the invention in prokaryotic (e.g., E. cein, dansylchloride or phycoerythrin; an example of alumi coli) or eukaryotic cells (e.g., insect cells using baculovirus nescent material includes luminol; examples of biolumines expression vectors}, yeast cells or mammalian cells). Suit cent materials include luciferase, luciferin, and aequorin, and able host cells are discussed further in Goeddel, supra. Alter examples of suitable radioactive material include 'I. 'I, natively, the recombinant expression vector can be tran SS or H. scribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. V. Recombinant Expression Vectors and Host Cells 0.195 Expression of proteins in prokaryotes is most often 0.192 Another aspect of the invention pertains to vectors, carried out in E. coli with vectors containing constitutive or preferably expression vectors, containing a nucleic acid inducible promoters directing the expression of either fusion encoding a polypeptide corresponding to a marker of the or non-fusion proteins. Fusion vectors add a number of amino invention (or a portion of such a polypeptide). As used herein, acids to a protein encoded therein, usually to the amino ter the term “vector” refers to a to nucleic acid molecule capable minus of the recombinant protein. Such fusion vectors typi US 2009/0297536A1 Dec. 3, 2009 cally serve three purposes: 1) to increase expression of 40. For other suitable expression systems for both prokaryotic recombinant protein; 2) to increase the solubility of the and eukaryotic cells see chapters 16 and 17 of Sambrook et recombinant protein; and 3) to aid in the purification of the al., Supra. recombinant protein by acting as a ligand in affinity purifica 0201 In another embodiment, the recombinant mamma tion. Often, in fusion expression vectors, a proteolytic cleav lian expression vector is capable of directing expression of age site is introduced at the junction of the fusion moiety and the nucleic acid preferentially in a particular cell type (e.g., the recombinant protein to enable separation of the recombi tissue-specific regulatory elements are used to express the nant protein from the fusion moiety Subsequent to purifica nucleic acid). Tissue-specific regulatory elements are known tion of the fusion protein. Such enzymes, and their cognate in the art. Non-limiting examples of Suitable tissue-specific recognition sequences, include Factor Xa, thrombin and promoters include the albumin promoter (liver-specific: Pink enterokinase. Typical fusion expression vectors include ert et al., 1987, Genes Dev. 1:268-277), lymphoid-specific pGEX (Pharmacia Biotech Inc.; Smith and Johnson, 1988, promoters (Calame and Eaton, 1988, Adv. Immunol. 43:235 Gene 67:31-40), pMAL (New England Biolabs, Beverly, 275), in particular promoters of T cell receptors (Winoto and Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse Baltimore, 1989, EMBO.J. 8:729-733) and immunoglobulins glutathione S-transferase (GST), maltose E binding protein, (Banerji et al., 1983, Cell 33:729-740; Queen and Baltimore, or protein A, respectively, to the target recombinant protein. 1983, Cell 33:741-748), neuron-specific promoters (e.g., the 0196. Examples of suitable inducible non-fusion E. coli neurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl. expression vectors include pTrc (Amann et al., 1988, Gene Acad. Sci. USA 86:5473-5477), pancreas-specific promoters 69:301-315) and pET 11d (Studier et al., p. 60-89, In Gene (Edlund et al., 1985, Science 230:912-916), and mammary Expression Technology Methods in Enzymology Vol. 185, gland-specific promoters (e.g., milk whey promoter, U.S. Pat. Academic Press, San Diego, Calif., 1991). Target gene No. 4,873.316 and European Application Publication No. expression from the pTrc vector relies on host RNA poly 264,166). Developmentally-regulated promoters are also merase transcription from a hybrid trp-lac fusion promoter. encompassed, for example the murine hoX promoters (Kessel Target gene expression from the pET 11d vector relies on and Gruss, 1990, Science 249:374-379) and the C-fetoprotein transcription from a T7 gn 10-lac fusion promoter mediated promoter (Camper and Tilghman, 1989, Genes Dev: 3:537 by a co-expressed viral RNA polymerase (T7 gn1). This viral 546). polymerase is supplied by host strains BL21 (DE3) or 0202 The invention further provides a recombinant HMS174(DE3) from a resident prophage harboring a T7 gn1 expression vector comprising a DNA molecule of the inven gene under the transcriptional control of the lacUV 5 pro tion cloned into the expression vector in an antisense orien moter. tation: That is, the DNA molecule is operably linked to a 0197) One strategy to maximize recombinant protein regulatory sequence in a manner which allows for expression expression in E. coli is to express the protein in a host bacteria (by transcription of the DNA molecule) of an RNA molecule with an impaired capacity to proteolytically cleave the recom which is antisense to the mRNA encoding a polypeptide of binant protein (Gottesman, p. 119-128. In Gene Expression the invention. Regulatory sequences operably linked to a Technology: Methods in Enzymology Vol. 185, Academic nucleic acid cloned in the antisense orientation can be chosen Press, San Diego, Calif., 1990. Another strategy is to alter the which direct the continuous expression of the antisense RNA nucleic acid sequence of the nucleic acid to be inserted into an molecule in a variety of cell types, for instance viral promot expression vector so that the individual codons for each ers and/or enhancers, or regulatory sequences can be chosen amino acid are those preferentially utilized in E. coli (Wada et which direct constitutive, tissue-specific or cell type specific al., 1992, Nucleic Acids Res. 20:2111-2118). Such alteration expression of antisense RNA. The antisense expression vec of nucleic acid sequences of the invention can be carried out tor can be in the form of a recombinant plasmid, phagemid, or by standard DNA synthesis techniques. attenuated virus in which antisense nucleic acids are pro 0198 In another embodiment, the expression vector is a duced under the control of a high efficiency regulatory region, yeast expression vector. Examples of vectors for expression the activity of which can be determined by the cell type into in yeast S. cerevisiae include pYepSec1 (Baldariet al., 1987, which the vector is introduced. For a discussion of the regu EMBO.J. 6:229-234), pMFa (Kurjan and Herskowitz, 1982, lation of gene expression using antisense genes see Weintraub Cell 30:933-943), p.JRY88 (Schultz et al., 1987, Gene et al., 1986, Trends in Genetics, Vol. 1 (1). 54:113-123), pYES2 (Invitrogen Corporation, San Diego, 0203. Another aspect of the invention pertains to host cells Calif.), and pPicz (Invitrogen Corp., San Diego, Calif.). into which a recombinant expression vector of the invention 0199 Alternatively, the expression vector is a baculovirus has been introduced. The terms "host cell' and “recombinant expression vector. Baculovirus vectors available for expres host cell are used interchangeably herein. It is understood sion of proteins in cultured insect cells (e.g., Sf 9 cells) that such terms refer not only to the particular subject cell but include the p Ac series (Smith et al., 1983, Mol. Cell Biol. to the progeny or potential progeny of Such a cell. Because 3:2156-2165) and the pVL series (Lucklow and Summers, certain modifications may occur in Succeeding generations 1989, Virology 170:31-39). due to either mutation or environmental influences, such 0200. In yet another embodiment, a nucleic acid of the progeny may not, in fact, be identical to the parent cell, but are invention is expressed in mammalian cells using a mamma still included within the scope of the term as used herein. lian expression vector. Examples of mammalian expression 0204. A host cell can be any prokaryotic (e.g., E. coli) or vectors include pCDM8 (Seed, 1987, Nature 329:840) and eukaryotic cell (e.g., insect cells, yeast or mammalian cells). pMT2PC (Kaufman et al., 1987, EMBO.J. 6:187-195). When 0205 Vector DNA can be introduced into prokaryotic or used in mammalian cells, the expression vector's control eukaryotic cells via conventional transformation or transfec functions are often provided by viral regulatory elements. For tion techniques. As used herein, the terms “transformation' example, commonly used promoters are derived from and “transfection' are intended to refer to a variety of art polyoma, Adenovirus 2, cytomegalovirus and Simian Virus recognized techniques for introducing foreign nucleic acid US 2009/0297536A1 Dec. 3, 2009 22 into a host cell, including calcium phosphate or calcium chlo duced into a cell of the animal, e.g., an embryonic cell of the ride co-precipitation, DEAE-dextran-mediated transfection, animal, prior to development of the animal. Transgenic ani lipofection, or electroporation. Suitable methods for trans mals also include inducible transgenic animals. Such as those forming or transfecting host cells can be found in Sambrook, described in, for example, Chan I. T., et al. (2004) J Clin et al. (Supra), and other laboratory manuals. Invest. 113(4):528-38 and Chin L. et al (1999) Nature 400 0206 For stable transfection of mammalian cells, it is (6743):468-72. known that, depending upon the expression vector and trans 0210. A transgenic animal of the invention can be created fection technique used, only a small fraction of cells may by introducing a nucleic acid encoding a polypeptide corre integrate the foreign DNA into their genome. In order to sponding to a marker of the invention into the male pronuclei identify and select these integrants, a gene that encodes a of a fertilized oocyte, e.g., by microinjection, retroviral infec selectable marker (e.g., for resistance to antibiotics) is gen tion, and allowing the oocyte to develop in a pseudopregnant erally introduced into the host cells along with the gene of female foster animal. Intronic sequences and polyadenylation interest. Preferred selectable markers include those which signals can also be included in the transgene to increase the confer resistance to drugs, such as G418, hygromycin and efficiency of expression of the transgene. A tissue-specific methotrexate. Cells stably transfected with the introduced regulatory sequence(s) can be operably linked to the trans nucleic acid can be identified by drug selection (e.g., cells that gene to direct expression of the polypeptide of the invention have incorporated the selectable marker gene will survive, to particular cells. Methods for generating transgenic animals while the other cells die). via embryo manipulation and microinjection, particularly 0207. A host cell of the invention, such as a prokaryotic or animals such as mice, have become conventional in the art eukaryotic host cell in culture, can be used to produce a and are described, for example, in U.S. Pat. Nos. 4.736,866 polypeptide corresponding to a marker of the invention. and 4,870,009, U.S. Pat. No. 4,873,191 and in Hogan, Accordingly, the invention further provides methods for pro Manipulating the Mouse Embryo, Cold Spring Harbor Labo ducing a polypeptide corresponding to a marker of the inven ratory Press, Cold Spring Harbor, N.Y., 1986. Similar meth tion using the host cells of the invention. In one embodiment, ods are used for production of other transgenic animals. A the method comprises culturing the host cell of invention transgenic founder animal can be identified based upon the (into which a recombinant expression vector encoding a presence of the transgene in its genome and/or expression of polypeptide of the invention has been introduced) in a Suit mRNA encoding the transgene in tissues or cells of the ani able medium such that the marker is produced. In another mals. A transgenic founder animal can then be used to breed embodiment, the method further comprises isolating the additional animals carrying the transgene. Moreover, trans marker polypeptide from the medium or the host cell. genic animals carrying the transgene can further be bred to 0208. The host cells of the invention can also be used to other transgenic animals carrying other transgenes. produce nonhuman transgenic animals. For example, in one 0211 To create an homologous recombinant animal, a embodiment, a host cell of the invention is a fertilized oocyte vector is prepared which contains at least a portion of a gene or an embryonic stem cell into which sequences encoding a encoding a polypeptide corresponding to a marker of the polypeptide corresponding to a marker of the invention have invention into which a deletion, addition or substitution has been introduced. Such host cells can then be used to create been introduced to thereby alter, e.g., functionally disrupt, the non-human transgenic animals in which exogenous gene. In a preferred embodiment, the vector is designed Such sequences encoding a marker protein of the invention have that, upon homologous recombination, the endogenous gene been introduced into their genome or homologous recombi is functionally disrupted (i.e., no longer encodes a functional nant animals in which endogenous gene(s) encoding a protein; also referred to as a “knock out vector). Alterna polypeptide corresponding to a marker of the invention tively, the vector can be designed such that, upon homologous sequences have been altered. Such animals are useful for recombination, the endogenous gene is mutated or otherwise studying the function and/or activity of the polypeptide cor altered but still encodes functional protein (e.g., the upstream responding to the marker, for identifying and/or evaluating regulatory region can be altered to thereby alter the expres modulators of polypeptide activity, as well as in pre-clinical sion of the endogenous protein). In the homologous recom testing of therapeutics or diagnostic molecules, for marker bination vector, the altered portion of the gene is flanked at its discovery or evaluation, e.g., therapeutic and diagnostic 5' and 3' ends by additional nucleic acid of the gene to allow marker discovery or evaluation, or as Surrogates of drug effi for homologous recombination to occur between the exog cacy and specificity. enous gene carried by the vector and an endogenous gene in 0209. As used herein, a “transgenic animal' is a non an embryonic stem cell. The additional flanking nucleic acid human animal, preferably a mammal, more preferably a sequences are of sufficient length for Successful homologous rodent such as a rat or mouse, in which one or more of the cells recombination with the endogenous gene. Typically, several of the animal includes a transgene. Other examples of trans kilobases of flanking DNA (both at the 5' and 3' ends) are genic animals include non-human primates, sheep, dogs, included in the vector (see, e.g., Thomas and Capecchi, 1987, cows, goats, chickens, amphibians, etc. A transgene is exog Cell 51:503 for a description of homologous recombination enous DNA which is integrated into the genome of a cell from vectors). The vector is introduced into an embryonic stem cell which a transgenic animal develops and which remains in the line (e.g., by electroporation) and cells in which the intro genome of the mature animal, thereby directing the expres duced gene has homologously recombined with the endog sion of an encoded gene product in one or more cell types or enous gene are selected (see, e.g., Lietal., 1992, Cell 69:915). tissues of the transgenic animal. As used herein, an "homolo The selected cells are then injected into a blastocyst of an gous recombinant animal' is a non-human animal, preferably animal (e.g., a mouse) to form aggregation chimeras (see, a mammal, more preferably amouse, in which an endogenous e.g., Bradley, Teratocarcinomas and Embryonic Stem Cells. gene has been altered by homologous recombination between A Practical Approach, Robertson, Ed., IRL, Oxford, 1987, the endogenous gene and an exogenous DNA molecule intro pp. 113-152). A chimeric embryo can then be implanted into US 2009/0297536A1 Dec. 3, 2009 a Suitable pseudopregnant female foster animal and the ited by inhibiting amount, e.g., expression or protein level. embryo brought to term. Progeny harboring the homolo and/or activity of those markers. Likewise, cancer character gously recombined DNA in their germ cells can be used to ized by a decrease in the amount and/or activity, or a change breed animals in which all cells of the animal contain the in the structure, of one or more markers listed in Table 4 (e.g., homologously recombined DNA by germline transmission of a marker that is shown to be decreased in cancer), can be the transgene. Methods for constructing homologous recom inhibited by enhancing amount, e.g., expression or protein bination vectors and homologous recombinant animals are level, and/or activity of those markers. described further in Bradley (1991) Current Opinion in Bio/ 0216. Accordingly, another aspect of the invention per Technology 2:823-829 and in PCT Publication NOS. WO tains to methods for treating a Subject Suffering from cancer. 90/11354, WO 91/01140, WO92/0968, and WO 93/04169. These methods involve administering to a subject a com 0212. In another embodiment, transgenic non-human ani pound which modulates amount and/or activity of one or mals can be produced which contain selected systems which more markers of the invention. For example, methods of allow for regulated expression of the transgene. One example treatment or prevention of cancer include administering to a of such a system is the cre/loxP recombinase system of bac Subject a compound which decreases the amount and/or activ teriophage P1. For a description of the cre/loxP recombinase ity of one or more markers listed in Table 5 (e.g., a marker that system, see, e.g., LakSo et al. (1992) Proc. Natl. Acad. Sci. was shown to be increased in cancer). Compounds, e.g., USA 89:6232-6236. Another example of a recombinase sys antagonists, which may be used to inhibit amount and/or tem is the FLP recombinase system of Saccharomyces cer activity of a marker listed in Table 5, to thereby treat or evisiae (O'Gorman et al., 1991, Science 251:1351-1355). If a prevent cancer include antibodies (e.g., conjugated antibod cre/loXP recombinase system is used to regulate expression of ies), Small molecules, RNA interfering agents, e.g., siRNA the transgene, animals containing transgenes encoding both molecules, ribozymes, and antisense oligonucleotides. In one the Cre recombinase and a selected protein are required. Such embodiment, an antibody used for treatment is conjugated to animals can be provided through the construction of “double' a toxin, a chemotherapeutic agent, or radioactive particles. transgenic animals, e.g., by mating two transgenic animals, 0217 Methods of treatment or prevention of cancer also one containing a transgene encoding a selected protein and include administering to a Subject a compound which the other containing a transgene encoding a recombinase. increases the amount and/or activity of one or more markers 0213 Clones of the non-human transgenic animals listed in Table 4 (e.g., a marker that was shown to be described herein can also be produced according to the meth decreased in cancer). Compounds, e.g., agonists, which may ods described in Wilmut etal. (1997) Nature 385:810-813 and be used to increase expression or activity of a marker listed in PCT Publication NOS. WO 97/O7668 and WO 97/O7669. Table 4, to thereby treat or prevent cancer include small molecules, peptides, peptoids, peptidomimetics, and VI. Methods of Treatment polypeptides. 0218. Small molecules used in the methods of the inven 0214. The present invention provides for both prophylac tion include those which inhibit a protein-protein interaction tic and therapeutic methods of treating a subject, e.g., a and thereby either increase or decrease marker amount and/or human, who has or is at risk of (or Susceptible to) cancer, e.g., activity. Furthermore, modulators, e.g., Small molecules, pancreatic cancer. As used herein, “treatment of a subject which cause re-expression of silenced genes, e.g., tumor Sup includes the application or administration of a therapeutic pressors, are also included herein. For example, such mol agent to a Subject, or application or administration of a thera ecules include compounds which interfere with DNA binding peutic agent to a cell or tissue from a Subject, who has a or methyltransferas activity. diseases or disorder, has a symptom of a disease or disorder, 0219. An aptamer may also be used to modulate, e.g., or is at risk of (or susceptible to) a disease or disorder, with the increase or inhibit expression or activity of a marker of the purpose of curing, inhibiting, healing, alleviating, relieving, invention to thereby treat, prevent or inhibit cancer. Aptamers altering, remedying, ameliorating, improving, or affecting are DNA or RNA molecules that have been selected from the disease or disorder, the symptom of the disease or disor der, or the risk of (or susceptibility to) the disease or disorder. random pools based on their ability to bind other molecules. As used herein, a “therapeutic agent' or “compound Aptamers may be selected which bind nucleic acids or pro includes, but is not limited to, Small molecules, peptides, teins. peptidomimetics, polypeptides, RNA interfering agents, e.g., VII. Screening Assays siRNA molecules, antibodies, ribozymes, and antisense oli gonucleotides. 0220. The invention also provides methods (also referred 0215. As described herein, cancer in subjects is associated to herein as 'screening assays”) for identifying modulators, with a change, e.g., an increase in the amount and/or activity, i.e., candidate or test compounds or agents (e.g., proteins, or a change in the structure, of one or more markers listed in peptides, peptidomimetics, peptoids, Small molecules or Table 5 (e.g., a marker that was shown to be increased in other drugs) which (a) bind to the marker, or (b) have a cancer), and/or a decrease in the amount and/or activity, or a modulatory (e.g., stimulatory or inhibitory) effect on the change in the structure of one or more markers listed in Table activity of the marker or, more specifically, (c) have a modu 4 (e.g. a marker that was shown to be decreased in cancer). latory effect on the interactions of the marker with one or While, as discussed above, Some of these changes in amount, more of its natural Substrates (e.g., peptide, protein, hormone, structure, and/or activity, result from occurrence of the can co-factor, or nucleic acid), or (d) have a modulatory effect on cer, others of these changes induce, maintain, and promote the the expression of the marker. Such assays typically comprise cancerous state of cancer, cells. Thus, cancer, characterized a reaction between the marker and one or more assay com by an increase in the amount and/or activity, or a change in the ponents. The other components may be either the test com structure, of one or more markers listed in Table 5 (e.g., a pound itself, or a combination of test compound and a natural marker that is shown to be increased in cancer), can be inhib binding partner of the marker. Compounds identified via US 2009/0297536A1 Dec. 3, 2009 24 assays Such as those described herein may be useful, for For the purposes of this discussion, Such cellular and extra example, for modulating, e.g., inhibiting, ameliorating, treat cellular molecules are referred to herein as “binding partners' ing, or preventing cancer. or marker “substrate'. 0221) The test compounds of the present invention may be 0226 One necessary embodiment of the invention in order obtained from any available source, including systematic to facilitate Such screening is the use of the marker to identify libraries of natural and/or synthetic compounds. Test com its natural in vivo binding partners. There are many ways to pounds may also be obtained by any of the numerous accomplish this which are known to one skilled in the art. One approaches in combinatorial library methods known in the example is the use of the marker protein as “bait protein’ in a art, including: biological libraries; peptoid libraries (libraries two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. of molecules having the functionalities of peptides, but with a No. 5,283,317; Zervos et al., 1993, Cell 72:223-232; Madura novel, non-peptide backbone which are resistant to enzymatic et al., 1993, J. Biol. Chem. 268: 12046-12054: Bartel et al, degradation but which nevertheless remain bioactive; see, 1993, Biotechniques 14:920-924; Iwabuchietal, 1993. Onco e.g., Zuckermann et al., 1994, J. Med. Chem. 37:2678-85); gene 8:1693-1696; Brent WO94/10300) in order to identify spatially addressable parallel solid phase or Solution phase other proteins which bind to or interact with the marker (bind libraries; synthetic library methods requiring deconvolution; ing partners) and, therefore, are possibly involved in the natu the one-bead one-compound library method; and synthetic ral function of the marker. Such marker binding partners are library methods using affinity chromatography selection. The also likely to be involved in the propagation of signals by the biological library and peptoid library approaches are limited marker or downstream elements of a marker-mediated sig to peptide libraries, while the other four approaches are appli naling pathway. Alternatively, such marker binding partners cable to peptide, non-peptide oligomer or Small molecule may also be found to be inhibitors of the marker. libraries of compounds (Lam, 1997, Anticancer Drug Des. 0227. The two-hybrid system is based on the modular 12:145). nature of most transcription factors, which consist of sepa 0222 Examples of methods for the synthesis of molecular rable DNA-binding and activation domains. Briefly, the assay libraries can be found in the art, for example in: DeWitt et al. utilizes two different DNA constructs. In one construct, the (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) gene that encodes a marker protein fused to a gene encoding Proc. Natl. Acad. Sci. USA 91:11422, Zuckermann et al. the DNA binding domain of a known transcription factor (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science (e.g., GAL-4). In the other construct, a DNA sequence, from 261: 1303: Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. a library of DNA sequences, that encodes an unidentified 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. protein (“prey” or “sample') is fused to a gene that codes for 33:2061; and in Gallop et al. (1994).J. Med. Chem. 37:1233. the activation domain of the known transcription factor. If the 0223 Libraries of compounds may be presented in solu “bait' and the “prey’ proteins are able to interact, in vivo, tion (e.g., Houghten, 1992, Biotechniques 13:412-421), or on forming a marker-dependent complex, the DNA-binding and beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, activation domains of the transcription factor are brought into Nature 364:555-556), bacteria and/or spores, (Ladner, U.S. close proximity. This proximity allows transcription of a Pat. No. 5.223.409), plasmids (Cullet al., 1992, Proc Natl reporter gene (e.g., Lacz) which is operably linked to a tran AcadSci USA 89:1865-1869) or on phage (Scott and Smith, Scriptional regulatory site responsive to the transcription fac 1990, Science 249:386-390; Devlin, 1990, Science 249:404 tor. Expression of the reporter gene can be readily detected 406; Cwirla-etal, 1990, Proc. Natl. Acad. Sci. 87:6378-6382: and cell colonies containing the functional transcription fac Felici, 1991, J. Mol. Biol. 222:301-310; Ladner, supra.). tor can be isolated and used to obtain the cloned gene which 0224. In one embodiment, the invention provides assays encodes the protein which interacts with the marker protein. for Screening candidate or test compounds which are Sub 0228. In a further embodiment, assays may be devised strates of a marker or biologically active portion thereof. In through the use of the invention for the purpose of identifying another embodiment, the invention provides assays for compounds which modulate (e.g., affect either positively or screening candidate or test compounds which bind to a negatively) interactions between a marker and its Substrates marker or biologically active portion thereof. Determining and/or binding partners. Such compounds can include, but are the ability of the test compound to directly bind to a marker not limited to, molecules Such as antibodies, peptides, hor can be accomplished, for example, by coupling the com mones, oligonucleotides, nucleic acids, and analogs thereof. pound with a radioisotope or enzymatic label Such that bind Such compounds may also be obtained from any available ing of the compound to the marker can be determined by Source, including systematic libraries of natural and/or syn detecting the labeled marker compound in a complex. For thetic compounds. The preferred assay components for use in example, compounds (e.g., marker Substrates) can be labeled this embodiment is a cancer, marker identified herein, the with ‘I, S, C, or H, either directly or indirectly, and the known binding partner and/or Substrate of same, and the test radioisotope detected by direct counting of radioemission or compound. Test compounds can be supplied from any source. by Scintillation counting. Alternatively, assay components 0229. The basic principle of the assay systems used to can be enzymatically labeled with, for example, horseradish identify compounds that interfere with the interaction peroxidase, alkaline phosphatase, or luciferase, and the enzy between the marker and its binding partner involves preparing matic label detected by determination of conversion of an a reaction mixture containing the marker and its binding appropriate Substrate to product. partner under conditions and for a time sufficient to allow the 0225. In another embodiment, the invention provides two products to interact and bind, thus forming a complex. In assays for screening candidate or test compounds which order to test an agent for inhibitory activity, the reaction modulate the activity of a marker or a biologically active mixture is prepared in the presence and absence of the test portion thereof. In all likelihood, the marker can, in vivo, compound. The test compound can be initially included in the interact with one or more molecules, such as, but not limited reaction mixture, or can be added at a time Subsequent to the to, peptides, proteins, hormones, cofactors and nucleic acids. addition of the marker and its binding partner. Control reac US 2009/0297536A1 Dec. 3, 2009

tion mixtures are incubated without the test compound or partner can be immobilized utilizing conjugation of biotin with a placebo. The formation of any complexes between the and streptavidin. Biotinylated marker protein or target mol marker and its binding partner is then detected. The formation ecules can be prepared from biotin-NHS(N-hydroxy-succin of a complex in the control reaction, but less or no Such imide) using techniques known in the art (e.g., biotinylation formation in the reaction mixture containing the test com kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the pound, indicates that the compound interferes with the inter wells of streptavidin-coated 96 well plates (Pierce Chemical). action of the marker and its binding partner. Conversely, the In certain embodiments, the protein-immobilized surfaces formation of more complex in the presence of compound than can be prepared in advance and stored. in the control reaction indicates that the compound may 0233. In order to conduct the assay, the corresponding enhance interaction of the marker and its binding partner. The partner of the immobilized assay component is exposed to the assay for compounds that interfere with the interaction of the coated surface with or without the test compound. After the marker with its binding partner may be conducted in a het reaction is complete, unreacted assay components are erogeneous or homogeneous format. Heterogeneous assays removed (e.g., by washing) and any complexes formed will involve anchoring either the marker or its binding partner remain immobilized on the solid surface. The detection of onto a solid phase and detecting complexes anchored to the complexes anchored on the Solid Surface can be accomplished Solid phase at the end of the reaction. In homogeneous assays, in a number of ways. Where the non-immobilized component the entire reaction is carried out in a liquid phase. In either is pre-labeled, the detection of label immobilized on the sur approach, the order of addition of reactants can be varied to face indicates that complexes were formed. Where the non obtain different information about the compounds being immobilized component is not pre-labeled, an indirect label tested. For example, test compounds that interfere with the can be used to detect complexes anchored on the Surface; e.g., interaction between the markers and the binding partners using a labeled antibody specific for the initially non-immo (e.g., by competition) can be identified by conducting the bilized species (the antibody, in turn, can be directly labeled reaction in the presence of the test Substance, i.e., by adding or indirectly labeled with, e.g., a labeled anti-Ig antibody). the test Substance to the reaction mixture prior to or simulta Depending upon the order of addition of reaction compo neously with the marker and its interactive binding partner. nents, test compounds which modulate (inhibit or enhance) Alternatively, test compounds that disrupt preformed com complex formation or which disrupt preformed complexes plexes, e.g. compounds with higher binding constants that can be detected. displace one of the components from the complex, can be 0234. In an alternate embodiment of the invention, a tested by adding the test compound to the reaction mixture homogeneous assay may be used. This is typically a reaction, after complexes have been formed. The various formats are analogous to those mentioned above, which is conducted in a briefly described below. liquid phase in the presence or absence of the test compound. 0230. In a heterogeneous assay system, either the marker The formed complexes are then separated from unreacted or its binding partner is anchored onto a solid Surface or components, and the amount of complex formed is deter matrix, while the other corresponding non-anchored compo mined. As mentioned for heterogeneous assay systems, the nent may be labeled, either directly or indirectly. In practice, order of addition of reactants to the liquid phase can yield microtitre plates are often utilized for this approach. The information about which test compounds modulate (inhibitor anchored species can be immobilized by a number of meth enhance) complex formation and which disrupt preformed ods, either non-covalent or covalent, that are typically well complexes. known to one who practices the art. Non-covalent attachment 0235. In such a homogeneous assay, the reaction products can often be accomplished simply by coating the Solid Surface may be separated from unreacted assay components by any of with a solution of the marker or its binding partner and drying. a number of standard techniques, including but not limited to: Alternatively, an immobilized antibody specific for the assay differential centrifugation, chromatography, electrophoresis component to be anchored can be used for this purpose. Such and immunoprecipitation. In differential centrifugation, com Surfaces can often be prepared in advance and stored. plexes of molecules may be separated from uncomplexed 0231. In related embodiments, a fusion protein can be molecules through a series of centrifugal steps, due to the provided which adds a domain that allows one or both of the different sedimentation equilibria of complexes based on assay components to be anchored to a matrix. For example, their different sizes and densities (see, for example, Rivas, G., glutathione-S-transferase/marker fusion proteins or glu and Minton, A. P., Trends Biochem Sci 1993 August; 18(8): tathione-S-transferase/binding partner can be adsorbed onto 284-7). Standard chromatographic techniques may also be glutathione Sepharose beads (Sigma Chemical, St. Louis, utilized to separate complexed molecules from uncomplexed Mo.) or glutathione derivatized microtiter plates, which are ones. For example, gel filtration chromatography separates then combined with the test compound or the test compound molecules based on size, and through the utilization of an and either the non-adsorbed marker or its binding partner, and appropriate gel filtration resin in a column format, for the mixture incubated under conditions conducive to complex example, the relatively larger complex may be separated from formation (e.g., physiological conditions). Following incuba the relatively smaller uncomplexed components. Similarly, tion, the beads or microtiter plate wells are washed to remove the relatively different charge properties of the complex as any unbound assay components, the immobilized complex compared to the uncomplexed molecules may be exploited to assessed either directly or indirectly, for example, as differentially separate the complex from the remaining indi described above. Alternatively, the complexes can be disso vidual reactants, for example through the use of ion-exchange ciated from the matrix, and the level of marker binding or chromatography resins. Such resins and chromatographic activity determined using standard techniques. techniques are well known to one skilled in the art (see, e.g., 0232 Other techniques for immobilizing proteins on Heegaard, 1998, J. Mol. Recognit. 11:141-148; Hage and matrices can also be used in the screening assays of the Tweed, 1997, J. Chromatogr: B. Biomed. Sci. Appl., 699:499 invention. For example, either a marker or a marker binding 525). Gel electrophoresis may also be employed to separate US 2009/0297536A1 Dec. 3, 2009 26 complexed molecules from unbound species (see, e.g., the presence of the candidate compound is compared to the Ausubeletal (eds.). In: Current Protocols in Molecular Biol level of expression of mRNA or protein in the absence of the ogy, J. Wiley & Sons, New York. 1999). In this technique, candidate compound. The candidate compound can then be protein or nucleic acid complexes are separated based on size identified as a modulator of marker expression based on this or charge, for example. In order to maintain the binding comparison. For example, when expression of marker mRNA interaction during the electrophoretic process, nondenaturing or protein is greater (statistically significantly greater) in the gels in the absence of reducing agent are typically preferred, presence of the candidate compound than in its absence, the but conditions appropriate to the particular interactants will candidate compound is identified as a stimulator of marker be well known to one skilled in the art. Immunoprecipitation mRNA or protein expression. Conversely, when expression is another common technique utilized for the isolation of a of marker mRNA or protein is less (statistically significantly protein-protein complex from Solution (see, e.g., Ausubel et less) in the presence of the candidate compound than in its al (eds.), In: Current Protocols in Molecular Biology, J. Wiley absence, the candidate compound is identified as an inhibitor & Sons, New York. 1999). In this technique, all proteins of marker mRNA or protein expression. The level of marker binding to an antibody specific to one of the binding mol mRNA or protein expression in the cells can be determined by ecules are precipitated from solution by conjugating the anti methods described herein for detecting marker mRNA or body to a polymer bead that may be readily collected by protein. centrifugation. The bound assay components are released 0238. In another aspect, the invention pertains to a com from the beads (through a specific proteolysis event or other bination of two or more of the assays described herein. For technique well known in the art which will not disturb the example, a modulating agent can be identified using a cell protein-protein interaction in the complex), and a second based or a cell free assay, and the ability of the agent to immunoprecipitation step is performed, this time utilizing modulate the activity of a marker protein can be further con antibodies specific for the correspondingly different interact firmed in vivo, e.g., in a whole animal model for cancer, ing assay component. In this manner, only formed complexes cellular transformation and/or tumorigenesis. An animal should remain attached to the beads. Variations in complex model for pancreatic cancer is described in, for example, formation in both the presence and the absence of a test Aguirre A., et al. (2003) Genes Dev. December 15; 17(24): compound can be compared, thus offering information about 3112-26, the contents of which are expressly incorporated the ability of the compound to modulate interactions between herein by reference. Additional animal based models of can the marker and its binding partner. cer are well known in the art (reviewed in Animal Models of 0236 Also within the scope of the present invention are Cancer Predisposition Syndromes, Hiai, Hand Hino, O (eds.) methods for direct detection of interactions between the 1999, Progress in Experimental Tumor Research, Vol. 35: marker and its natural binding partner and/or a test compound Clarke A R Carcinogenesis (2000) 21:435-41) and include, in a homogeneous or heterogeneous assay system without for example, carcinogen-induced tumors (Rithidech, Ketal. further sample manipulation. For example, the technique of Mutat Res (1999) 428:33-39; Miller, ML et al. Environ Mol fluorescence energy transfer may be utilized (see, e.g., Mutagen (2000) 35:319-327), injection and/or transplanta Lakowicz et al., U.S. Pat. No. 5,631,169; Stavrianopoulos et tion of tumor cells into an animal, as well as animals bearing al, U.S. Pat. No. 4,868,103). Generally, this technique mutations in growth regulatory genes, for example, onco involves the addition of a fluorophore label on a first donor genes (e.g., ras) (Arbeit, J M et al. Am J Pathol (1993) 142: molecule (e.g., marker or test compound) such that its emitted 1187-1197: Sinn, Eetal. Cell (1987) 49:465-475; Thorgeirs fluorescent energy will be absorbed by a fluorescent label on son, S S et al. Toxicol Lett (2000) 112-113:553-555) and a second, acceptor molecule (e.g., marker or test com tumor Suppressor genes (e.g. p53) (Vooijs, Metal. Oncogene pound), which in turn is able to fluoresce due to the absorbed (1999) 18:5293-5303: Clark A R Cancer Metast Rev (1995) energy. Alternately, the donor protein molecule may simply 14:125-148; Kumar, T Retal.J Intern Med (1995) 238:233 utilize the natural fluorescent energy of tryptophan residues. 238; Donehower, LA et al. (1992) Nature 356215-221). Fur Labels are chosen that emit different wavelengths of light, thermore, experimental model systems are available for the such that the acceptor molecule label may be differentiated study of for example, ovarian cancer (Hamilton, T C et al. from that of the donor. Since the efficiency of energy trans Semin Oncol (1984) 11:285-298; Rahman, NAetal. Mol Cell fer between the labels is related to the distance separating the Endocrinol (1998) 145:167-174; Beamer, W G et al. Toxicol molecules, spatial relationships between the molecules can Pathol (1998) 26:704-710), gastric cancer (Thompson, Jetal. be assessed. In a situation in which binding occurs between IntJ Cancer (2000) 86:863-869: Fodde, Ret al. Cytogenet the molecules, the fluorescent emission of the acceptor mol Cell Genet (1999) 86:105-111), breast cancer (Li, M et al. ecule label in the assay should be maximal. An FET binding Oncogene (2000) 19:1010-1019; Green, J E et al. Oncogene event can be conveniently measured through standard fluo (2000) 19:1020-1027), melanoma (Satyamoorthy, K et al. rometric detection means well known in the art (e.g., using a Cancer Metast Rev (1999) 18:401-405), and prostate cancer fluorimeter). A test substance which either enhances or hin (Shirai, Tet al. Mutat Res (2000) 462:219-226: Bostwick, D ders participation of one of the species in the preformed G. et al. Prostate (2000) 43:286-294). Animal models complex will result in the generation of a signal variant to that described in, for example, Chin L. et al (1999) Nature 400 of background. In this way, test Substances that modulate (6743):468-72, may also be used in the methods of the inven interactions between a marker and its binding partner can be tion. identified in controlled assays. 0239. This invention further pertains to novel agents iden 0237. In another embodiment, modulators of marker tified by the above-described screening assays. Accordingly, expression are identified in a method wherein a cell is con it is within the scope of this invention to further use an agent tacted with a candidate compound and the expression of identified as described herein in an appropriate animal model. mRNA or protein, corresponding to a marker in the cell, is For example, an agent identified as described herein (e.g., a determined. The level of expression of mRNA or protein in marker modulating agent, a small molecule, an antisense US 2009/0297536A1 Dec. 3, 2009 27 marker nucleic acid molecule, a ribozyme, a marker-specific than about 10,000 grams per mole, organic or inorganic com antibody, or fragment thereof, a marker protein, a marker pounds having a molecular weight less than about 5,000 nucleic acid molecule, an RNA interfering agent, e.g., an grams per mole, organic or inorganic compounds having a siRNA molecule targeting a marker of the invention, or a molecular weight less than about 1,000 grams per mole, marker-binding partner) can be used in an animal model to organic or inorganic compounds having a molecular weight determine the efficacy, toxicity, or side effects of treatment less than about 500 grams per mole, and salts, esters, and with Such an agent. Alternatively, an agent identified as other pharmaceutically acceptable forms of such compounds. described herein can be used in an animal model to determine 0243 Exemplary doses of a small molecule include mil the mechanism of action of such an agent. Furthermore, this ligram or microgram amounts per kilogram of Subject or invention pertains to uses of novel agents identified by the sample weight (e.g. about 1 microgram per kilogram to about above-described screening assays for treatments as described 500 milligrams per kilogram, about 100 micrograms per kilo herein. gram to about 5 milligrams per kilogram, or about 1 micro gram per kilogram to about 50 micrograms per kilogram). VIII. Pharmaceutical Compositions 0244 As defined herein, a therapeutically effective 0240. The small molecules, peptides, peptoids, peptido amount of an RNA interfering agent, e.g., siRNA, (i.e., an mimetics, polypeptides, RNA interfering agents, e.g., siRNA effective dosage) ranges from about 0.001 to 3,000 mg/kg molecules, antibodies, ribozymes, and antisense oligonucle body weight, preferably about 0.01 to 2500 mg/kg body otides (also referred to herein as “active compounds” or weight, more preferably about 0.1 to 2000, about 0.1 to 1000 “compounds') corresponding to a marker of the invention can mg/kg body weight, 0.1 to 500 mg/kg body weight, 0.1 to 100 be incorporated into pharmaceutical compositions suitable mg/kg body weight, 0.1 to 50 mg/kg body weight, 0.1 to 25 for administration. Such compositions typically comprise the mg/kg body weight, and even more preferably about 1 to 10 Small molecules, peptides, peptoids, peptidomimetics, mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 polypeptides, RNA interfering agents, e.g., siRNA mol mg/kg body weight. Treatment of a Subject with a therapeu ecules, antibodies, ribozymes, or antisense oligonucleotides tically effective amount of an RNA interfering agent can and a pharmaceutically acceptable carrier. As used herein the include a single treatment or, preferably, can include a series language “pharmaceutically acceptable carrier' is intended to of treatments. In a preferred example, a subject is treated with include any and all solvents, dispersion media, coatings, anti an RNA interfering agent in the range of between about 0.1 to bacterial and antifungal agents, isotonic and absorption 20 mg/kg body weight, one time per week for between about delaying agents, and the like, compatible with pharmaceuti 1 to 10 weeks, preferably between 2 to 8 weeks, more pref cal administration. The use of Such media and agents for erably between about 3 to 7 weeks, and even more preferably pharmaceutically active Substances is well known in the art. for about 4, 5, or 6 weeks. Except insofar as any conventional media or agent is incom 0245 Exemplary doses of a protein or polypeptide include patible with the active compound, use thereof in the compo gram, milligram or microgram amounts per kilogram of Sub sitions is contemplated. Supplementary active compounds ject or sample weight (e.g. about 1 microgram per kilogram to can also be incorporated into the compositions. about 5 grams per kilogram, about 100 micrograms per kilo 0241 The invention includes methods for preparing phar gram to about 500 milligrams per kilogram, or about 1 mil maceutical compositions for modulating the expression or ligram per kilogram to about 50 milligrams per kilogram). It activity of a polypeptide or nucleic acid corresponding to a is furthermore understood that appropriate doses of one of marker of the invention. Such methods comprise formulating these agents depend upon the potency of the agent with a pharmaceutically acceptable carrier with an agent which respect to the expression or activity to be modulated. Such modulates expression or activity of a polypeptide or nucleic appropriate doses can be determined using the assays acid corresponding to a marker of the invention. Such com described herein. When one or more of these agents is to be positions can further include additional active agents. Thus, administered to an animal (e.g. a human) in order to modulate the invention further includes methods for preparing a phar expression or activity of a polypeptide or nucleic acid of the maceutical composition by formulating a pharmaceutically invention, a physician, Veterinarian, or researcher can, for acceptable carrier with an agent which modulates expression example, prescribe a relatively low dose at first, Subsequently or activity of a polypeptide or nucleic acid corresponding to a increasing the dose until an appropriate response is obtained. marker of the invention and one or more additional active In addition, it is understood that the specific dose level for any compounds. particular animal Subject will depend upon a variety of factors 0242. It is understood that appropriate doses of small mol including the activity of the specific agent employed, the age, ecule agents and protein or polypeptide agents depends upon body weight, general health, gender, and diet of the Subject, a number of factors within the knowledge of the ordinarily the time of administration, the route of administration, the skilled physician, Veterinarian, or researcher. The dose(s) of rate of excretion, any drug combination, and the degree of these agents will vary, for example, depending upon the iden expression or activity to be modulated. tity, size, and condition of the Subject or sample being treated, 0246 A pharmaceutical composition of the invention is further depending upon the route by which the composition is formulated to be compatible with its intended route of admin to be administered, if applicable, and the effect which the istration. Examples of routes of administration include practitioner desires the agent to have upon the nucleic acid parenteral, e.g., intravenous, intradermal, Subcutaneous, oral molecule or polypeptide of the invention. Small molecules (e.g., inhalation), transdermal (topical), transmucosal, and include, but are not limited to, peptides, peptidomimetics, rectal administration. Solutions or Suspensions used for amino acids, amino acid analogs, polynucleotides, poly parenteral, intradermal, or Subcutaneous application can nucleotide analogs, nucleotides, nucleotide analogs, organic include the following components: a sterile diluent Such as or inorganic compounds (i.e., including heteroorganic and water for injection, Saline Solution, fixed oils, polyethylene organometallic compounds) having a molecular weight less glycols, glycerine, propylene glycol or other synthetic Sol US 2009/0297536A1 Dec. 3, 2009 28 vents; antibacterial agents such as benzyl alcohol or methyl similar nature: a binder Such as microcrystalline cellulose, parabens; antioxidants such as ascorbic acid or sodium gum tragacanth or gelatin; an excipient such as starch or bisulfite; chelating agents such as ethylenediamine-tetraace lactose, a disintegrating agent such as alginic acid, Primogel, tic acid; buffers such as acetates, citrates or phosphates and or corn starch; a lubricant such as magnesium Stearate or agents for the adjustment oftonicity Such as Sodium chloride Sterotes; a glidant Such as colloidal silicon dioxide; a Sweet or dextrose. pH can be adjusted with acids or bases, such as ening agent such as Sucrose or saccharin; or a flavoring agent hydrochloric acid or sodium hydroxide. The parenteral prepa Such as peppermint, methyl salicylate, or orange flavoring. ration can be enclosed in ampules, disposable syringes or 0251 For administration by inhalation, the compounds multiple dose vials made of glass or plastic. are delivered in the form of an aerosol spray from a pressur 0247 Pharmaceutical compositions suitable for injectable ized container or dispenser which contains a suitable propel use include sterile aqueous Solutions (where water soluble) or lant, e.g. a gas such as carbon dioxide, or a nebulizer. dispersions and sterile powders for the extemporaneous 0252 Systemic administration can also be by transmu preparation of sterile injectable solutions or dispersions. For cosal or transdermal means. For transmucosal or transdermal intravenous administration, Suitable carriers include physi administration, penetrants appropriate to the barrier to be ological saline, bacteriostatic water, Cremophor EL (BASF: permeated are used in the formulation. Such penetrants are Parsippany, N.J.) or phosphate buffered saline (PBS). In all generally known in the art, and include, for example, for cases, the composition must be sterile and should be fluid to transmucosal administration, detergents, bile salts, and the extent that easy syringability exists. It must be stable fusidic acid derivatives. Transmucosal administration can be under the conditions of manufacture and storage and must be accomplished through the use of nasal sprays or Supposito preserved against the contaminating action of microorgan ries. For transdermal administration, the active compounds isms such as bacteria and fungi. The carrier can be a solvent are formulated into ointments, salves, gels, or creams as gen or dispersion medium containing, for example, water, etha erally known in the art. nol, polyol (for example, glycerol, propylene glycol, and 0253) The compounds can also be prepared in the form of liquid polyethylene glycol, and the like), and Suitable mix Suppositories (e.g., with conventional Suppository bases Such tures thereof. The proper fluidity can be maintained, for as cocoa butter and other glycerides) or retention enemas for example, by the use of a coating Such as lecithin, by the rectal delivery. maintenance of the required particle size in the case of dis 0254. In one embodiment, the active compounds are pre persion and by the use of surfactants. Prevention of the action pared with carriers that will protect the compound against of microorganisms can be achieved by various antibacterial rapid elimination from the body, such as a controlled release and antifungal agents, for example, parabens, chlorobutanol, formulation, including implants and microencapsulated phenol, ascorbic acid, thimerosal, and the like. In many cases, delivery systems. Biodegradable, biocompatible polymers it will be preferable to include isotonic agents, for example, can be used, such as ethylene vinyl acetate, polyanhydrides, Sugars, polyalcohols such as mannitol, Sorbitol, or sodium polyglycolic acid, collagen, polyorthoesters, and polylactic chloride in the composition. Prolonged absorption of the acid. Methods for preparation of such formulations will be injectable compositions can be brought about by including in apparent to those skilled in the art. The materials can also be the composition an agent which delays absorption, for obtained commercially from Alza Corporation and Nova example, aluminum monostearate and gelatin. Pharmaceuticals, Inc. Liposomal Suspensions (including 0248 Sterile injectable solutions can be prepared by liposomes having monoclonal antibodies incorporated incorporating the active compound (e.g., a polypeptide or therein or thereon) can also be used as pharmaceutically antibody) in the required amount in an appropriate solvent acceptable carriers. These can be prepared according to meth with one or a combination of ingredients enumerated above, ods known to those skilled in the art, for example, as as required, followed by filtered sterilization. Generally, dis described in U.S. Pat. No. 4,522,811. persions are prepared by incorporating the active compound 0255. It is especially advantageous to formulate oral or into a sterile vehicle which contains a basic dispersion parenteral compositions in dosage unit form for ease of medium, and then incorporating the required other ingredi administration and uniformity of dosage. Dosage unit form as ents from those enumerated above. In the case of sterile used herein refers to physically discrete units Suited as unitary powders for the preparation of sterile injectable solutions, the dosages for the Subject to be treated; each unit containing a preferred methods of preparation are vacuum drying and predetermined quantity of active compound calculated to pro freeze-drying which yields a powder of the active ingredient duce the desired therapeutic effect in association with the plus any additional desired ingredient from a previously ster required pharmaceutical carrier. The specification for the dos ile-filtered solution thereof. age unit forms of the invention are dictated by and directly 0249 Oral compositions generally include an inert diluent dependent on the unique characteristics of the active com or an edible carrier. They can be enclosed in gelatin capsules pound and the particular therapeutic effect to be achieved, and or compressed into tablets. For the purpose of oral therapeutic the limitations inherent in the art of compounding Such an administration, the active compound can be incorporated active compound for the treatment of individuals. with excipients and used in the form of tablets, troches, or 0256 For antibodies, the preferred dosage is 0.1 mg/kg to capsules. Oral compositions can also be prepared using a fluid 100 mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). carrier for use as a mouthwash, wherein the compound in the If the antibody is to act in the brain, a dosage of 50 mg/kg to fluid carrier is applied orally and swished and expectorated or 100 mg/kg is usually appropriate. Generally, partially human swallowed. antibodies and fully human antibodies have a longer half-life 0250 Pharmaceutically compatible binding agents, and/ within the human body than other antibodies. Accordingly, or adjuvant materials can be included as part of the compo lower dosages and less frequent administration is often pos sition. The tablets, pills, capsules, troches, and the like can sible. Modifications such as lipidation can be used to stabilize contain any of the following ingredients, or compounds of a antibodies and to enhance uptake and tissue penetration (e.g., US 2009/0297536A1 Dec. 3, 2009 29 into the epithelium). A method for lipidation of antibodies is 0265 Methods for evaluating copy number of encoding described by Cruikshank et al. (1997) J Acquired Immune nucleic acid in a sample include, but are not limited to, Deficiency Syndromes and Human Retrovirology 14:193. hybridization-based assays. For example, one method for 0257 The nucleic acid molecules corresponding to a evaluating the copy number of encoding nucleic acid in a marker of the invention can be inserted into vectors and used sample involves a Southern Blot. In a Southern Blot, the as gene therapy vectors. Gene therapy vectors can be deliv genomic DNA (typically fragmented and separated on an ered to a subject by, for example, intravenous injection, local electrophoretic gel) is hybridized to a probe specific for the administration (U.S. Pat. No. 5,328,470), or by stereotactic target region. Comparison of the intensity of the hybridiza injection (see, e.g., Chen et al., 1994, Proc. Natl. Acad. Sci. tion signal from the probe for the target region with control USA 91:3054-3057). The pharmaceutical preparation of the probe signal from analysis of normal genomic DNA (e.g., a gene therapy vector can include the gene therapy vector in an non-amplified portion of the same or related cell, tissue, acceptable diluent, or can comprise a slow release matrix in organ, etc.) provides an estimate of the relative copy number which the gene delivery vehicle is imbedded. Alternatively, of the target nucleic acid. where the complete gene delivery vector can be produced 0266. An alternative means for determining the copy num intact from recombinant cells, e.g. retroviral vectors, the ber is in situ hybridization (e.g., Angerer (1987) Meth. Enzy pharmaceutical preparation can include one or more cells mol 152: 649). Generally, in situ hybridization comprises the which produce the gene delivery system. following steps: (1) fixation of tissue or biological structure to 0258. The RNA interfering agents, e.g., siRNAs used in be analyzed; (2) prehybridization treatment of the biological the methods of the invention can be inserted into vectors. structure to increase accessibility of target DNA, and to These constructs can be delivered to a subject by, for example, reduce nonspecific binding; (3) hybridization of the mixture intravenous injection, local administration (see U.S. Pat. No. of nucleic acids to the nucleic acid in the biological structure 5.328,470) or by stereotactic injection (see e.g., Chen et al. or tissue; (4) post-hybridization washes to remove nucleic (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The phar acid fragments not bound in the hybridization and (5) detec maceutical preparation of the vector can include the RNA tion of the hybridized nucleic acid fragments. The reagent interfering agent, e.g., the siRNA vector in an acceptable used in each of these steps and the conditions for use vary diluent, or can comprise a slow release matrix in which the depending on the particular application. gene delivery vehicle is imbedded. Alternatively, where the 0267 Preferred hybridization-based assays include, but complete gene delivery vector can be produced intact from are not limited to, traditional “direct probe' methods such as recombinant cells, e.g., retroviral vectors, the pharmaceutical Southern blots or in situ hybridization (e.g., FISH), and preparation can include one or more cells which produce the “comparative probe' methods such as comparative genomic gene delivery system. hybridization (CGH), e.g., cDNA-based or oligonucleotide 0259. The pharmaceutical compositions can be included based CGH. The methods can be used in a wide variety of in a container, pack, or dispenser together with instructions formats including, but not limited to, Substrate (e.g. mem for administration. brane or glass) bound methods or array-based approaches. 0268. In a typical in situ hybridization assay, cells are fixed IX. Predictive Medicine to a solid Support, typically a glass slide. If a nucleic acid is to 0260 The present invention also pertains to the field of be probed, the cells are typically denatured with heat or alkali. predictive medicine in which diagnostic assays, prognostic The cells are then contacted with a hybridization solutionata assays, pharmacogenomics, and monitoring clinical trails are moderate temperature to permit annealing of labeled probes used for prognostic (predictive) purposes to thereby treat an specific to the nucleic acid sequence encoding the protein. individual prophylactically. Accordingly, one aspect of the The targets (e.g., cells) are then typically washed at a prede present invention relates to diagnostic assays for determining termined stringency or at an increasing stringency until an the amount, structure, and/or activity of polypeptides or appropriate signal to noise ratio is obtained. nucleic acids corresponding to one or more markers of the 0269. The probes are typically labeled, e.g., with radioiso invention, in order to determine whether an individual is at topes or fluorescent reporters. Preferred probes are suffi risk of developing cancer. Such assays can be used for prog ciently long so as to specifically hybridize with the target nostic or predictive purposes to thereby prophylactically treat nucleic acid(s) under Stringent conditions. The preferred size an individual prior to the onset of the cancer. range is from about 200 bases to about 1000 bases. 0261 Yet another aspect of the invention pertains to moni 0270. In some applications it is necessary to block the toring the influence of agents (e.g., drugs or other compounds hybridization capacity of repetitive sequences. Thus, in some administered either to inhibit cancer, or to treat or prevent any embodiments, tRNA, human genomic DNA, or Cot-I DNA is other disorder {i.e. in order to understand any carcinogenic used to block non-specific hybridization. effects that such treatment may have}) on the amount, struc 0271. In CGH methods, a first collection of nucleic acids ture, and/or activity of a marker of the invention in clinical (e.g. from a sample, e.g., a possible tumor) is labeled with a trials. These and other agents are described in further detail in first label, while a second collection of nucleic acids (e.g. a the following sections. control, e.g., from a healthy cell/tissue) is labeled with a 0262 A. Diagnostic Assays second label. The ratio of hybridization of the nucleic acids is 0263. 1. Methods for Detection of Copy Number determined by the ratio of the two (first and second) labels 0264 Methods of evaluating the copy number of a particu binding to each fiber in the array. Where there are chromo lar marker or chromosomal region (e.g., an MCR) are well somal deletions or multiplications, differences in the ratio of known to those of skill in the art. The presence or absence of the signals from the two labels will be detected and the ratio chromosomal gain or loss can be evaluated simply by a deter will provide a measure of the copy number. Array-based CGH mination of copy number of the regions or markers identified may also be performed with single-color labeling (as opposed herein. to labeling the control and the possible tumor sample with two US 2009/0297536A1 Dec. 3, 2009 30 different dyes and mixing them prior to hybridization, which Expression of a marker of the invention may be assessed by will yield ratio due to competitive hybridization to probes on any of a wide variety of well known methods for detecting the arrays). In single color CGH, the control is labeled and expression of a transcribed molecule or protein. Non-limiting hybridized to one array and absolute signals are read, and the examples of Such methods include immunological methods possible tumor sample is labeled and hybridized to a second for detection of secreted, cell-surface, cytoplasmic, or nuclear array (with identical content) and absolute signals are read. proteins, protein purification methods, protein function or Copy number difference is calculated based on absolute sig activity assays, nucleic acid hybridization methods, nucleic nals from the two arrays. Hybridization protocols suitable for acid reverse transcription methods, and nucleic acid amplifi use with the methods of the invention are described, e.g., in cation methods. Albertson (1984) EMBO J. 3: 1227-1234: Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 0279. In preferred embodiments, activity of a particular 430, 402: Methods in Molecular Biology, Vol. 33: In Situ gene is characterized by a measure of gene transcript (e.g. Hybridization Protocols, Choo, ed., Humana Press, Totowa, mRNA), by a measure of the quantity of translated protein, or N.J. (1994), etc. In one embodiment, the hybridization pro by a measure of gene product activity. Marker expression can tocol of Pinkel et al. (1998) Nature Genetics 20: 207-211, or be monitored in a variety of ways, including by detecting of Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321 mRNA levels, protein levels, or protein activity, any of which 5325 (1992) is used. can be measured using standard techniques. Detection can 0272. The methods of the invention are particularly well involve quantification of the level of gene expression (e.g., suited to array-based hybridization formats. Array-based genomic DNA, cDNA, mRNA, protein, or enzyme activity), CGH is described in U.S. Pat. No. 6,455,258, the contents of or, alternatively, can be a qualitative assessment of the level of which are incorporated herein by reference. gene expression, in particular in comparison with a control 0273. In still another embodiment, amplification-based level. The type of level being detected will be clear from the assays can be used to measure copy number. In Such ampli COInteXt. fication-based assays, the nucleic acid sequences act as a 0280 Methods of detecting and/or quantifying the gene template in an amplification reaction (e.g., Polymerase Chain transcript (mRNA or cDNA made therefrom) using nucleic Reaction (PCR). In a quantitative amplification, the amount acid hybridization techniques are known to those of skill in of amplification product will be proportional to the amount of the art (see Sambrook et al. Supra). For example, one method template in the original sample. Comparison to appropriate for evaluating the presence, absence, or quantity of cDNA controls, e.g. healthy tissue, provides a measure of the copy involves a Southern transfer as described above. Briefly, the number. mRNA is isolated (e.g. using an acid guanidinium-phenol 0274 Methods of “quantitative' amplification are well chloroform extraction method, Sambrook et al. Supra.) and known to those of skill in the art. For example, quantitative reverse transcribed to produce cDNA. The cDNA is then PCR involves simultaneously co-amplifying a known quan optionally digested and run on a gel in buffer and transferred tity of a control sequence using the same primers. This pro to membranes. Hybridization is then carried out using the vides an internal standard that may be used to calibrate the nucleic acid probes specific for the target clNA. PCR reaction. Detailed protocols for quantitative PCR are 0281. A general principle of such diagnostic and prognos provided in Innis et al. (1990) PCR Protocols, A Guide to tic assays involves preparing a sample or reaction mixture that Methods and Applications, Academic Press, Inc. N.Y.). Mea may contain a marker, and a probe, under appropriate condi surement of DNA copy number at microsatellite loci using tions and for a time sufficient to allow the marker and probe to quantitative PCR analysis is described in Ginzonger, et al interact and bind, thus forming a complex that can be (2000) Cancer Research 60:5405-5409. The known nucleic removed and/or detected in the reaction mixture. These acid sequence for the genes is sufficient to enable one of skill assays can be conducted in a variety of ways. in the art to routinely select primers to amplify any portion of 0282 For example, one method to conduct such an assay the gene. Fluorogenic quantitative PCR may also be used in would involve anchoring the marker or probe onto a solid the methods of the invention. In fluorogenic quantitative phase Support, also referred to as a Substrate, and detecting PCR, quantitation is based on amount of fluorescence signals, target marker/probe complexes anchored on the solid phase at e.g., TaqMan and Sybr green. the end of the reaction. In one embodiment of such a method, 0275 Other suitable amplification methods include, but a sample from a subject, which is to be assayed for presence are not limited to, ligase chain reaction (LCR) (see Wu and and/or concentration of marker, can be anchored onto a car Wallace (1989) Genomics 4: 560, Landegren et al. (1988) rier or Solid phase Support. In another embodiment, the Science 241: 1077, and Barringer et al. (1990) Gene 89: 117, reverse situation is possible, in which the probe can be transcription amplification (Kwoh et al. (1989) Proc. Natl. anchored to a solid phase and a sample from a Subject can be Acad. Sci. USA 86: 1173), self-sustained sequence replica allowed to react as an unanchored component of the assay. tion (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 0283. There are many established methods for anchoring 1874), dot PCR, and linker adapter PCR, etc. assay components to a solid phase. These include, without 0276 Loss of heterozygosity (LOH) mapping (Wang Z. C. limitation, marker or probe molecules which are immobilized et al. (2004) Cancer Res 64(1):64-71; Seymour, A. B., et al. through conjugation of biotin and streptavidin. Such biotiny (1994) Cancer Res 54, 2761-4; Hahn, S.A., et al. (1995) lated assay components can be prepared from biotin-NHS(N- Cancer Res 55, 4670-5; Kimura, M., et al. (1996) Genes hydroxy-Succinimide) using techniques known in the art Chromosomes Cancer 17, 88-93) may also be used to identify (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and regions of amplification or deletion. immobilized in the wells of streptavidin-coated 96 well plates (0277 2. Methods for Detection of Gene Expression (Pierce Chemical). In certain embodiments, the surfaces with 0278 Marker expression level can also be assayed as a immobilized assay components can be prepared in advance method for diagnosis of cancer or risk for developing cancer. and stored. US 2009/0297536A1 Dec. 3, 2009

0284. Other suitable carriers or solid phase supports for plexed components by any of a number of Standard tech Such assays include any material capable of binding the class niques, including but not limited to: differential centrifuga of molecule to which the marker or probe belongs. Well tion, chromatography, electrophoresis and known Supports or carriers include, but are not limited to, immunoprecipitation. In differential centrifugation, marker/ glass, polystyrene, nylon, polypropylene, nylon, polyethyl probe complexes may be separated from uncomplexed assay ene, dextran, amylases, natural and modified celluloses, poly components through a series of centrifugal steps, due to the acrylamides, gabbros, and magnetite. different sedimentation equilibria of complexes based on 0285. In order to conduct assays with the above mentioned their different sizes and densities (see, for example, Rivas, G., approaches, the non-immobilized component is added to the and Minton, A. P., 1993, Trends Biochem Sci. 18(8):284-7). Solid phase upon which the second component is anchored. Standard chromatographic techniques may also be utilized to After the reaction is complete, uncomplexed components separate complexed molecules from uncomplexed ones. For may be removed (e.g., by washing) under conditions such that example, gel filtration chromatography separates molecules any complexes formed will remain immobilized upon the based on size, and through the utilization of an appropriate gel solid phase. The detection of marker/probe complexes filtration resin in a column format, for example, the relatively anchored to the Solid phase can be accomplished in a number larger complex may be separated from the relatively smaller of methods outlined herein. uncomplexed components. Similarly, the relatively different 0286. In a preferred embodiment, the probe, when it is the charge properties of the marker/probe complex as compared unanchored assay component, can be labeled for the purpose to the uncomplexed components may be exploited to differ of detection and readout of the assay, either directly or indi entiate the complex from uncomplexed components, for rectly, with detectable labels discussed herein and which are example through the utilization of ion-exchange chromatog well-known to one skilled in the art. raphy resins. Such resins and chromatographic techniques are 0287. It is also possible to directly detect marker/probe well known to one skilled in the art (see, e.g., Heegaard, N.H., complex formation without further manipulation or labeling 1998, J. Mol. Recognit. Winter 11 (1-6): 141-8; Hage, D. S., of either component (marker or probe), for example by uti and Tweed, S. A.J. Chromatogr B Biomed Sci Appl 1997 Oct. lizing the technique of fluorescence energy transfer (see, for 10: 699 (1-2):499-525). Gel electrophoresis may also be example, Lakowicz et al., U.S. Pat. No. 5,631, 169; Stavrian employed to separate complexed assay components from opoulos, et al., U.S. Pat. No. 4,868,103). A fluorophore label unbound components (see, e.g., Ausubel et al., ed., Current on the first, donor molecule is selected such that, upon Protocols in Molecular Biology, John Wiley & Sons, New excitation with incident light of appropriate wavelength, its York, 1987-1999). In this technique, protein or nucleic acid emitted fluorescent energy will be absorbed by a fluorescent complexes are separated based on size or charge, for example. label on a second acceptor molecule, which in turn is able to In order to maintain the binding interaction during the elec fluoresce due to the absorbed energy. Alternately, the donor trophoretic process, non-denaturing gel matrix materials and protein molecule may simply utilize the natural fluorescent conditions in the absence of reducing agent are typically energy of tryptophan residues. Labels are chosen that emit preferred. Appropriate conditions to the particular assay and different wavelengths of light, such that the acceptor mol components thereof will be well known to one skilled in the ecule label may be differentiated from that of the donor. art Since the efficiency of energy transfer between the labels is 0290. In a particular embodiment, the level of mRNA cor related to the distance separating the molecules, spatial rela responding to the marker can be determined both by in situ tionships between the molecules can be assessed. In a situa and by in vitro formats in a biological sample using methods tion in which binding occurs between the molecules, the known in the art. The term “biological sample' is intended to fluorescent emission of the acceptor molecule label in the include tissues, cells, biological fluids and isolates thereof, assay should be maximal. An FET binding event can be isolated from a subject, as well as tissues, cells and fluids conveniently measured through standard fluorometric detec present within a Subject. Many expression detection methods tion means well known in the art (e.g., using a fluorimeter). use isolated RNA. For in vitro methods, any RNA isolation 0288. In another embodiment, determination of the ability technique that does not select against the isolation of mRNA of a probe to recognize a marker can be accomplished without can be utilized for the purification of RNA from cells (see, labeling either assay component (probe or marker) by utiliz e.g., Ausubel et al.,ed., Current Protocols in Molecular Biol ing a technology Such as real-time Biomolecular Interaction ogy, John Wiley & Sons, NewYork 1987-1999). Additionally, Analysis (BIA) (see, e.g., Solander, S, and Urbaniczky, C., large numbers of tissue samples can readily be processed 1991, Anal. Chem. 63:2338-2345 and Szabo et al., 1995, using techniques well known to those of skill in the art. Such Curr. Opin. Struct. Biol. 5:699-705). As used herein, “BIA' as, for example, the single-step RNA isolation process of or 'Surface plasmon resonance' is a technology for studying Chomczynski (1989, U.S. Pat. No. 4,843,155). biospecific interactions in real time, without labeling any of 0291. The isolated nucleic acid can be used in hybridiza the interactants (e.g., BIAcore). Changes in the mass at the tion or amplification assays that include, but are not limited binding Surface (indicative of a binding event) result in alter to, Southern or Northern analyses, polymerase chain reaction ations of the refractive index of light near the surface (the analyses and probe arrays. One preferred diagnostic method optical phenomenon of Surface plasmon resonance (SPR)), for the detection of mRNA levels involves contacting the resulting in a detectable signal which can be used as an isolated mRNA with a nucleic acid molecule (probe) that can indication of real-time reactions between biological mol hybridize to the mRNA encoded by the gene being detected. ecules. The nucleic acid probe can be, for example, a full-length 0289 Alternatively, in another embodiment, analogous cDNA, or a portion thereof. Such as an oligonucleotide of at diagnostic and prognostic assays can be conducted with least 7, 15, 30, 50, 100, 250 or 500 nucleotides in length and marker and probe as Solutes in a liquid phase. In Suchanassay, Sufficient to specifically hybridize under Stringent conditions the complexed marker and probe are separated from uncom to a mRNA or genomic DNA encoding a marker of the US 2009/0297536A1 Dec. 3, 2009 32 present invention. Other suitable probes for use in the diag Suitable genes for normalization include housekeeping genes nostic assays of the invention are described herein. Hybrid Such as the actin gene, or epithelial cell-specific genes. This ization of an mRNA with the probe indicates that the marker normalization allows the comparison of the expression level in question is being expressed. in one sample, e.g., a Subject sample, to another sample, e.g., 0292. In one format, the mRNA is immobilized on a solid a non-cancerous sample, or between samples from different Surface and contacted with a probe, for example by running SOUCS. the isolated mRNA on an agarose gel and transferring the 0296 Alternatively, the expression level can be provided mRNA from the gel to a membrane, such as nitrocellulose. In as a relative expression level. To determine a relative expres an alternative format, the probe(s) are immobilized on a solid sion level of a marker, the level of expression of the marker is surface and the mRNA is contacted with the probe(s), for determined for 10 or more samples of normal versus cancer example, in an Affymetrix gene chip array. A skilled artisan cell isolates, preferably 50 or more samples, prior to the can readily adapt known mRNA detection methods for use in determination of the expression level for the sample in ques detecting the level of mRNA encoded by the markers of the tion. The mean expression level of each of the genes assayed present invention. in the larger number of samples is determined and this is used 0293. The probes can be full length or less than the full as a baseline expression level for the marker. The expression length of the nucleic acid sequence encoding the protein. level of the marker determined for the test sample (absolute Shorter probes are empirically tested for specificity. Prefer level of expression) is then divided by the mean expression ably nucleic acid probes are 20 bases or longer in length. (See, value obtained for that marker. This provides a relative e.g., Sambrook et al. for methods of selecting nucleic acid expression level. probe sequences for use in nucleic acid hybridization.) Visu 0297 Preferably, the samples used in the baseline deter alization of the hybridized portions allows the qualitative mination will be from cancer cells or normal cells of the same determination of the presence or absence of cDNA. tissue type. The choice of the cell source is dependent on the 0294. An alternative method for determining the level of a use of the relative expression level. Using expression found in transcript corresponding to a marker of the present invention normal tissues as a mean expression score aids in validating in a sample involves the process of nucleic acid amplification, whether the marker assayed is specific to the tissue from e.g., by rtPCR (the experimental embodiment set forth in which the cell was derived (versus normal cells). In addition, Mullis, 1987, U.S. Pat. No. 4,683.202), ligase chain reaction as more data is accumulated, the mean expression value can (Barany, 1991, Proc. Natl. Acad. Sci. USA, 88:189-193), self be revised, providing improved relative expression values sustained sequence replication (Guatelli et al., 1990, Proc. based on accumulated data. Expression data from normal Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplifi cells provides a means for grading the severity of the cancer cation system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA State. 86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/ 0298. In another preferred embodiment, expression of a Technology 6: 1197), rolling circle replication (Lizardi et al., marker is assessed by preparing genomic DNA or mRNA/ U.S. Pat. No. 5,854,033) or any other nucleic acid amplifica cDNA (i.e. a transcribed polynucleotide) from cells in a sub tion method, followed by the detection of the amplified mol ject sample, and by hybridizing the genomic DNA or mRNA/ ecules using techniques well known to those of skill in the art. cDNA with a reference polynucleotide which is a Fluorogenic rtPCR may also be used in the methods of the complement of a polynucleotide comprising the marker, and invention. In fluorogenic rtPCR, quantitation is based on fragments thereof. cDNA can, optionally, be amplified using amount of fluorescence signals, e.g., TaqMan and Sybr green. any of a variety of polymerase chain reaction methods prior to These detection schemes are especially useful for the detec hybridization with the reference polynucleotide. Expression tion of nucleic acid molecules if such molecules are present in of one or more markers can likewise be detected using quan very low numbers. As used herein, amplification primers are titative PCR (QPCR) to assess the level of expression of the defined as being a pair of nucleic acid molecules that can marker(s). Alternatively, any of the many known methods of anneal to 5' or 3' regions of a gene (plus and minus strands, detecting mutations or variants (e.g. single nucleotide poly respectively, or Vice-versa) and contain a short region in morphisms, deletions, etc.) of a marker of the invention may between. In general, amplification primers are from about 10 be used to detect occurrence of a mutated marker in a Subject. to 30 nucleotides in length and flanka region from about 50 to 0299. In a related embodiment, a mixture of transcribed 200 nucleotides in length. Under appropriate conditions and polynucleotides obtained from the sample is contacted with a with appropriate reagents, such primers permit the amplifi Substrate having fixed thereto a polynucleotide complemen cation of a nucleic acid molecule comprising the nucleotide tary to or homologous with at least a portion (e.g. at least 7. sequence flanked by the primers. For in situ methods, mRNA 10, 15, 20, 25, 30, 40, 50, 100, 500, or more nucleotide does not need to be isolated from the cells prior to detection. residues) of a marker of the invention. If polynucleotides In Such methods, a cellor tissue sample is prepared/processed complementary to or homologous with are differentially using known histological methods. The sample is then immo detectable on the substrate (e.g. detectable using different bilized on a Support, typically a glass slide, and then contacted chromophores or fluorophores, or fixed to different selected with a probe that can hybridize to mRNA that encodes the positions), then the levels of expression of a plurality of marker. markers can be assessed simultaneously using a single Sub 0295. As an alternative to making determinations based on strate (e.g. a 'gene chip' microarray of polynucleotides fixed the absolute expression level of the marker, determinations at selected positions). When a method of assessing marker may be based on the normalized expression level of the expression is used which involves hybridization of one marker. Expression levels are normalized by correcting the nucleic acid with another, it is preferred that the hybridization absolute expression level of a marker by comparing its be performed under Stringent hybridization conditions. expression to the expression of a gene that is not a marker, 0300. In another embodiment, a combination of methods e.g., a housekeeping gene that is constitutively expressed. to assess the expression of a marker is utilized. US 2009/0297536A1 Dec. 3, 2009

0301 Because the compositions, kits, and methods of the include glass, polystyrene, polypropylene, polyethylene, invention rely on detection of a difference in expression levels dextran, nylon, amylases, natural and modified celluloses, or copy number of one or more markers of the invention, it is polyacrylamides, gabbros, and magnetite. preferable that the level of expression or copy number of the 0308 One skilled in the art will know many other suitable marker is significantly greater than the minimum detection carriers for binding antibody or antigen, and will be able to limit of the method used to assess expression or copy number adapt Such Support for use with the present invention. For in at least one of normal cells and cancerous cells. example, protein isolated from cells can be run on a polyacry 0302) 3. Methods for Detection of Expressed Protein lamide gel electrophoresis and immobilized onto a solid 0303. The activity or level of a marker protein can also be phase Support such as nitrocellulose. The Support can then be detected and/or quantified by detecting or quantifying the washed with suitable buffers followed by treatment with the expressed polypeptide. The polypeptide can be detected and detectably labeled antibody. The solid phase support can then quantified by any of a number of means well known to those be washed with the buffer a second time to remove unbound of skill in the art. These may include analytic biochemical antibody. The amount of bound label on the solid support can methods such as electrophoresis, capillary electrophoresis, then be detected by conventional means. Means of detecting high performance liquid chromatography (HPLC), thin layer proteins using electrophoretic techniques are well known to chromatography (TLC), hyperdiffusion chromatography, and those of skill in the art (see generally, R. Scopes (1982) the like, or various immunological methods such as fluid or Protein Purification, Springer-Verlag, N.Y.; Deutscher, gel precipitin reactions, immunodiffusion (single or double), (1990) Methods in Enzymology Vol. 182: Guide to Protein immunoelectrophoresis, radioimmunoassay (RIA), enzyme Purification, Academic Press, Inc., N.Y.). linked immunosorbent assays (ELISAS), immunofluorescent (0309. In another preferred embodiment, Western blot (im assays, western blotting, and the like. A skilled artisan can munoblot) analysis is used to detect and quantify the presence readily adapt known protein/antibody detection methods for of a polypeptide in the sample. This technique generally use in determining whether cells express a marker of the comprises separating sample proteins by gel electrophoresis present invention. on the basis of molecular weight, transferring the separated 0304. A preferred agent for detecting a polypeptide of the proteins to a Suitable solid Support, (such as a nitrocellulose invention is an antibody capable of binding to a polypeptide filter, a nylon filter, orderivatized nylon filter), and incubating corresponding to a marker of the invention, preferably an the sample with the antibodies that specifically bind a antibody with a detectable label. Antibodies can be poly polypeptide. The anti-polypeptide antibodies specifically clonal, or more preferably, monoclonal. An intactantibody, or bind to the polypeptide on the solid support. These antibodies a fragment thereof (e.g., Fab or F(ab')) can be used. The term may be directly labeled or alternatively may be subsequently “labeled, with regard to the probe or antibody, is intended to detected using labeled antibodies (e.g., labeled sheep anti encompass direct labeling of the probe or antibody by cou mouse antibodies) that specifically bind to the anti-polypep pling (i.e., physically linking) a detectable Substance to the tide. probe or antibody, as well as indirect labeling of the probe or 0310. In a more preferred embodiment, the polypeptide is antibody by reactivity with another reagent that is directly detected using an immunoassay. As used herein, an immu labeled. Examples of indirect labeling include detection of a noassay is an assay that utilizes an antibody to specifically primary antibody using a fluorescently labeled secondary bind to the analyte. The immunoassay is thus characterized by antibody and end-labeling of a DNA probe with biotin such detection of specific binding of a polypeptide to an anti that it can be detected with fluorescently labeled streptavidin. antibody as opposed to the use of other physical or chemical 0305. In a preferred embodiment, the antibody is labeled, properties to isolate, target, and quantify the analyte. e.g. a radio-labeled, chromophore-labeled, fluorophore-la 0311. The polypeptide is detected and/or quantified using beled, or enzyme-labeled antibody). In another embodiment, any of a number of well recognized immunological binding an antibody derivative (e.g. an antibody conjugated with a assays (see, e.g., U.S. Pat. Nos. 4.366,241; 4,376,110; 4.517, Substrate or with the protein or ligand of a protein-ligand pair 288; and 4.837,168). For a review of the general immunoas {e.g. biotin-streptavidin), or an antibody fragment (e.g. a says, see also Asai (1993) Methods in Cell Biology Volume single-chain antibody, an isolated antibody hyperVariable 37: Antibodies in Cell Biology, Academic Press, Inc. New domain, etc.) which binds specifically with a protein corre York; Stites & Terr (1991) Basic and Clinical Immunology sponding to the marker, such as the protein encoded by the 7th Edition. open reading frame corresponding to the marker or such a 0312 Immunological binding assays (or immunoassays) protein which has undergone all or a portion of its normal typically utilize a "capture agent” to specifically bind to and post-translational modification, is used. often immobilize the analyte (polypeptide or Subsequence). 0306 Proteins from cells can be isolated using techniques The capture agent is a moiety that specifically binds to the that are well known to those of skill in the art. The protein analyte. In a preferred embodiment, the capture agent is an isolation methods employed can, for example, be such as antibody that specifically binds a polypeptide. The antibody those described in Harlow and Lane (Harlow and Lane, 1988, (anti-peptide) may be produced by any of a number of means Antibodies: A Laboratory Manual, Cold Spring Harbor Labo well known to those of skill in the art. ratory Press, Cold Spring Harbor, N.Y.). 0313 Immunoassays also often utilize a labeling agent to 0307. In one format, antibodies, or antibody fragments, specifically bind to and label the binding complex formed by can be used in methods such as Western blots or immunof the capture agent and the analyte. The labeling agent may luorescence techniques to detect the expressed proteins. In itself be one of the moieties comprising the antibody/analyte such uses, it is generally preferable to immobilize either the complex. Thus, the labeling agent may be a labeled polypep antibody or proteins on a solid Support. Suitable Solid phase tide or a labeled anti-antibody. Alternatively, the labeling Supports or carriers include any Support capable of binding an agent may be a third moiety, Such as another antibody, that antigen or an antibody. Well-known Supports or carriers specifically binds to the antibody/polypeptide complex. US 2009/0297536A1 Dec. 3, 2009 34

0314. In one preferred embodiment, the labeling agent is a 0323. In vivo techniques for detection of a biomarker pro second human antibody bearing a label. Alternatively, the tein include introducing into a subject a labeled antibody second antibody may lack a label, but it may, in turn, bebound directed against the protein. For example, the antibody can be by a labeled third antibody specific to antibodies of the spe labeled with a radioactive marker whose presence and loca cies from which the second antibody is derived. The second tion in a subject can be detected by standard imaging tech can be modified with a detectable moiety, e.g. as biotin, to niques. which a third labeled molecule can specifically bind, such as 0324 Certain markers identified by the methods of the enzyme-labeled streptavidin. invention may be secreted proteins. It is a simple matter for 0315. Other proteins capable of specifically binding the skilled artisan to determine whether any particular marker immunoglobulin constant regions, such as protein A or pro protein is a secreted protein. In order to make this determina tein G may also be used as the label agent. These proteins are tion, the marker protein is expressed in, for example, a mam normal constituents of the cell walls of streptococcal bacteria. malian cell, preferably a human cell line, extracellular fluid is They exhibit a strong non-immunogenic reactivity with collected, and the presence or absence of the protein in the immunoglobulin constant regions from a variety of species extracellular fluid is assessed (e.g. using a labeled antibody (see, generally Kronval, et al. (1973).J. Immunol. 111: 1401 which binds specifically with the protein). 1406, and Akerstrom (1985) J. Immunol., 135: 2589-2542). 0325 The following is an example of a method which can 0316. As indicated above, immunoassays for the detection be used to detect secretion of a protein. About 8x10 293T and/or quantification of a polypeptide can take a wide variety cells are incubated at 37° C. in wells containing growth of formats well known to those of skill in the art. medium (Dulbecco's modified Eagle's medium (DMEM} 0317 Preferred immunoassays for detecting a polypep supplemented with 10% fetal bovine serum) under a 5%(v/v) tide are either competitive or noncompetitive. Noncompeti CO2, 95% air atmosphere to about 60-70% confluence. The tive immunoassays are assays in which the amount of cap cells are then transfected using a standard transfection mix tured analyte is directly measured. In one preferred ture comprising 2 micrograms of DNA comprising an expres 'sandwich' assay, for example, the capture agent (anti-pep sion vector encoding the protein and 10 microliters of Lipo tide antibodies) can be bound directly to a solid substrate fectAMINETM (GIBCO/BRL Catalog no. 18342-012) per where they are immobilized. These immobilized antibodies well. The transfection mixture is maintained for about 5 then capture polypeptide present in the test sample. The hours, and then replaced with fresh growth medium and polypeptide thus immobilized is then bound by a labeling maintained in an air atmosphere. Each well is gently rinsed agent, such as a second human antibody bearing a label. twice with DMEM which does not contain methionine or 0318. In competitive assays, the amount of analyte cysteine (DMEM-MC; ICN Catalog no. 16-424–54). About 1 (polypeptide) present in the sample is measured indirectly by milliliter of DMEM-MC and about 50 microcuries of Trans measuring the amount of an added (exogenous) analyte S' reagent (ICN Catalog no. 51006) are added to each (polypeptide) displaced (or competed away) from a capture well. The wells are maintained under the 5% CO atmosphere agent (anti peptide antibody) by the analyte present in the described above and incubated at 37°C. for a selected period. sample. In one competitive assay, a known amount of, in this Following incubation, 150 microliters of conditioned case, a polypeptide is added to the sample and the sample is medium is removed and centrifuged to remove floating cells then contacted with a capture agent. The amount of polypep and debris. The presence of the protein in the Supernatant is an tide bound to the antibody is inversely proportional to the indication that the protein is secreted. concentration of polypeptide present in the sample. 0326. It will be appreciated that subject samples, e.g., a 0319. In one particularly preferred embodiment, the anti sample containing tissue, whole blood, serum, plasma, buccal body is immobilized on a solid substrate. The amount of scrape, saliva, cerebrospinal fluid, urine, stool, bile, pancre polypeptide bound to the antibody may be determined either atic juice, and pancreatic tissue, may contain cells therein, by measuring the amount of polypeptidepresentina polypep particularly when the cells are cancerous, and, more particu tide? antibody complex, or alternatively by measuring the larly, when the cancer is metastasizing, and thus may be used amount of remaining uncomplexed polypeptide. The amount in the methods of the present invention. The cell sample can, of polypeptide may be detected by providing a labeled of course, be subjected to a variety of well-known post polypeptide. collection preparative and storage techniques (e.g., nucleic 0320. The assays of this invention are scored (as positive acid and/or protein extraction, fixation, storage, freezing, or negative or quantity of polypeptide) according to standard ultrafiltration, concentration, evaporation, centrifugation, methods well known to those of skill in the art. The particular etc.) prior to assessing the level of expression of the marker in method of scoring will depend on the assay format and choice the sample. Thus, the compositions, kits, and methods of the of label. For example, a Western Blot assay can be scored by invention can be used to detect expression of markers corre visualizing the colored product produced by the enzymatic sponding to proteins having at least one portion which is label. A clearly visible colored band or spot at the correct displayed on the surface of cells which express it. It is a molecular weight is scored as a positive result, while the simple matter for the skilled artisan to determine whether the absence of a clearly visible spot or band is scored as a nega protein corresponding to any particular marker comprises a tive. The intensity of the band or spot can provide a quanti cell-surface protein. For example, immunological methods tative measure of polypeptide. may be used to detect such proteins on whole cells, or well 0321) Antibodies for use in the various immunoassays known computer-based sequence analysis methods (e.g. the described herein, can be produced as described below. SIGNALP program; Nielsen et al., 1997, Protein Engineer 0322. In another embodiment, level (activity) is assayed ing 10:1-6) may be used to predict the presence of at least one by measuring the enzymatic activity of the gene product. extracellular domain (i.e. including both secreted proteins Methods of assaying the activity of an enzyme are well known and proteins having at least one cell-surface domain). Expres to those of skill in the art. sion of a marker corresponding to a protein having at least one US 2009/0297536A1 Dec. 3, 2009

portion which is displayed on the surface of a cell which tion 7:244. In one embodiment, a chip comprises all the allelic expresses it may be detected without necessarily lysing the variants of at least one polymorphic region of a gene. The cell (e.g. using a labeled antibody which binds specifically Solid phase Support is then contacted with a test nucleic acid with a cell-surface domain of the protein). and hybridization to the specific probes is detected. Accord 0327. The invention also encompasses kits for detecting ingly, the identity of numerous allelic variants of one or more the presence of a polypeptide or nucleic acid corresponding to genes can be identified in a simple hybridization experiment. a marker of the inventionina biological sample, e.g., a sample For example, the identity of the allelic variant of the nucle containing tissue, whole blood, serum, plasma, buccal scrape, otide polymorphism in the 5' upstream regulatory element saliva, cerebrospinal fluid, urine, stool, bile, pancreatic juice, can be determined in a single hybridization experiment. and pancreatic tissue. Such kits can be used to determine if a Subject is Suffering from or is at increased risk of developing 0333. In other detection methods, it is necessary to first cancer. For example, the kit can comprise a labeled com amplify at least a portion of a marker prior to identifying the pound or agent capable of detecting a polypeptide or an allelic variant. Amplification can be performed, e.g., by PCR mRNA encoding a polypeptide corresponding to a marker of and/or LCR (see Wu and Wallace (1989) Genomics 4:560), the invention in a biological sample and means for determin according to methods known in the art. In one embodiment, ing the amount of the polypeptide or mRNA in the sample genomic DNA of a cell is exposed to two PCR primers and (e.g., an antibody which binds the polypeptide or an oligo amplification for a number of cycles sufficient to produce the nucleotide probe which binds to DNA or mRNA encoding the required amount of amplified DNA. In preferred embodi polypeptide). Kits can also include instructions for interpret ments, the primers are located between 150 and 350 base ing the results obtained using the kit. pairs apart. 0328. For antibody-based kits, the kit can comprise, for 0334 Alternative amplification methods include: self sus example: (1) a first antibody (e.g., attached to a solid Support) tained sequence replication (Guatelli, J. C. et al., (1990) Proc. which binds to a polypeptide corresponding to a marker of the Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplifi invention; and, optionally, (2) a second, different antibody cation system (Kwoh, D. Y. et al., (1989) Proc. Natl. Acad. which binds to either the polypeptide or the first antibody and Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et is conjugated to a detectable label. al., (1988) Bio/Technology 6:1197), and self-sustained 0329. For oligonucleotide-based kits, the kit can com sequence replication (Guatelli et al., (1989)Proc. Nat. Acad. prise, for example: (1) an oligonucleotide, e.g., a detectably Sci. 87: 1874), and nucleic acid based sequence amplification labeled oligonucleotide, which hybridizes to a nucleic acid (NABSA), or any other nucleic acid amplification method, sequence encoding a polypeptide corresponding to a marker followed by the detection of the amplified molecules using of the invention or (2) a pair of primers useful for amplifying techniques well known to those of skill in the art. These a nucleic acid molecule corresponding to a marker of the detection schemes are especially useful for the detection of invention. The kit can also comprise, e.g., a buffering agent, a nucleic acid molecules if such molecules are present in very preservative, or a protein stabilizing agent. The kit can further low numbers. comprise components necessary for detecting the detectable 0335. In one embodiment, any of a variety of sequencing label (e.g., an enzyme or a Substrate). The kit can also contain reactions known in the art can be used to directly sequence at a control sample or a series of control samples which can be least a portion of a marker and detect allelic variants, e.g., assayed and compared to the test sample. Each component of mutations, by comparing the sequence of the sample the kit can be enclosed within an individual container and all sequence with the corresponding reference (control) of the various containers can be within a single package, sequence. Exemplary sequencing reactions include those along with instructions for interpreting the results of the based on techniques developed by Maxamand Gilbert (Proc. assays performed using the kit. Natl. AcadSci USA (1977) 74:560) or Sanger (Sanger et al. 0330. 4. Method for Detecting Structural Alterations (1977) Proc. Nat. Acad. Sci. 74:5463). It is also contemplated 0331. The invention also provides a method for assessing that any of a variety of automated sequencing procedures may whether a subject is afflicted with cancer or is at risk for be utilized when performing the subject assays (Biotech developing cancer by comparing the structural alterations, niques (1995) 19:448), including sequencing by mass spec e.g., mutations or allelic variants, of a marker in a cancer trometry (see, for example, U.S. Pat. No. 5,547,835 and inter sample with the structural alterations, e.g., mutations of a national patent application Publication Number WO marker in a normal, e.g., control sample. The presence of a 94/16101, entitled DNA Sequencing by Mass Spectrometry structural alteration, e.g., mutation or allelic variant in the by H. Köster; U.S. Pat. No. 5,547,835 and international patent marker in the cancer sample is an indication that the Subject is application Publication Number WO 94/21822 entitled afflicted with cancer. “DNA Sequencing by Mass Spectrometry Via Exonuclease 0332 A preferred detection method is allele specific Degradation” by H. Köster), and U.S. Pat. No. 5,605,798 and hybridization using probes overlapping the polymorphic site International Patent Application No. PCT/US96/03651 and having about 5, 10, 20, 25, or 30 nucleotides around the entitled DNA Diagnostics Based on Mass Spectrometry by H. polymorphic region. In a preferred embodiment of the inven Köster; Cohen et al. (1996) Adv Chromatogr36:127-162; and tion, several probes capable of hybridizing specifically to Griffinet al. (1993) Appl Biochem Biotechnol 38:147-159). It allelic variants are attached to a solid phase Support, e.g., a will be evident to one skilled in the art that, for certain “chip”. Oligonucleotides can be bound to a solid support by a embodiments, the occurrence of only one, two or three of the variety of processes, including lithography. For example a nucleic acid bases need be determined in the sequencing chip can hold up to 250,000 oligonucleotides (GeneChip, reaction. For instance, A-track or the like, e.g., where only AffymetrixTM). Mutation detection analysis using these chips one nucleotide is detected, can be carried out. comprising oligonucleotides, also termed “DNA probe 0336 Yet other sequencing methods are disclosed, e.g., in arrays” is described e.g., in Croninet al. (1996) Human Muta U.S. Pat. No. 5,580,732 entitled “Method of DNA sequencing US 2009/0297536A1 Dec. 3, 2009 36 employing a mixed DNA-polymer chain probe' and U.S. Pat. with labeled probes. The sensitivity of the assay may be No. 5,571,676 entitled “Method for mismatch-directed in enhanced by using RNA (rather than DNA), in which the vitro DNA sequencing.” secondary structure is more sensitive to a change in sequence. 0337. In some cases, the presence of a specificallele of a In another preferred embodiment, the subject method utilizes marker in DNA from a subject can be shown by restriction heteroduplex analysis to separate double stranded heterodu enzyme analysis. For example, a specific nucleotide polymor plex molecules on the basis of changes in electrophoretic phism can result in a nucleotide sequence comprising a mobility (Keen et al. (1991) Trends Genet. 7:5). restriction site which is absent from the nucleotide sequence 0341. In yet another embodiment, the identity of an allelic of another allelic variant. variant of a polymorphic region is obtained by analyzing the 0338. In a further embodiment, protection from cleavage movement of a nucleic acid comprising the polymorphic agents (such as a nuclease, hydroxylamine or osmium tetroX region in polyacrylamide gels containing a gradient of dena ide and with piperidine) can be used to detect mismatched turant is assayed using denaturing gradient gel electrophore bases in RNA/RNA DNA/DNA, or RNA/DNA heterodu sis (DGGE) (Myers et al. (1985) Nature 313:495). When plexes (Myers, et al. (1985) Science 230:1242). In general, DGGE is used as the method of analysis, DNA will be modi the technique of "mismatch cleavage' starts by providing fied to insure that it does not completely denature, for heteroduplexes formed by hybridizing a control nucleic acid, example by adding a GC clamp of approximately 40 bp of which is optionally labeled, e.g., RNA or DNA, comprising a high-melting GC-rich DNA by PCR. In a further embodi nucleotide sequence of a marker allelic variant with a sample ment, a temperature gradient is used in place of a denaturing nucleic acid, e.g., RNA or DNA, obtained from a tissue agent gradient to identify differences in the mobility of con sample. The double-stranded duplexes are treated with an trol and sample DNA (Rosenbaum and Reissner (1987) Bio agent which cleaves single-stranded regions of the duplex phy's Chem 265:1275). Such as duplexes formed based on basepair mismatches 0342. Examples of techniques for detecting differences of between the control and sample strands. For instance, RNA/ at least one nucleotide between two nucleic acids include, but DNA duplexes can be treated with RNase and DNA/DNA are not limited to, selective oligonucleotide hybridization, hybrids treated with S1 nuclease to enzymatically digest the selective amplification, or selective primer extension. For mismatched regions. In other embodiments, either DNA/ example, oligonucleotide probes may be prepared in which DNA or RNA/DNA duplexes can be treated with hydroxy the known polymorphic nucleotide is placed centrally (allele lamine or osmium tetroxide and with piperidine in order to specific probes) and then hybridized to target DNA under digest mismatched regions. After digestion of the mis conditions which permit hybridization only if a perfect match matched regions, the resulting material is then separated by is found (Saiki et al. (1986) Nature 324:163): Saiki et al size on denaturing polyacrylamide gels to determine whether (1989) Proc. Natl. Acad. Sci. USA 86:6230; and Wallace etal. the control and sample nucleic acids have an identical nucle (1979) Nucl. Acids Res. 6:3543). Such allele specific oligo otide sequence or in which nucleotides they are different. See, nucleotide hybridization techniques may be used for the for example, Cotton et al (1988) Proc. Natl AcadSci USA simultaneous detection of several nucleotide changes in dif 85:4397: Saleeba et al (1992) Methods Enzymol. 217:286 ferent polylmorphic regions of marker. For example, oligo 295. In a preferred embodiment, the control or sample nucleic nucleotides having nucleotide sequences of specific allelic acid is labeled for detection. variants are attached to a hybridizing membrane and this 0339. In another embodiment, an allelic variant can be membrane is then hybridized with labeled sample nucleic identified by denaturing high-performance liquid chromatog acid. Analysis of the hybridization signal will then reveal the raphy (DHPLC) (Oefner and Underhill, (1995) Am. J. Human identity of the nucleotides of the sample nucleic acid. Gen. 57:Suppl. A266). DHPLC uses reverse-phase ion-pair 0343 Alternatively, allele specific amplification technol ing chromatography to detect the heteroduplexes that are ogy which depends on selective PCR amplification may be generated during amplification of PCR fragments from indi used in conjunction with the instant invention. Oligonucle viduals who are heterozygous at a particular nucleotide locus otides used as primers for specific amplification may carry the within that fragment (Oefner and Underhill (1995) Am. J. allelic variant of interest in the center of the molecule (so that Human Gen. 57:Suppl. A266). In general, PCR products are amplification depends on differential hybridization) (Gibbs et produced using PCR primers flanking the DNA of interest. al (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme DHPLC analysis is carried out and the resulting chromato 3' end of one primer where, under appropriate conditions, grams are analyzed to identify base pair alterations or dele mismatch can prevent, or reduce polymerase extension tions based on specific chromatographic profiles (see (Prossner (1993) Tibtech 11:238; Newton et al. (1989) Nucl. O'Donovan et al. (1998) Genomics 52:44-49). Acids Res. 17:2503). This technique is also termed “PROBE 0340. In other embodiments, alterations in electrophoretic for Probe Oligo Base Extension. In addition it may be desir mobility is used to identify the type of marker allelic variant. able to introduce a novel restriction site in the region of the For example, single strand conformation polymorphism mutation to create cleavage-based detection (Gasparini etal (SSCP) may be used to detect differences in electrophoretic (1992) Mol. Cell. Probes 6:1). mobility between mutant and wildtype nucleic acids (Orita et 0344. In another embodiment, identification of the allelic al. (1989) Proc Natl. Acad. Sci. USA 86:2766, see also Cotton variant is carried out using an oligonucleotide ligation assay (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet (OLA), as described, e.g., in U.S. Pat. No. 4,998,617 and in Anal Tech Appl 9:73-79). Single-stranded DNA fragments of Landegren, U. et al., (1988) Science 241:1077-1080. The sample and control nucleic acids are denatured and allowed to OLA protocol uses two oligonucleotides which are designed renature. The secondary structure of single-stranded nucleic to be capable of hybridizing to abutting sequences of a single acids varies according to sequence, the resulting alteration in Strand of a target. One of the oligonucleotides is linked to a electrophoretic mobility enables the detection of even a single separation marker, e.g., biotinylated, and the other is detect base change. The DNA fragments may be labeled or detected ably labeled. If the precise complementary sequence is found US 2009/0297536A1 Dec. 3, 2009 37 in a target molecule, the oligonucleotides will hybridize Such 18:3671; Syvanen, A.-C., et al., (990) Genomics 8:684-692; that their termini abut, and create a ligation Substrate. Liga Kuppuswamy, M. N. et al., (1991) Proc. Natl. Acad. Sci. tion then permits the labeled oligonucleotide to be recovered (U.S.A.) 88:1143-1147: Prezant, T. R. et al., (1992) Hum. using avidin, or another biotin ligand. Nickerson, D. A. et al. Mutat. 1:159-164; Ugozzoli, L. et al., (1992) GATA 9:107 have described a nucleic acid detection assay that combines 112: Nyren, P. (1993) et al., Anal Biochem. 208:171-175). attributes of PCR and OLA (Nickerson, D. A. et al., (1990) These methods differ from GBATM in that they all rely on the Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927. In this method, incorporation of labeled deoxynucleotides to discriminate PCR is used to achieve the exponential amplification of target between bases at a polymorphic site. In Such a format, since DNA, which is then detected using OLA. the signal is proportional to the number of deoxynucleotides 0345 The invention further provides methods for detect incorporated, polymorphisms that occur in runs of the same ing single nucleotide polymorphisms in a marker. Because nucleotide can result in signals that are proportional to the single nucleotide polymorphisms constitute sites of variation length of the run (Syvanen, A. C., et al., (1993) Amer. J. Hum. flanked by regions of invariant sequence, their analysis Genet. 52:46-59). requires no more than the determination of the identity of the 0350 For determining the identity of the allelic variant of single nucleotide present at the site of variation and it is a polymorphic region located in the coding region of a unnecessary to determine a complete gene sequence for each marker, yet other methods than those described above can be subject. Several methods have been developed to facilitate the used. For example, identification of an allelic variant which analysis of Such single nucleotide polymorphisms. encodes a mutated marker can be performed by using an 0346. In one embodiment, the single base polymorphism antibody specifically recognizing the mutant protein in, e.g., can be detected by using a specialized exonuclease-resistant immunohistochemistry or immunoprecipitation. Antibodies nucleotide, as disclosed, e.g., in Mundy, C. R. (U.S. Pat. No. to wild-type marker or mutated forms of markers can be 4,656.127). According to the method, a primer complemen prepared according to methods known in the art. tary to the allelic sequence immediately 3' to the polymorphic 0351 Alternatively, one can also measure an activity of a site is permitted to hybridize to a target molecule obtained marker, Such as binding to a marker ligand. Binding assays from a particular animal or human. If the polymorphic site on are known in the art and involve, e.g., obtaining cells from a the target molecule contains a nucleotide that is complemen Subject, and performing binding experiments with a labeled tary to the particular exonuclease-resistant nucleotide deriva ligand, to determine whether binding to the mutated form of tive present, then that derivative will be incorporated onto the the protein differs from binding to the wild-type of the pro end of the hybridized primer. Such incorporation renders the tein. primer resistant to exonuclease, and thereby permits its detec 0352 B. Pharmacogenomics tion. Since the identity of the exonuclease-resistant derivative 0353 Agents or modulators which have a stimulatory or of the sample is known, a finding that the primer has become inhibitory effect on amount and/or activity of a marker of the resistant to exonucleases reveals that the nucleotidepresent in invention can be administered to individuals to treat (prophy the polymorphic site of the target molecule was complemen lactically ortherapeutically) cancer in the Subject. In conjunc tary to that of the nucleotide derivative used in the reaction. tion with Such treatment, the pharmacogenomics (i.e., the This method has the advantage that it does not require the study of the relationship between an individual’s genotype determination of large amounts of extraneous sequence data. and that individual's response to a foreign compound or drug) 0347 In another embodiment of the invention, a solution of the individual may be considered. Differences in metabo based method is used for determining the identity of the lism of therapeutics can lead to severe toxicity or therapeutic nucleotide of a polymorphic site. Cohen, D. et al. (French failure by altering the relation between dose and blood con Patent 2,650,840; PCT Applin. No. WO91/02087). As in the centration of the pharmacologically active drug. Thus, the Mundy method of U.S. Pat. No. 4,656,127, a primer is pharmacogenomics of the individual permits the selection of employed that is complementary to allelic sequences imme effective agents (e.g., drugs) for prophylactic or therapeutic diately 3' to a polymorphic site. The method determines the treatments based on a consideration of the individual's geno identity of the nucleotide of that site using labeled dideoxy type. Such pharmacogenomics can further be used to deter nucleotide derivatives, which, if complementary to the nucle mine appropriate dosages and therapeutic regimens. Accord otide of the polymorphic site will become incorporated onto ingly, the amount, structure, and/or activity of the invention in the terminus of the primer. an individual can be determined to thereby select appropriate 0348 An alternative method, known as Genetic Bit Analy agent(s) for therapeutic or prophylactic treatment of the indi sis or GBATM is described by Goelet, Petal. (PCT Applin. No. vidual. 92/15712). The method of Goelet, P. et al. uses mixtures of 0354 Pharmacogenomics deals with clinically significant labeled terminators and a primer that is complementary to the variations in the response to drugs due to altered drug dispo sequence 3' to a polymorphic site. The labeled terminator that sition and abnormal action in affected persons. See, e.g., is incorporated is thus determined by, and complementary to, Linder (1997) Clin. Chem. 43(2):254-266. In general, two the nucleotide present in the polymorphic site of the target types of pharmacogenetic conditions can be differentiated. molecule being evaluated. In contrast to the method of Cohen Genetic conditions transmitted as a single factor altering the et al. (French Patent 2,650,840; PCT Applin. No. WO91/ way drugs act on the body are referred to as “altered drug 02087) the method of Goelet, P. et al. is preferably a hetero action. Genetic conditions transmitted as single factors alter geneous phase assay, in which the primer or the target mol ing the way the body acts on drugs are referred to as “altered ecule is immobilized to a solid phase. drug metabolism'. These pharmacogenetic conditions can 0349. Several primer-guided nucleotide incorporation occur either as rare defects or as polymorphisms. For procedures for assaying polymorphic sites in DNA have been example, glucose-6-phosphate dehydrogenase (G6PD) defi described (Komher, J. S. et al., (1989) Nucl. Acids. Res. ciency is a common inherited enzymopathy in which the main 17:7779-7784: Sokolov, B. P., (1990) Nucl. Acids Res. clinical complication is hemolysis after ingestion of oxidant US 2009/0297536A1 Dec. 3, 2009 drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) agent can be desirable to increase amount and/or activity of and consumption of fava beans. the marker(s) to higher levels than detected, i.e., to increase 0355. As an illustrative embodiment, the activity of drug the effectiveness of the agent. Alternatively, decreased metabolizing enzymes is a major determinant of both the administration of the agent can be desirable to decrease intensity and duration of drug action. The discovery of amount and/or activity of the marker(s) to lower levels than genetic polymorphisms of drug metabolizing enzymes (e.g., detected, i.e., to decrease the effectiveness of the agent. N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explana EXAMPLES tion as to why some subjects do not obtain the expected drug effects or show exaggerated drug response and serious toxic 0359. This invention is further illustrated by the following ity after taking the standard and safe dose of a drug. These examples which should not be construed as limiting. The polymorphisms are expressed in two phenotypes in the popu contents of all references, figures. Sequence Listing, patents lation, the extensive metabolizer (EM) and poor metabolizer and published patent applications cited throughout this appli (PM). The prevalence of PM is different among different cation are hereby incorporated by reference. populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identi Example 1 fied in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 A. Materials and Methods quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite Primary Tumors and Cell Lines is the active therapeutic moiety, a PM will show notherapeu tic response, as demonstrated for the analgesic effect of 0360 All cell lines were acquired from the AmericanType codeine mediated by its CYP2D6-formed metabolite mor Culture Collection (ATCC) or the German Collection of phine. The other extreme are the so called ultra-rapid metabo Microorganisms and Cell Cultures (DSMZ). All fresh-frozen lizers who do not respond to standard doses. Recently, the specimens of primary pancreatic ductal adenocarcinoma molecular basis of ultra-rapid metabolism has been identified were obtained from the Memorial Sloan-Kettering Cancer to be due to CYP2D6 gene amplification. Center tumor bank and histology was confirmed by hema 0356. Thus, the amount, structure, and/or activity of a toxylin and eosin (H&E) staining prior to inclusion in the marker of the invention in an individual can be determined to study (Table 2). Table 2 lists the cell lines analyzed by array thereby select appropriate agent(s) for therapeutic or prophy CGH and expression profiling in the study. lactic treatment of the individual. In addition, pharmacoge Array-CGH Profiling on cDNA Microarrays netic studies can be used to apply genotyping of polymorphic 0361 Genomic DNA was fragmented and random-prime alleles encoding drug-metabolizing enzymes to the identifi labeled according to published protocol (Pollack, J. R. et al. cation of an individual's drug responsiveness phenotype. This (1999) Nat Genet. 23, 41-6) with modifications. Labeled knowledge, when applied to dosing or drug selection, can DNAs were hybridized to human cDNA microarrays contain avoid adverse reactions or therapeutic failure and thus ing 14,160 cDNA clones (Agilent TechnologiesTM. Human 1 enhance therapeutic or prophylactic efficiency when treating clone set), for which approximately 9.420 unique map posi a subject with a modulator of amount, structure, and/or activ tions were defined (NCBI. Build 33). The median interval ity of a marker of the invention. between mapped elements is 100.1 kilobase, 92.8% of inter 0357 C. Monitoring Clinical Trials vals are less than 1 megabase, and 98.6% are less than 3 0358 Monitoring the influence of agents (e.g., drug com megabases. pounds) on amount, structure, and/or activity of a marker of 0362 Fluorescence ratios of scanned images of the arrays the invention can be applied not only in basic drug screening, were calculated and the raw array-CGH profiles were pro but also in clinical trials. For example, the effectiveness of an cessed to identify statistically significant transitions in copy agent to affect marker amount, structure, and/or activity can number using a segmentation algorithm which employs per be monitored in clinical trials of subjects receiving treatment mutation to determine the significance of change points in the for cancer. In a preferred embodiment, the present invention raw data (Olshen, A. B., and Venkatraman, E. S. (2002) ASA provides a method for monitoring the effectiveness of treat Proceedings of the Joint Statistical Meetings, 2530-2535: ment of a subject with an agent (e.g., an agonist, antagonist, Ginzinger, D. G. (2002) Exp Hematol 30, 503-12). Each peptidomimetic, protein, peptide, antibody, nucleic acid, segment was assigned a Log ratio that is the median of the antisense nucleic acid, ribozyme, small molecule, RNA inter contained probes. The data was centered by the tallest mode fering agent, or other drug candidate) comprising the steps of in the distribution of the segmented values. After mode-cen (i) obtaining a pre-administration sample from a subject prior tering, gains and losses were defined as Log ratios of greater to administration of the agent; (ii) detecting the amount, than or equal to +0.13 or -0.13 (+/-4 standard deviations of structure, and/or activity of one or more selected markers of the middle 50% quantile of data), and amplification and dele the invention in the pre-administration sample; (iii) obtaining tion as ratio greater than 0.52 or less than -0.58, respectively one or more post-administration samples from the subject; (i.e., 97% or 3% quantiles) (FIG. 4). (iv) detecting the amount, structure, and/or activity of the marker(s) in the post-administration samples; (V) comparing Automated Locus Definition the level of expression of the marker(s) in the pre-adminis tration sample with the amount, structure, and/or activity of 0363 Loci were defined by an automated algorithm the marker(s) in the post-administration sample or samples: applied to the segmented databased on the following rules: and (vi) altering the administration of the agent to the subject 0364 1. Segments above 97" percentile or below 3" per accordingly. For example, increased administration of the centile were identified as altered. US 2009/0297536A1 Dec. 3, 2009 39

0365 2. If two or more altered segments are adjacent in a number value. In order to maximize detection of focal CNAs, single profile or separated by less than 500KB, the entire two separate interpolations were calculated: one selecting the region spanned by the segments was considered to be an higher bounding CGH probe and one choosing the lower. For altered span. each gene position, the samples were grouped based on 0366 3. Highly altered segments or spans that were whether array-CGH shows altered copy number or not based shorter than 20 MB were retained as “informative spans' for on interpolated CGH value. The effect of gene dosage on defining discrete locus boundaries. Longer regions were not expression was measured by calculating a gene weight discarded, but were not included in defining locus bound defined as the difference of the means of the expression value aries. in the altered and unaltered sample groups divided by the Sum 0367 4. Informative spans were compared across samples of the standard deviations of the expression values in altered to identify overlapping groups of positive-value or negative and unaltered sample groups (Hyman, E., et al. (2002) Can value segments; each group defined a locus. cer Res 62,6240-5). The significance of the weight for each 0368 5. Minimal common regions (MCRs) were defined gene was estimated by permuting the sample labels 10,000 as contiguous spans having at least 75% of the peak recur times and applying an alpha threshold of 0.05. rence as calculated by counting the occurrence of highly altered segments. If two MCRs were separated by a gap of B. Results only one probe position, they were joined. If there were more 0373 Comprehensive Catalogue of CNAs in the Pancre than 3 MCRs in a locus, the whole region was reported as a atic Adenocarcinoma Genome single complex MCR. 0374 From a total of 75 primary pancreatic tumor speci MCR Characterization mens, 13 samples were identified that possessed greater than 60% neoplastic cellularity. Genomic DNAs from these pri 0369 For each MCR, the peak segment value was identi mary tumor samples, along with DNAs derived from 24 fied. Recurrence of gain or loss was calculated across all established pancreatic cancer cell lines, e.g., pancreatic samples based on the lower thresholds previously defined adenocarcinoma cell lines (Table 2), were subjected to (~+/-0.13). As an additional measure of recurrence indepen genome-wide array-CGH profiling using a cDNA-based dent of thresholds for segment value or length, Median Aber array platform that offers a median resolution of 100 kB. To ration (MA) was calculated for each probe position by taking facilitate identification of significant copy number events in the median of all segment values above Zero for amplified these array-CGH profiles, a modified version of the circular regions, below zero for deleted regions. This pair of values binary segmentation methodology developed by Olshen and was compared to the distribution of values obtained after colleagues was employed (Olshen, A. B., and Venkatraman, permuting the probe labels independently in each sample E. S. (2002)ASA Proceedings of the Joint Statistical Meetings profile. Where the magnitude of the MA exceeds 95% of the 2530-2535; Ginzinger, D.G. (2002) Exp Hematol 30,503-12; permuted averages, the region was marked as significantly Golub, T. R., et al. (1999) Science 286,531-7: Hyman, E., et gained or lost, and this was used in the Voting system for al. (2002) Cancer Res 62, 6240-5; Lucito, R., et al. (2003) prioritization. The number of known genes and GENSCAN Genome Res 13, 2291-305). This algorithm applies nonpara model predicted genes were counted based on the April 2003 metric statistical testing to identify and distinguish discrete human assembly at UCSC (genome.ucsc.edu). copy number transition points from chance noise events in the primary dataset. As shown in FIG. 1A, the segmented array QPCRVerification CGH profiles readily identified large regional changes that 0370 PCR primers were designed to amplify products of are typically of low amplitude, hereafter referred to as gain 100-150 bp within target and control sequences. Primers for or loss. Similarly, focal high amplitude alterations repre control sequences in each cell line were designed within a senting amplification or deletion are evident in both pri region of euploid copy number as shown by array-CGH mary tumor specimens and tumor cell lines (FIG. 1). Recur analysis. Quantitative PCR was performed by monitoring in rence frequencies of the CNAs reported here match the real-time the increase in fluorescence of SYBR Green dye frequencies described in the published literature (Solinas (QiagenTM) with an ABI 7700 sequence detection system Toldo, S., et al. (1996) Cancer Res 56,3803-7: Mahlamaki, E. (PerkinElmer Life SciencesTM). Relative gene copy number H., et al. (1997) Genes Chromosomes Cancer 20, 383-91; was calculated by the comparative C, method (Ginzinger, D. Mahlamaki, E. H., et al. (2002) Genes Chromosomes Cancer G. (2002) Exp Hematol 30, 503-12). 35, 353-8: Fukushige, S., et al. (1997) Genes Chromosomes Cancer 19:161-9; Curtis, L. J., et al. (1998) Genomics 53, Expression Profiling on Affymetrix GeneChipTM 42-55; Ghadimi, B.M., et al. (1999) Am J Pathol 154,525-36: 0371 Biotinylated target cFNA was generated from total Armengol, G., et al. (2000) Cancer Genet Cytogenet 116, sample RNA and hybridized to human oligonucleotide probe 133-41) (FIG. 1B). There is also strong concordance between arrays (U133A, AffymetrixTM, Santa Clara, Calif.) according primary tumors and cell lines with respect to gains on 3q, 8q. to standard protocols (Golub, T. R., et al. (1999) Science 286, and 20q and losses on I p, 3p, 6q 9p. 17p and 18q (see FIG. 531-7). Expression values for each gene were standardized by 5 and FIG. 6). However, some differences were evident Log2 ratio to a middle value for the sample set, defined as the between primary tumor and cell line datasets and are likely midpoint between 25% and 75% quantiles, and were mapped attributable to the cellular heterogeneity within primary to genomic positions based on NCBI Build 33 of the human tumor samples and/or culture-induced genetic adaptation in genome. the cell lines. 0375. The identification of many novel CNAs, along with Integrated Copy Number and Expression Analysis the high degree of structural complexity within each CNA, 0372 Array-CGH data was interpolated such that each prompted the implementation of objective criteria to define expression value can be mapped to its corresponding copy and prioritize CNAs across the dataset. To that end, a locus US 2009/0297536A1 Dec. 3, 2009 40 identification algorithm was developed that defines informa Notably, genes known to play important roles in the patho tive CNAS on the basis of size and achievement of a high genesis of pancreatic adenocarcinoma the p16' and significance threshold for the amplitude of change. Overlap TP53 tumor suppressors and the MYC, KRAS2 and AKT2 ping CNAS from multiple profiles are then merged in an oncogenes—were present within these high-confidence loci automated fashion to define a discrete "locus of regional (Table 1). Within the prioritized MCRs, there was an average copy number change, the bounds of which represent the com recurrence rate for gain/loss of 38% across the entire dataset bined physical extent of these overlapping CNAs (FIG. 1C). and the maxima or minima absolute Log values for 34 of these 64 MCRs are greater than 1.0, placing them signifi Each locus is characterized by a peak profile, the width and cantly above the threshold defined for amplification or dele amplitude of which reflect the contour of the most prominent tion (FIG. 4). In the majority of cases, the peak profile of a amplification or deletion for that locus. Furthermore, within locus coincided with one of the MCRs (47 of 54 Loci, Table each locus, one or more minimal common region (MCRs) can 1) (Albertson, D. G., et al. (2000) Nat Genet. 25, 144-6). The be identified across multiple tumor samples (FIG. 1C), with median size of these 64 prioritized MCRs is 2.7 Mb, with 21 each MCR potentially harboring a distinct cancer-relevant MCRs (33%) spanning 1 Mb or less (Table 1). Residing gene targeted for copy number alteration across the sample within these 21 highly focal MCRs with a median size of 0.33 Set. Mb, there are on average 15 annotated and 8 GENSCAN 0376. The locus identification algorithm is highly effec predicted genes, rendering them highly attractive for target tive in delineating more discrete CNAs within previously identification. described larger regions of gain or loss. For example, chro 0379 The confidence-level ascribed to these prioritized mosome 6q has been reported as one of the most frequently loci was further validated by real-time quantitative PCR deleted regions in pancreatic adenocarcinoma, but no vali (QPCR), which demonstrated 100% concordance with 16 dated tumor Suppressor gene has yet been assigned to this selected MCRs defined by array-CGH (Table 1). For locus. Analysis of 6q loss in the dataset presented herein has example, the MCR of an amplified locus at 7q21.11-7q32.2 identified 5 distinct MCRs that range in size from 2.4 to 12.8 was readily confirmed by QPCR (FIG. 2A). Furthermore, Mb, raising the possibility that there may be multiple targets QPCR analyses also verified the structural details of complex for 6q loss. Notably, two of these MCRs (Table 3, Locus #75 CNAs reported by array-CGH. As shown in FIG. 2B, QPCR and #76) coincide with previously identified regions of com precisely mirrored each component of the complex 9p21 mon allelic loss (Abe, T., et al. (1999) Genes Chromosomes locus in HUP-T3, including homozygous deletion of Cancer 25, 60-4), an observation that provides further vali p16'', the known target for this CNA. Such detailed struc dation for the analytical approach described herein. tural information will prove useful in dissecting the mecha nisms responsible for the genesis of these cancer-associated Selection of High-Priority Loci chromosomal aberrations. (0380. When high-priority MCRs in Table 1 were com 0377 The above locus-identification algorithm defined bined with an additional 81 moderate-priority MCRs (within 287 discrete MCRs (from 256 independent autosomal loci) 66 distinct loci) satisfying 2 out of 4 criteria, a genomic within this dataset and annotated each in terms of recurrence, characterization produced a list of 145 MCRs within 121 amplitude of change and representation in both cell lines and independent loci (Table 3). Table 3 shows the combined list of primary tumors. Based upon extensive experience with this 145 MCRs in 121 independent loci, including 64 high-confi platform across many tumor types, recurrence across multiple dence MCRs (23 votes) and 81 moderate-priority (2 votes) independent samples and high amplitude signals are the two MCRs, that were prioritized by the automated algorithm as features most predictive of verification by independent described herein. Each locus is assigned to a cytogenetic assays. Hence, these discrete MCRs were prioritized based on band, while the actual extent of the locus is defined more four criteria that include (1) recurrence of high-threshold precisely by its physical location on the chromosome (in Mb) amplification or deletion (above 97" percentile or below 3" (NCBI, Built 33). The overall contour of the locus is reflected percentile) in at least two specimens, (2) presence of a high by the maxima/minima profile, which defines the most promi threshold event in at least one primary tumor specimen, (3) nent point of amplification or deletion within the locus by its statistically significant Median Aberration (see M&M), and width (defined in physical Mb) and amplitude. Each locus is (4) a peak amplitude of equal to or greater than absolute Log furthered defined by one or more Minimal Common Regions value of 0.8 in either a cell line or primary tumor (beyond (MCRs), depending on the cytogenetic complexity of the 0.5% quantiles). locus. Each MCR is defined in a similar manner. In addition, 0378. Implementation of this prioritization scheme the number of known and predicted (GENSCAN) transcripts yielded 64 MCRs within 54 independent loci that satisfied at as well as the total number of Affymetrix-represented genes least three of the four criteria (Table 1). In Table 1, the high and those with p-value-0.05 are shown for each MCR. MCR confidence recurrent CNAS in pancreatic adenocarcinoma are recurrence is denoted as percentage of the total dataset. The depicted. For each MCR, the numbers of known (“K”) and number of primary tumors (T) or cell lines (C) with gain or predicted (“P”) (GenScan) transcripts (“Trspt') are indicated. loss (90% and 10% quantiles, respectively) is listed. Further Of these, some are represented on AffymetrixTM U133A chip more, the subset of these tumors with gain/loss that meet the (“Total), a subset of which show statistical significance threshold for amplification or deletion (97% and 3% quan (p<0.05) for copy number correlation ("Sig"). MCR recur tiles, respectively) are also indicated. The boundaries of the rence is denoted as percentage of the total dataset. The num MCRs of each locus have been defined based on conservative bers of primary tumors (T) or cell lines (C) with gain or loss, parameters. and amplification or deletion, are listed, respectively. Candi date genes within a locus for which there is literature support Integrated Analysis of Copy Number and Expression Infor for involvement in pancreatic cancer are listed. Black dia mation. monds denote the loci where the peak did not fall within a 0381 Copy number aberrations and their associated defined MCR. MCRs in bold have been validated by QPCR. impact on gene expression patterns represent a common US 2009/0297536A1 Dec. 3, 2009 mechanism of oncogene activation and tumor suppressor correlation between gene expression and gene dosage (p<0. inactivation. Indeed, integration of copy number and tran 05). The chromosome, physical position in Mb, Gene Weight Scriptional profile datasets revealed a consistent influence of (see M&M) and p-value are listed. Affymetrix probe(s) num gene dosage on mRNA expression globally across the ber(s) corresponding to each GenBank Accession Number genome (FIG. 6) (Hyman, E., et al. (2002) Cancer Res 62, (“GI' Number) and UniGene ID are listed, along with SEQ 6240-5: Pollack, J. R., et al. (2002) Proc Natl AcadSci USA ID NOs for each nucleic acid and protein sequence. P-Values 99, 12963-8). Conversely, as previously demonstrated were calculated by permutation analysis of a gene weight (Platzer, P., et al. (2002) Cancer Res 62, 1134-8), only a Subset of genes within any given CNA show copy-number statistic. Of these, 336 are located within regions of amplifi driven expression changes—a feature that provides a first cations and 267 within regions of deletions. Importantly, pass means of distinguishing bystanders from potential can among these 603 genes were known pancreatic cancer genes cer gene targets within the CNA. As a case in point, a novel such as MYC (13), p16'-' (Rozenblum, E., et al. (1997) locus of amplification on chromosome 17 in the cell line Cancer Res 57, 1731-4; Caldas, C., et al. (1994) Nat Genet. 8, Hup-T3 (Locus # 21, Table 1) contains 455 genes of which 27-32) and DUSP6 (Furukawa, T., et al. (2003) Am J Pathol 151 are present on the Affymetrix U133A array. Of these 151 162, 1807-15) (Tables 4 and 5), thus reinforcing the value of genes, only 19 exhibited increased transcript levels above integrating both copy number and expression information. 2-fold. Moreover, these 19 genes reside within the peak of this 0383 While incomplete representation of known and pre locus (FIG. 3A). Similar correlations can be established in dicted genes on the Affymetrix U133A expression array pre regions of deletion. For example, the 9p21 deletion locus in cluded assessment of all possible target genes, the comple the BXPC-3 cell line demonstrated that only 5 out of 91 genes mentary analysis of array-CGH and expression profiles residing within the MCR show undetectable or decreased presented herein serves to prioritize the list of available can expression below 2-fold (FIG.3B). Examination of p16''', cer gene candidates and provides a basis for focus on a Subset the known target for deletion, across the entire sample set of high-probability candidates. In addition, integrating shows that 11 of 24 cell lines show low or absent expression, genomic datasets across species may also prove effective in of which 5 showed homozygous deletion while the remaining facilitating cancer gene identification. A particularly produc 6 were present at the DNA level (FIG. 3C). In the latter, epigenetic silencing is the presumed mechanism of p16'''“ tive path for oncogene identification may be the analysis of inactivation. common integration sites (CISS) present in retrovirally-pro 0382. In many cancer types, including pancreatic adeno moted leukemias and lymphomas (Neil, J. C. & Cameron, E. carcinoma, the true cell-of-origin remains unknown and thus R. (2002) Cancer Cell 2, 253-5). Consistent with the para a premalignant physiological frame-of-reference is not avail digm that proviral integration primarily serves to activate able. In the examples above, a model for interfacing copy endogenous proto-oncogene (Neil, J. C. & Cameron, E. R. number and expression profiles by midpoint-centering the (2002) Cancer Cell 2, 253-5), syntenic mapping of 232 CISs expression data and calculating a weighted Statistic for to the (Akagi K., et al. (2004) Nucl. Acids Res assignment of significance values to genes with correlated 32) uncovered 19 CISs residing within MCRs of amplified copy number and expression was applied (Golub, T. R., et al. loci in Table 1, whereas only 10 would be expected by chance (1999) Science 286,531-7: Hyman, E., et al. (2002) Cancer alone (p<0.006). On the contrary, MCRs within regions of Res 62, 6240-5) (see M&M). Using this approach, the genes loss or deletion contained only 16 CISs whereas 14.4 would residing within the 64 high-confidence MCRs (Table 1) were have been expected by chance alone. Thus, the abundance of prioritized based on the correlation of their expression with CIS's mapping to amplified loci may represent genes with gene dosage. While only a Subset of genes are represented, the pathogenetic relevance in mouse models of tumor progres AffymetrixTM U133A array permitted inclusion of 1,926 sion as well as in human cancer, although there may be genes out of a total of 4,742 genes residing within these possible cell-type specific roles for these candidate genes. MCRs for this analysis. By weighing each of these 1,926 genes based on the magnitude of its expression alteration and EQUIVALENTS its representation within CNAS across the dataset, the inte grated copy number and expression analysis yielded a list of 0384 Those skilled in the art will recognize, or be able to 603 genes that show a statistically significant association ascertain using no more than routine experimentation, many between gene copy number and mRNA expression (p<0.05, equivalents to the specific embodiments of the invention Tables 4 and 5). Tables 4 and 5 show the genes located within described herein. Such equivalents are intended to be encom high-priority MCRs (Table 1) and having highly significant passed by the following claims.

TABLE 1. List of High-Confidence MCRs. Minimal Common Regions (MCRs) Cytogenetic Locus Locus Peak Profile Size # Band Boundary (Mb) Pos (MB) Max/Min Position (MB) (MB) Gain and Amplification

1 1 p13.1-p12 116.83-119.49 119.07-119.2 1.56 116.83-119.49 2.65 2 5p15.33-p15.31 O.28-6.69 O.28-051 0.79 O.28-6.69 6.4 3 5q31.1-q31.1 133.51-134.33 133.58-133.95 0.97 133.53-133.56 0.04

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TABLE 1-continued List of High-Confidence MCRs.

O 1 O 14 1 4 1 1 15 3 4 1 29 1 9 O 4 NOTCH4 16 6 7 2 23 2 6 1 1 45 53 O 6 S1 4 14 O 7 23 25 3 1 37 1 12 1 2 12 22 7 S 34 1 11 1 1 356 282 186 42 46 4 12 1 5 10 31 14 4 8 37 1 12 O 2 FGFRI 4 5 O 31 1 10 O 2 11 15 4 O 49 4 13 1 1 12 317 461 11S 41 66 6 17 O 7 MYC 13 39 23 8 9 20 1 6 O 3 14 18 26 9 O 46 O 16 O 2 15 5 3 1 26 2 7 2 2 KRAS2 16 15 13 1 O 23 2 6 2 17 43 74 9 3 20 2 5 2 18 14 8 3 1 29 4 6 1 19 36 61 4 7 34 4 8 2 133 18O 68 6 31 1 10 O 4 2O 474 3O8 21S 70 46 2 14 O 7 HER2 21 14 4 5 3 34 3 9 2 22 26 37 1 6 29 4 6 3 3 23 124 77 35 6 26 4 S 2 4 OZF, AKT2 24 732 396 282 6S 29 3 7 5 25 26 31 O 1 40 3 11 6 129 138 57 9 46. 3 13 O 7 AIB1, STK15 26 7 8 2 23 3 5 3 3 27 12 7 5 2 26 3 6 1 Loss and Deletion

28 -0.94 14 7 2 1 46 4 12 2 -0.73 151 67 64 8 49 3 14 1 29 -0.77 28 28 17 7 37 3 O 2 -0.87 28 29 10 4 37 3 O 2 30 1 O O 49 4 13 5 31 O 1 1 26 4 6 2 1 32 12 1 O 23 2 6 1 33 13 15 6 3 34 4 8 O 2 34 18 39 6 2 51 4 14 O 4 24 40 14 O 51 4 14 4 35 94 94 40 8 S4 6 13 O 2 36 24 3 1 20 1 6 1 37 133 81 56 21 51 5 3 O 6 38 98 44 38 8 46 S 1 4 39 22 62 8 2 46 S 1 3 40 27 57 12 4 S4 2 17 O 11 FEZ1 60 31 28 16 46 1 5 O 11 NRG1 41 15 38 8 3 60 8 13 2 9 129 216 55 15 57 7 13 11 INK4A 42 23 36 13 O 6 1 1 1 43 123 29 68 18 29 2 8 1 44 116 70 70 19 34 3 9 1 82 69 60 4 34 2 10 1 45 57 96 34 13 31 3 8 1 46 63 11 27 9 37 4 9 2 4 DUSP6 47 34 17 15 7 29 1 9 2 48 18 34 14 O 57 5 5 5 TP53, MKK4 49 21 94 16 S 54 6 13 O 7 50 98 287 23 8 60 7 14 O 7 51 55 9 12 2 31 4 7 2 52 8 3 O 60 S 6 2 53 3 2 2 34 S 7 O 3 1 6 1 40 S 9 O 3 S4 424 3SO 243 6S S4 S 14 O 7 US 2009/0297536A1 Dec. 3, 2009 44

TABLE 2 List of cell lines and corresponding references used in this study

Name Source Reference PA-TU-8988T DSMZ Elsasser et al. Virchows Arch B Cell Pathol 61 (5): 295-306. 1992 PA-TU-8988S DSMZ Elsasser et al. Virchows Arch B Cell Pathol 61 (5): 295-306. 1992 PA-TU-8902 DSMZ Elsasser et al. Virchows Arch B Cell Pathol. 64: 201-207. 1993 DAN-G DSMZ Not Published HUP-T4 DSMZ Nishimura et al., Int. J. Pancreatol 13:31–41. 1993 HUP-T3 DSMZ Nishimura et al., Int. J. Pancreatol 13:31–41. 1993 Panic 10.05 ATCC affee E Metal. Cancer J Sci Am 4: 194-203, 1998. PL45 ATCC affee E Metal. Cancer J Sci Am 4: 194-203, 1998. Aspc-1 ATCC Chen et al. In Vitro. 1982 January; 18(1): 24-34. Mpanc-96 ATCC Peiper Metal. Int. J. Cancer 71:993-999, 1997. BXPC-3 ATCC Tan M Hetal. Cancer Invest. 4: 15-23, 1986. Capan-1 ATCC Fogh et al. JNCI 58: 209-214. 1977 Capan-2 ATCC Fogh et al. JNCI 58: 209-214. 1977 CFPAC-1 ATCC Schoumacher et al. PNAS 87: 4012-4016. 1990 HPAF-II ATCC Kim YW et al. Pancreas 4:353-362. 1989. HS766T ATCC Owens R. B. et al. JNCI 56: 843-849, 1976. Panc-1 ATCC Lieber Metal. Int J. Cancer 15: 741-747. 1975 SW 1990 ATCC Kyriazis AP et al. Cancer Res.43:4393-4401. 1983 MIA PaCa-2 ATCC Yunis AA et al. Int. J. Cancer 19:128-135. 1977 HPAC ATCC Gower WR et al. In vitro Cell Dev Biol. 30A: 151-161. 1994 Panic O2O3 ATCC affee E Metal. Cancer J Sci Am 4: 194-203, 1998. Panic O2.13 ATCC affee E Metal. Cancer J Sci Am 4: 194-203, 1998. Panic 3.27 ATCC affee E Metal. Cancer J Sci Am 4: 194-203, 1998. Panic O8.13 ATCC affee E Metal. Cancer J Sci Am 4: 194-203, 1998.

TABLE 3 List of High and Moderal Confidence MCRs. Locus Minimal Common Regions (MCRs) MCRRecorrance Locus Peak Profits # on Ampf Cytogenetic Boundary Max. Size Max # Trapt U133A Gain Loss Del # Band (Mb) Pos (MB) Min Position (MB) (MB) Min K P Total Sig. 96 T C T C 1 1 p13.1-p12 11553-119.49 11907-119.2 55 116.83-119.48 26S 1.SS 23 47 15 2 23 2 8 2 2 2 2p11.2-p11.1 85.23-91.48 85.78-85.78 85.75-85.85 O.1 10 5 7 2 14 O S O 2 3 3p11.1-q12.3 87.99-102.85 95.13-102.38 O.93 90.13-99.58 945 0.93 18 37 5 O 29 1 9 O 2 101.35-101.63 O48 0.93 5 7 5 5 17 O 8 O 2 4 5p15.33-p15.31 0.28-6.69 O.28-051 O.79 0.28-8.69 6.4 OS 57 164 19 8 43 1. 14 O 5 5 5q23.1-q31.1 115.81-132.47 13144-132.27 O8 115.81-132.47 16.88 1.08 110 182 44 8 28 1 8 O 1 6 5q31.1-q31.1 133.51-134.33 133.56-133.95 O.97 133.53-133.56 O.O4 O.97 O O 1 O 14 1 4 1 1 7 5q31.3-q31.3 139.21-140.22 13948-140.22 O.6S 139.21-140.04 O.83 0.65 38 16 10 O 31 2 9 O 2 9 8p22.1-p21.32 28.12-32.72 31.98-32.44 38 33.98-32.11 O.13 1.38 15 3 14 1 29 1 9 O 4 32.19-32.21 O.O2 1.36 O O 2 O 29 1 9 O 3 10 8p21.1-p21.1 42.91-43.19 42.98-43.15 O.54 42.91-43.03 O.12 O.64 16 8 7 2 23 2 8 1 1 11 8q24.3-q25.1 145.88-151.63 130.09-151.19 4S 150.09-151.12 1.03 1.45 16 32 4 1 17 O 8 O 2 12 7p22.3-p22.1 O.72-4.53 O.72-246 O.93 0.72-2.28 1.58 0.93 46 63 10 6 S1 4 14 O 7 13 7p21.3-p21.2 739-1444 7.97-1444 O-58 7.39-1444 7.04 O.8 2O 58 13 3 40 2 12 1 2 14 7p15.1-p14.3 30.12-31.58 30.5-30.81 O.82. 30.12-31.58 144 O.82 23 25 13 1 37 1 2 1 2 15 7p13-p11.2 44.93-54.88 43.64-47.71 23 44.93-54.88 9.9S 1.23 41 143 27 13 34 O 12 O 2 16 7p11.21-q21.11 64.54-77.18 54.95-54.95 O6 6495-65.65 O.9 O8 12 22 7 S 34 1 1 1 1 17 7q21.11-q32.2 79.45-129.48 97.88-98. SS 3.06 92.33-112.27 1994 3.06 358 282 168 42 45 4 12 1 S 18 7q34-q38. 1427-15022 143.26-143.38 O.94 143.38-147.82 4.44 O.94 6 38 4 O 17 O 6 O 2 19 8p23.1-p22 8.75-12.74 11.05-11.67 2.OS 6.76-12.74 5.95 2.05 45 74 14 3 11 O 4 O 1 2O 8p12-p11.21 37.7-41.78 37.72-38.02 .71 37.7-28.45 0.75 1.71 31 14 14 8 37 1 2 O 2 38.68-39.52 O.84 1.44 3 4 5 O 31 1 O O 2 21 8q12.1-q12.3 59.09-63.88 59.23-82.28 O.S8 S9.23-60.62 1.S. O.S8 1 15 4 O 49 4 13 1. 1 22 8q12.3-q13.1 83.7-67. OS 65.44-88.81 O.87 63.7-67.06 3.37 0.87 2O 43 6 O 43 3 2 O 1 23 8q21.3-q24.3 90.7-145.83 133.72-13416 .78 11897-145.83 26.88 1.78 317 461 11S 41 68 6 17 O 7 24 8p21.3-p13.2 23.88-37.87 35.68-36.56 O.88 35.8-38.56 O.96 O.85 39 23 18 9 20 1 6 O 3 25 10q25.2-q25.3 1124-113.62 1124-11277 O.85 1124-115.62 3.22 O.85 2O 61 15 3 28 2 7 O 1 26 10q26.13-q28.2 124.92-129.83 126.14-126.14 O.85 124.92-126.41 149 0.85 9 30 7 O 23 1 7 O 2 27 11 p12-q13.6 40.18-77.26 77.77.15 * O.9 60.11-61.14 1.03 0.81 32 19 13 O 40 1 3 O 3 28 11q14.1-q14.2 78.15-80.74 82.76-85.89 O.9 83.39-86.21 2.82 0.9 18 26 9 O 46 O 6 O 2

US 2009/0297536A1 Dec. 3, 2009 48

TABLE 3-continued List of High and Moderal Confidence MCRs. 82 3 8 17O 82 9p24.3-9p21(2) TRUE 0.47-27.18 20.77-21.31 -2.53 O.47-3.39 2.92 18 6S 171 82 9p24.3-9q21.1(2) FALSE 0.47-27.18 20.77-21.31 -2.63 6.3-23.88 18.39 83 O O 272 83 9q13-9q21.1(2) TRUE 65.19-55.51 65.26-65.51 -1.27 65.19-SS.S1 1.32 84 2 13 297 84 9q22.33-9q3(2) TRUE 97.63-101.94 97.84-101.81 -0.81 97.63-101.94 4.31 85 8 13 250 85 9q31.2-9q31(2) TRUE 105.59-109.28, 108.59-107.89 -1 106.59-108.83 2.04 86 5 10 28S 86 9q34.11-9q3(2) TRUE 127.73-128.55 127.75-127.81 -0.91 12773-128.53 O6 87 3 7 258 87 Op15.3-10p (2) TRUE 0.29-117 O.29-1.08 -0.61 O.29-117 O.88 88 4 24 173 88 Op12.33-10p (2) TRUE (27.91-25.66 18.09-18.32 -3.3 17.91-26.88 8.75 89 9 20 298 89 Op12.1-10p (2) TRUE 27.19-33.36 27.83-32.71 -0.62 27.19-33.88 6.17 90 O 1 174 90 0q22.1-10q.(2) TRUE 73.46-73.72 73.46-73.47 -3.23 73.48-73.72 O.28 91 15 34 221 91 Oq22.3-10q.(2) 92 4 10 175 92 1p 15.4-11p(2) TRUE 10.84-13 12.02-12.O2 -3.72 10.54-13 2.16 93 4 27 195 93 1p15.2-11p(2) TRUE 1458-15.47 17.38-1843 -O.92 14.58-1847 3.59 94 7 23 198 94 1p15.1-11p(2) TRUE 20.11-32.17 2042-31.85 -1.2 2011-32.17 12O6 95 O 11 204 95 1a14.1-11a(2) TRUE 78.15-85.59 82.76-83.39 -0.87 78.15-85.69 7:44 96 O 13 222 96 1a14.2-11a(2) TRUE (2).59-69.24 85.21-89.24 -0.9 85.59-59.24 3.35 97 8 75 223 97 1q21-11q23(2) TRUE 95.74-114.13 95.74-114.13 -O.75 95.74-114.13 1830 98 1 4 178 98 1q23.3-11a(2) TRUE 110.73-134.28 117.1-134.27 -O.7 12033-121.54 1.21 99 18 55 290 99 2d 12-12q13(2) TRUE 40.08-49.24 41.04-49.21 -0.71 40.O6-49.24 9.16 OO 19 70 291 100 2a13.12-12(2) TRUE 49.55-58.82 SO.O1-53.06 -O.7 49.SS-63.08 3.41 4 80 292 100 2a13.12-12(2) FALSE 49.55-55.82 SO.O1-53.08 -O.7 53.17-55.82 2.85 O1 13 34 293 101 2a14.1-12a(2) TRUE 57.92-58.77 62.76-68.27 -0.5 62.76-68.77 O.O1 O2 9 27 197 102 2a21.2-12a(2) TRUE 77.19-133.4 81.03-85.63 -1.19 77.19-91.44 14.25 O3 1 2 294 103 4q24.2-14d (2) TRUE 71.88-72.03 71.72-71.73 -1.1 71.65-72.03 O.35 O4 7 15 242 104 6p13.3-16p (2) TRUE 0.03-23.97 O.7O-23.7 -0.67 2.24-2.(2)2 O.S8 05 8 24 192 105 6d12.1-16a (2) TRUE 48.37-55.26 (2).06-53.88 -1.1 48.37-53.88 5.52 O6 O 14 178 106 7p13.3-17q.(2) TRUE 0.02-25.81 8.16-1405 -O.99 10.36-12.8 2.44 O7 1 5 225 107 7a11.2-17a (2) TRUE 25.68-30.32 (). (2)-28.65 -O.83 27.8-28.97 1.17 O8 O 2 228 108 7p24.2-17q3) TRUE (2).73-76.58 66.96-71-88 -O.92 71.63-71.65 O.23 10 5 16 208 110 8q11.2-18a (2) TRUE 18.61-48.28 34.95-40.58 -1.19 34.18-43.14 8.99 11 8 23 213 111 8q22.1-18q(2) TRUE 604-77.53 7448-78.84 -1.83 (2.4-77.63 1723 12 2 6 196 112 19p13.3-19p (2) TRUE 4.62-9.11 C.C.C.C) -O.85 6.61-(2).88 O.24 13 3 4 158 113 9p13.2-19p (2) TRUE 12.83-13.18 12.84-13.11 -O.88 12.9-13.07 O.17 14 12 21, 159 114 9a13.2-19a (2) TRUE 44.87-63.76 59.99- C2).04 -1.31 50.65-51.57 O.92 2 12 150 114 9a13.2-19a (2) FALSE 44.57-63.76 59.99-60.04 -1.31 59.85-60.18 O.33 15 27 75 194 115 20p13-20q11(2) TRUE 0.33-30.89 8.81-25.63 -139 6.91-25.68 19.77 16 O 3 200 116 21 p11.2-21q.(2) TRUE 9.96-13.43 9.95-10.08 -1.24 995-13.43 3.47 17 O 3 208 117 21q22.11-21(2) TRUE 31.95-32.5 32.15-32.5 -1.16 31.9S-32.8 O.S3 18 O 4 214 118 21q22.11-20) TRUE 34.19-35.52 35.03-35.42 -1.09 34.81-35.42 O.61 19 2 5 215 119 21q22.15-21(2) TRUE 37.01-37.52 3701-37.02 -1.58 37.01-37.52 O.8 2O 2 2 243 120 21q22.2-21q.(2) TRUE 39.75-45.94 417-41.76 -4.09 45.08-45.17 O.09 1 6 244 120 21q22.2-21q.(2) FALSE 39.75-48.94 417-41.75 -4.09 48.77-46.94 O.17 21 65 243 179 121 22d 11.1-22d (2) TRUE 14:49-39.45 22.7-22.7 -1.32 20.64-39.45 1883 (2) indicates text missing or illegible when filed

TABLE 4

Markers of the invention which reside in MCRs of deletion and display decreased expression. POS Gene Minimum Chromosome (Mb) Weight p value Gene Description Gene Symbol GI UGIDhi 21.3 -0.84 0.005 alkaline phosphatase, liver bone/kidney ALPL gi: 28737 HS250769 22.8 -1.12 <.005 KIAA0601 protein KIAAO6O1 gi: 3043725 Hs.348515 23.3 -0.38 0.037 E2F transcription factor 2 E2F2 gi: 4758225 Hs.231444 23.5 -0.73 <.005 lysophospholipase II LYPLA2 gi: 9966.763 HS.413781 23.5 -0.39 0.049 galactose-4-epimerase, UDP- GALE gi:994.5333 Hs.76057 23.6 -0.56 0.012 3-hydroxymethyl-3-methylglutaryl- HMGCL gi: 4504426 Hs.444925 Coenzyme Alyase (hydroxymethylglutaricaciduria) 23.6 -0.80 <.005 lysophospholipase II LYPLA2 gi: 4376011 HS.413781 23.6 -0.42 O.O3 fucosidase, alpha-L-t, tissue FUCA1 gi: 4503802 Hs.576 24.3 -0.48 <.005 serinefarginine repetitive matrix 1 SRRM1 gi: 5032118 Hs. 18.192 24.5 -0.64 0.021 chloride intracellular channel 4 CL1C4 gi: 4588523 Hs.25035 24.9 -0.61 0.005 hypothetical protein dj465N24.2.1 DJ46SN24.2.1 gi: 12005626 Hs.25.9412 25.8 -0.87 <.005 stathmin Ioncoprotein 18 STMN1 gi: 13518023 Hs.209983 26.1 -0.45 0.037 zinc finger protein ZT86 gi: 7705661 Hs.102419 26.4 -0.44 0.021 high-mobility group nucleosomal binding HMGN2 gi: 13277559 Hs. 181163 domain 2 US 2009/0297536A1 Dec. 3, 2009 49

TABLE 4-continued

Markers of the invention which reside in MCRs of deletion and display decreased expression. 26.4 -0.86 ribosomal protein S6 kinase, 90 kDa, RPS6KA1 gi: 4506732 S.149957 polypeptide 1 26.7 -0.61 hypothetical protein FLJ20477 FLJ20477 gi: 8923441 S.259605 26.7 -0.84 hypothetical protein FLJ20477 FLJ20477 gi: 1799.134 S.259605 26.8 -2.26 hypothetical protein FLJ12455 FLJ12455 gi: 11545792 S.10903 28.5 -O.75 RNA selenocysteine associated protein SECP43 gi: 8923459 S.266935 28.6 -0.87 TAF12 RNA polymerase II, TATA box TAF12 gi: 1345403 S.421646 binding protein (TBP)-associated factor, 20 kDa 28.7 -0.63 glucocorticoid modulatory element GMEB1 gi: 13435376 binding protein 1 28.7 -0.78 high-glucose-regulated protein 8 HGRG8 gi: 77.05410 S.20993 29.1 -0.47 splicing factor, arginine?serine-rich 4 SFRS4 gi: 5032088 S.76122 29.2 -0.57 nuclear receptor binding factor 1 CGI-63 gi: 77.05776 S.183646 31.1 -0.49 hypothetical protein FLJ12650 FLJ126SO gi: 13375663 S.436090 32.7 -0.67 KIAA1522 protein KIAA1522 gi: 6588393 S.322735 32.9 -0.63 hypothetical protein FLJ90005 FLJ900OS gi: 6729581 S.S11807 32.9 -O.27 Homo sapiens transcribed sequence with gi: 6664283 S.416117 moderate similarity to protein ref: NP 060265.1 (H. sapiens) hypothetical protein FLJ20378 Homo Sapiens 33.3 -O.96 polyhomeotic-like 2 (Drosophila) PHC2 gi: 4758241 S.165263 14.1 -0.86 hypothetical protein MGC3222 MGC3222 gi: 1384.812 S.13O330 14.1 -1.30 Xeroderma pigmentosum, XPC gi: 475.156 S.32O complementation group C 14.2 -0.47 LSM3 homolog, U6 small nuclear RNA LSM3 gi: 7657314 S.111632 associated (S. cerevisiae) 53.2 -0.42 PET112-like (yeast) PET112L gi: 4758893 S.119316 : 53.8 -0.38 F-box and WD-40 domain protein 7 FBXW 7 gi: 8922851 S.312SO3 (archipslago homolog, Drosophila) 74.8 -0.42 UDP-N-acetyl-alpha-D- GALNT7 gi: 83934.08 S.156856 galactosamine:polypeptide N acetylgalactosaminyltransferase 7 (GalNAc-T7) 75 -0.42 Homo sapiens clone FLB9413 PRO2532 gi: 114.934S5 S383,372 mRNA, complete cds 75.1 -0.40 heart and neural crest derivatives HAND2 gi: 12545383 S388.245 expressed 2 84.5 -0.73 dCMP deaminase DCTD gi: S.76894 : 85 -0.70 collaborates, cooperates with ARF CARF gi: S.32922 (alternate reading frame) protein 86 -0.74 interferon regulatory factor 2 IRF2 gi: 47SS144 S.83.795 86.9 -0.38 hypothetical protein DKFZp761O0113 DKFZp761OO113 gi: 8922176 S.42768 87 -0.40 hypothetical protein FLJ1 1200 FLJ11200 gi: 8922937 S.368022 28.5 -2.23 CGI-111 protein CGI-111 gi: 77.05613 S.11085 O6.7 -0.63 APG5 autophagy 5-like (S. cerevisiae) APGSL gi: 7023451 S.11171 O7.1 -0.45 glutaminyl-tRNA synthase (glutamine- QRSL1 gi: 1205288.1 S4O6917 hydrolyzing)-like 1 -0.61 chromosome 6 open reading frame 210 C6orf210 gi: 996.6852 S.268733 -0.95 SEC63-like (S. cerevisiae) SEC63 gi: 5393231 S.330767 -0.48 Sorting nexin 3 SNX3 gi: 601.0168 S.12102 -0.53 Sorting nexin 3 SNX3 gi: 11765691 S.12102 -0.24 gi: 3821018 -O.75 CD164 antigen, sialomucin CD164 gi: 119433 SO S.43910 -0.77 sphingomyelin phosphodiesterase 2, SMPD2 gi: S101.461 S.SS235 neutral membrane (neutral sphingomyelinase) -0.76 NEDD9 interacting protein with calponin NICAL gi: 12232438 S.33476 homology and LIM domains O9.8 -0.54 Zinc finger protein 450 ZNF4SO gi: 7662127 S4O9876 10.1 -0.36 KIAAO274 KIAAO274 gi: 7662033 S.419998 1O.S -0.52 WAS protein family, member 1 WASF1 gi: S.758SO 1O.S -0.94 cell division cycle 40 homolog (yeast) CDC4O gi: S.116674 10.9 -1.03 cyclin-dependent kinase (CDC2-like) 11 CDK11 gi: 5100783 S.129836 11 -1.36 cyclin-dependent kinase (CDC2-like) 11 CDK11 gi: 5689392 S.129836 11.2 -0.41 adenosylmethionine decarboxylase 1 AMD1 gi: 178517 S.1591.18 11.7 -0.76 REV3-like, catalytic subunit of DNA REV3L gi: 4506482 S.232O21 polymerase Zeta (yeast) 14.3 -O.72 histone deacetylase 2 HDAC2 gi:4557640 S.3352 16.6 -0.84 TSPY-like TSPYL gi: 12052783 S.458358 66 19.2 -0.10 ASF1 anti-silencing function 1 homolog ASF1A gi: 766.1591 S.292316 A (S. cerevisiae) US 2009/0297536A1 Dec. 3, 2009 50

TABLE 4-continued

Markers of the invention which reside in MCRs of deletion and display decreased expression. 135.3 -0.65 HBS1-like (S. cerevisiae) HBS1L gi: 67.03779 S.221040 135.6 -0.57 Abelson helper integration stie AHI1 gi: 8923074 S.273294 136.9 -0.29 mitogen-activated protein kinase kinase MAP3KS gi: 1805499 S.151988 kinase 5 137.3 -0.58 interleukin 20 receptor, alpha L2ORA gi: 7657690 S.288240 138.7 -0.52 heme binding protein 2 HEBP2 gi: 76576O2 S.439081 139 -0.74 chromosome 6 open reading frame 80 C6orf&O gi: 126S3928 S.44468 139.4 -0.45 headcase homolog (Drosophila) HECA gi: 7706434 S.6679 146.5 -0.76 glutamate receptor, metabotropic 1 GRM1 gi: 6006005 S.32945 1698 -0.25 PHD finger protein 10 PHF10 gi: 11085906 S.435933 1698 -0.61 PHD finger protein 10 PHF10 gi: 8922799 S.435933 18 -0.44 N-acetyltransferase 1 (arylamine N NAT1 gi: 4SOS334 S.458430 acetyltransferase) 18.3 -0.65 hypothetical protein DKFZp761K1423 gi: 8922171 S.236438 18.5 -0.68 ADP-ribosylation factor guanine gi: 608S952 S.408.177 nucleotide factor 6 8 ATPase, H+ transporting, lysosomal gi: 4SO2310 S.295917 56/58 kDA, V1 subunit B, isoform 2 28.6 -0.40 exostoses (multiple)-like 3 EXTL3 gi: 2897904 S. 9018 28.6 -0.49 exostoses (multiple)-like 3:exostoses EXTL3 gi: 13623S12 s.9018; (multiple)-like 3 S. 9018 28.7 -O.80 hypothetical protein FLJ10871 FLJ10871 gi: 8922725 S.15562 29.9 -O.99 hypothetical protein MGC8721 MGC8721 gi: 77O6384 S.27992.1 30 -O.96 eptin receptor overlapping transcript-like 1 LEPROTL1 gi: 7662SO9 S.146585 30 -O.80 dynactin 6 DCTN6 gi: 573.01.15 S.158427 30.3 -0.22 RNA binding protein with multiple RBPMS gi: 5803140 S.1958.25 splicing -0.56 general transcription factor IIE, gi: 4504.194 S.771 OO polypeptide 2, beta 34 kDA 30.6 -0.84 glutathione reductase gi: 1083.5188 S.414334 30.6 -0.42 reproduction 8 gi: 1913786 S.153678 30.7 -0.46 protein phosphatase 2 (formerly 2A), gi: 4758.951 S.8O3SO catalytic Subunit, beta isoform 32.6 -0.08 neuregulin 1 NRG1 gi: 7669513 S.172816 33.4 -1.04 RNA binding protein; RNA binding LOC84549 gi: 136251.85 s.77135; protein S.77135 33.4 -0.53 hypothetical protein FLJ23263 FLU23263 gi: 13376690 S.288716 37.6 -O.75 chromosome 8 open reading frame 2: C8orf2 gi: 10241715 S.125849; chromosome 8 open reading frame 2 S.125849 37.6 -1.00 proline synthetase co-transcribed PROSC gi: S.301959 homolog (bacterial) 8 131.3 -0.17 Homo sapiens cDNA: FLJ23601 fis, gi: 10440343 S.306918 clone LNG15501 2.1 -0.36 SWISNF related, matrix associated, actin SMARCA2 gi: 6133361 S.396404 dependent regulator of chromatin, Subfamily a, member 2 2.6 -0.47 very low density lipoprotein receptor gi: 437386 S.370422 2.8 -1.06 minor histocompatibility antigen HA-8 gi: 7661865 S.443866 5.3 -0.77 chromosome 9 open reading frame 46 gi: 8923931 S.416649 5.5 -0.51 programmed cell death 1 ligand 2 gi: 13376849 S.61929 -1.15 RAN binding protein 6 gi: 3538.999 S.167496 14 -0.20 nuclear factor IB gi: 13410807 S.3O2690 14.2 -OSO nuclear factor IB NFIB gi: 4988418 S.3O2690 15.4 -0.56 Small nuclear RNA activating complex, SNAPC3 gi: 4507104 S.38OO92 polypeptide 3, 50 kDa 15.4 -0.65 PC4 and SFRS1 interacting protein 2 PSIP2 gi: 32833S1 S.3S1305 19 -0.94 Ras-related GTP binding A RRAGA gi: 5729998 S.432330 19 -0.43 hypothetical protein FLJ20060 FLJ20060 gi: 8923062 S.54617 19.3 -0.91 ribosomal protein S6 RPS6 gi: 4506.730 S.408073 20.8 -1.14 KIAA1797 KIAA1797 gi: 8923357 S.257696 21.3 -1.03 KIAA1354 protein KIAA1354 gi: 2185814 S.147717 21.9 cyclin-dependent kinase inhibitor 2A CDKN2A gi: 4502748 S.421349 (melanoma, p16, inhibits CDK4) 9 22 -0.15 methylthioadenosine phosphorylase MTAP gi: 4378719 S.459541 42.4 -1.31 PTK9 protein tyrosine kinase 9; PTK9 PTK9 gi: 4506274 s.189075; protein tyrosine kinase 9 S.18907S 12 43.8 -1.03 putative glycolipid transfer protein LOCS1054 gi: 770,5683 S.334649 12 44.6 -0.94 splicing factor, arginine?serine-rich 2, SFRS2IP gi: 4759171 S.210367 interacting protein 12 46.4 -1.06 hypothetical protein FLJ20489 FLU20489 gi: 8923451 S.438867 12 46.5 -2.40 vitamin D (1,25-dihydroxyvitamin D3) WDR gi: 2824O68 S.2O62 receptor 12 47.3 -0.54 O.036 hypothetical protein FLJ20436 FLU20436 gi: 10434303 S.2681.89 US 2009/0297536A1 Dec. 3, 2009 51

TABLE 4-continued

Markers of the invention which reside in MCRs of deletion and display decreased expression. 47.5 -O.89 calcium channel, Voltage-dependent, beta CACNB3 gi: 463890 3 subunit 47.5 -0.82 calcium channel, Voltage-dependent, beta CACNB3 gi: 463890 3 subunit 47.5 -0.58 DEAD (Asp-Glu-Ala-Asp) box DDX23 gi: polypeptide 23 47.6 -O.72 FK506 binding protein 11, 19 kDa gi: 7706130 S.438.695 : 47.6 -0.61 ADP-ribosylation factor 3 gi: 4502202 S.119.177 47.6 -1.26 ADP-ribosylation factor 3; ADP gi: 7898O s.1191.77; ribosylation factor 3 S.119.177 2 47.6 -0.48 protein kinase, AMP-activated, gamma 1 gi: S.3136 non-catalytic subunit 47.7 ipocalin-interacting membrane receptor gi: 8922462 S.272838 48.1 spermatogenesis associated, serine-rich 2 SPATS2 gi: 2751480 S.152982 48.2 microspherule protein 1 MCRS1 gi: S.253.13 48.4 estis enhanced gene transcript (BAX TEGT gi: S.3SOS2 inhibitor 1) 48.6 Rac GTPase activating protein 1 RACGAP1 gi: 1.015369 S.23900 : 49.6 Solute carrier family 11 (proton-coupled SLC11A2 gi: 1015990 S.S7435 divalent metalion transporters), member 2 49.8 transcription factor CP2 TFCP2 gi: S.154970 49.9 DAZ associated protein 2 DAZAP2 gi: S.369761 49.9 hypothetical protein from clone 643 LOCS7228 gi: 3097236 S.2O65O1 SO.6 activin A receptor, type IB ACVR1B gi: 2652986 S.371974 SO.6 activin A receptor, type IB ACVR1B gi: S912233 S.371974 50.7 Homo sapiens cDNA clone gi: 7152120 S.444433 MAGE: 5590288, partial cols 51.7 eukaryotic translation initiation factor 4B EIF4B gi: 4SO3S32 S.93379 51.8 retinoic acid receptor, gamma RARG gi: 307424 S.1497 51.9 hypothetical protein MGC11308 MGC11308 gi: 1197.5558 S.19210 51.9 brefoldin 5 PFDN5 gi: 4505742 S.288.856 51.9 chromosome 12 open reading frame 10 C12Orf10 gi: 11 OS 6017 S.40O8O1 52.6 homeobox C11 HOXC11 gi: 76571.65 S.127562 52.7 homeobox C6 HOXC6 gi: 6709275 S.820 52.8 single-strand selective monofunctional SMUG1 gi: 7657596 S.5212 uracil DNA glycosylase 52.9 Homo sapiens, clone IMAGE: 5288883, gi: 13284730 S.349283 mRNA 22 52.9 heterogeneous nuclear ribonucleoprotein HNRPA1 gi: 45.04444 S.356,721 A1 2 53 costomer protein complex, Subunit Zeta 1 COPZ1 gi: S.181271 S4.3 GCN5 general control of amino-acid GCNSL1 gi: S.94.672 synthesis 5-like 1 (yeast) 544 CD63 antigen (melanoma 1 antigen) CD63 gi: 4502678 S.445570 : 544 ORM1-like 2 (S. cerevisiae) ORMDL2 gi: 7661819 S.13144 63.1 exportin, tRNA (nuclear export receptor XPOT gi: S811224 S.8S951 for tRNAs) 2 63.1 TANK-binding kinase 1 TBK1 gi: 701.9S46 S.432466 6.3.3 glucosamine (N-acetyl)-6-sulfatase GNS gi: 10329021 S.334534 (Sanfilippo disease IIID) 2 63.4 glucosamine (N-acetyl)-6-sulfatase GNS gi: 4504060 S.334534 (Sanfilippo disease IIID) 63.8 integral inner nuclear membrane protein MAN1 gi: 77O6606 S.105234 64.8 CGI-119 protein CGI-119 gi: 77O6334 S.126372 65.9 TBP-interacting protein TIP12OA gi: 89 24259 S.512638 65.9 TBP-interacting protein TIP12OA gi: 424O146 S.512638 66.3 dual-specificity tyrosine-(Y)- DYRK2 gi: 1666.065 S.17313S phosphorylation regulated kinase 2 67.3 RAP1B, member of RAS oncogene RAP1B gi: 7661677 S.374418 family 674 -0.52 solute carrier family 35, member E2 SLC3SE3 gi: 8922O84 S.445043 : 68 -O.80 glioma-amplified sequence-41 GAS41 gi: 5729837 S4O29 68.2 -0.53 chaperonin containing TCP1, Subunit 2 CCT2 gi: S. 189772 (beta) 2 78.7 -0.98 protein phosphatase 1, regulatory PPP1R12A gi: 5436140 S.377908 (inhibitor) subunit 12A 81.2 -0.81 HSPC128 protein HSPC128 gi: 766.1789 S.90527 86.9 -0.86 hypothetical protein DKFZp434N2030 DKFZp434N2030 gi: 6708922 S.494204 87 -0.52 hypothetical protein FLJ13615 FLJ13615 : 12711597 S.28871S 87.4 -0.47 KIT ligand KITLG gi: 4505174 S.1048 88.2 -0.45 dual specificity phosphatase 6 DUSP6 gi: 1311 1942 S.298654 88.4 -O.88 ATPase, Ca++ transporting, plasma ATP2B1 gi: 7247996 S.20952 membrane 1 US 2009/0297536A1 Dec. 3, 2009 52

TABLE 4-continued

Markers of the invention which reside in MCRs of deletion and display decreased expression. 88.5 -0.73 ATPase, Ca++ transporting, plasma ATP2B1 i: 184269 S.20952 membrane 1 91 -0.65 B-cell translocation gene 1, anti gi: 45.02472 S.255935 proliferative 2.5 -0.60 ATPase, H+ transporting, lysosomal ATP6VOC gi: 4SO2312 S.389.107 16 kDa, VO subunit c 2.5 -0.62 O.019 ATPase, H+ transporting, lysosomal ATP6VOC gi: 189675 S.389.107 16 kDa, VO subunit c 2.5 -0.70 CGI-14 protein CGI-14 gi: 7705595 S.433499 2.5 -0.14 3-phosphoinositide dependent protein PDPK1 gi: 450S694 S.154729 kinase-1 2.5 3-phosphoinositide dependent protein PDPK1 gi: 24O7612 S.154729 kinase-1 2.8 -0.15 serinefarginine repetitive matrix 2 SRRM2 gi: 4739778 S.433343 2.8 -0.61 serinefarginine repetitive matrix 2 SRRM2 gi: 4531907 S.433343 45.4 -0.10 golgi SNAP receptor complex member 2 GOSR2 gi: 12711466 S.432552 37.8 -0.70 phosphoinositide-3-kinase, class 3 PIK3C3 gi: 4SOS800 S.418150 41.9 -0.70 ATP synthase, H+ transporting, ATPSA1 gi: 45.73764 S.298.28O mitochondrial F1 complex, alpha subunit, isoform 1, cardiac muscle 42.6 -0.41 MSX-interacting-Zinc finger gi: 1072O797 S.441069 42.6 -OSO MSX-interacting-Zinc finger gi: 3643114 S.441069 42.9 -0.60 HSPCO39 protein HSPCO39 gi: 777O186 S.406542 66.1 -0.42 Suppressor of cytokine signaling 4 SOCS4 gi: 4757991 S.44439 72.8 -0.48 myelin basic protein gi: 4SOS122 S.408543 75.3 -0.31 nuclear factor of activated T-cells, NFATC1 gi: 50.0631 S.S12591 cytoplasmic, calcineurin-dependent 1 38 75.5 -0.60 CTD (carboxy-terminal domain, RNA CTDP1 gi: 4758.093 polymerase II; polypeptide A) phosphatase, subunit 1 18 75.7 -0.58 hypothetical protein FLJ22378 FLJ22378 gi: 13376629 S.288284 18 75.8 -0.61 similar to S. pombe dim1 + DIM1 gi: 12654440 S.433683 18 75.8 -0.53 hypothetical protein FLJ21172 FL21172 gi: 133761.84 S.444.642 18 75.9 -0.28 KIAAO863 protein KIAAO863 gi: 10434228 S.131915 19 51.5 -0.81 protein phosphatase 5, catalytic Subunit PPP5C gi: 5453957 S.431861 19 59.3 -0.32 PRP31 pre-mRNA processing factor 31 PRPF31 gi: 7661653 S.312927 homolog (yeast) 19 59.9 -0.40 killer cell immunoglobulin-like receptor, KIR2DL1 gi: 897908 S.S12572 two domains, long cytoplasmic tail, 1 19 60.1 -0.18 natural cytotoxicity triggering receptor 1 NCR1 gi: 4758691 S.97084 21 45 -0.51 SMT3 Suppressor of miftwo 3 homolog 1 SMT3B1 gi: 590.2095 S.85119 (yeast) 21 45.1 -0.84 pituitary tumor-transforming 1 interacting PTTG1 IP gi: 11038670 S.369026 protein 21 46.9 -0.31 O.OS HMT1 hnRNP methyltransferase-like 1 HRMT1L1 gi: 4504494 S.1541.63 (S. cerevisiae) 22 21.3 -0.42 POM121 membrane glycoprotein-like 1 POM121L1 gi: 7657468 S.380370 (rat) 22 21.3 -0.57 immunoglobulin lambda joining 3 IGLJ3 gi: 13171335 S.1O2950 22 21.8 -OSO RAB36, member RAS oncogene family RAB36 gi: 60491.63 S.369557 22 21.9 -0.64 breakpoint cluster region BCR gi: 11038.638 S.446394 22 22.2 -0.65 immunoglobulin lambda-like polypeptide 1 IGLL1 gi: 13399297 S.348935 22 22.3 -0.52 Homo sapiens, clone IMAGE: 5728597, gi: 292400 S.2723O2 mRNA 22 22.4 -0.45 matrix metalloproteinase 11 (stromelysin MMP11 gi: 5177469 S.143751 3) 22 22.4 -1.28 SWISNF related, matrix associated, actin SMARCB1 gi: 4507076 S.S12700 dependent regulator of chromatin, Subfamily b, member 1 22 22.7 -1.44 glutathione S-transferase theta 1 GSTT1 gi: 4504184 S.268573 22 23.2 -0.58 Small nuclear ribonucleoprotein D3 SNRPD3 gi: 47591.59 S.356549 22 25.1 -0.48 Hermansky-Pudlak syndrome 4 HPS4 gi: 5420802 S.441481 22 25.1 -0.64 Hermansky-Pudlak syndrome 4 HPS4 gi: 11559920 S.441481 22 25.2 -0.15 tuftelin interacting protein 11 TFIP11 gi: 526,2598 S.2O225 22 25.3 -0.52 crystallin, beta A4 CRYBA4 gi: 4503058 S.S7690 22 26.4 -0.67 meningiorna (disrupted in balanced MN1 gi: 4SOS222 S.268515 translocation) 1 22 27.4 -0.73 CHK2 checkpoint homolog (S. pombe) CHEK2 gi: 13278893 S.146329 22 28.2 -O.O7 open reading frame 19 C22orf19 gi: 13177658 S.75361 22 28.5 -O.S.S ASC-1 complex subunit P100 ASC1p 100 gi: 5419897 S.4364O7 22 29 -0.84 splicing factor 3a, Subunit 1, 120 kDa SF3A1 gi: SO32O86 S.406277 22 29.1 -0.87 SEC14-like 2 (S. cerevisiae) SEC14L2 gi: 711071.4 S430576 22 30 -1.48 Zinc finger protein 278 ZNF278 gi: 99.54374 S.278O1 US 2009/0297536A1 Dec. 3, 2009 53

TABLE 4-continued

Markers of the invention which reside in MCRs of deletion and display decreased expression. 22 30.2 -0.73 <.005 KIAA0542 gene product KIAAOS42 gi: 6635200 Hs.62209 22 30.2 -1.14 <.005 KIAA0542 gene product KIAAOS42 gi: 3043607 Hs.62209 22 30.3 -0.66 <.005 phosphatidylserine decarboxylase PISD gi: 1348,9111 HS.8128 22 30.3 -0.84 <.005 KIAA0542 gene product KIAAOS42 gi:55.96770 Hs.62209 22 30.6 -O.72 <.005 KIAAO645 gene product KIAAO645 gi: 7662221 HS.435022 22 30.6 -0.45 0.021 tyrosine 3-monoxygenase/tryptophan 5- YWHAH gi:4507950 Hs.226755 monoxygenase activation protein, eta polypeptide 22 31.2 -O.72 0.011 Homo sapiens, clone IMAGE: 4818531, gi: 10030150 Hs.150167 mRNA 22 31.5 -OSO 0.027 tissue inhibitor of metalloproteinase 3 TIMP3 gi: 1519557 Hs.245.188 (Sorsby fundus dystrophy, pseudoinflammatory) 22 34 -0.37 0.047 target of myb1 (chicken) TOM1 gi: 4.885636 Hs.9482 22 34.3 -0.48 0.039 apolipoprotein L, 6 APOL6 gi: 13449280 Hs.257352 22 34.4 -0.76 <.005 RNA binding motif protein 9 RBM9 gi: 1267308 HS.433574 22 34.8 -0.62 0.013 apolipoprotein L, 3 APOL3 gi: 7656.972 Hs.241535 22 34.9 -0.66 <.005 apolipoprotein L, 2 APOL2 gi: 13325155 Hs.398,037 22 34.9 -0.44 0.032 myosin, heavy polypeptide 9, non-muscle MYH9 gi: 5448699 Hs.146550 22 35 -0.84 <.005 thioredoxin 2 TXN2 gi: 4200326 Hs.211929 22 35.1 -1.23 <.005 thioredoxin 2 TXN2 gi: 928.0552 Hs.211929 22 35.1 -1.11 <.005 eukaryotic translation initiation factor 3, EIF3S7 gi: 4503522 Hs.55682 subunit 7 zeta, 66/67 kDa 22 35.6 -0.37 0.046 thiosulfate Sulfurtransferase (rhodanese) TST gi: 1877030 Hs.351863 22 35.6 -0.64 0.005 mercaptopyruvate sulfurtransferase MPST gi: 1348,9090 Hs.248267 22 35.6 -0.58 0.01 hypothetical protein FLJ12242 FLJ12242 gi: 1348,9098 HS.94810 22 36.1 -0.53 0.033 manic fringe homolog (Drosophila) MFNG gi: 5175720 Hs.37.1768 22 36.1 -0.20 0.015 caspase recruitment domain family, CARD10 gi:5877877 Hs.57973 member 10 22 36.2 -1.08 &005 golgi associated, gamma adaptin ear GGA1 gi:9558728 Hs.405689 containing, ARF binding protein 22 36.2 -0.62 0.023 golgi associated, gamma adaptin ear GGA1 gi: 5858473 HS.405689 containing, ARF binding protein 22 36.2 -0.61 0.026 SH3-domain binding protein 1 SH3BP1 gi: 11545732 Hs.511954 22 36.4 -0.76 0.021 glycine C-acetyltransferase (2-amion-3- GCAT gi: 7657117 Hs.54609 ketobutyrate coenzyme Aligase) 22 36.5 -1.41 >.005 polymerase (RNA) II (DNA directed) POLR2F gi: 1309770 HS.46405 polypeptide F 22 36.9 -1.89 0.042 casein kinase 1, epsilon CSNK1E gi: 647.1575 Hs.355669 22 37.1 -O.88 <.005 KDEL (Lys-Asp-Glu-Leu) endoplasmic KDELR3 gi: 8051612 Hs.250696 reticulum protein retention receptor 3 22 37.1 -0.54 0.026 DMC1 dosage suppressor of mck DMC1 gi: 106600 HS.339396 homolog meiosis-specific homologens recombination (yeast) 22 37.3 -0.48 0.028 chromosome 22 open reading frame 2 C22orf2 gi: 7656941 Hs.334911 22 37.3 -0.47 0.038 transiocase of outer mitochondria TOMM22 gi: 991.0381 Hs.285.005 membrane 22 homolog (yeast) 22 37.3 -O.75 <.005 KIAAO063 gene product KIAAOO63 gi: 7661887 HS.3094 22 37.3 -0.41 0.036 unc-84 homolog B (C. elegans) UNC84B gi: 4582132 HS.406612 22 37.3 -OSO 0.016 GTP binding protein 1 GTPBP1 gi: 1916924 Hs.283677 22 37.3 -1.04 <.005 GTP binding protein 1 GTPBP1 gi: 7661735 Hs.283677 22 37.8 -0.43 0.008 platelet-derived growth factor beta PDGFB gi: 11012269 Hs. 1976 polypeptide (sitnian sarcoma viral (v-sis) oncogene homolog) 22 38 -0.54 0.042 mitogen-activated protein kinase kinase MAP3K7IP1 gi:5174702 Hs.403927 kinase 7 interacting protein 1 22 38.1 -0.56 0.047 mannosyl (beta-1,4-)-glycoprotein beta- MGAT3 gi: 6031184 Hs.276808 4-N-acetylglucosaminyltransferase 22 38.1 -0.64 0.03 hypothetical protein FLJ20232 FL2O232 gi: 12803520 Hs.505742 22 38.1 -0.62 0.018 hypothetical protein FLJ20232 FL2O232 gi: 1524716 Hs.505742 22 38.1 -0.48 0.041 activating transcription factor 4 (tax- ATF4 gi: 4502264 Hs. 181243 responsive enhancer element B67) 22 38.2 -0.52 0.043 mannosyl (beta-1,4-)-glycoprotein beta- MGAT3 gi: 4914501 Hs.276808 4-N-acetylglucosaminyltransferase 22 39 -0.26 0.022 hypothetical protein DJ1042K10.2 DJ 1042K102 gi: 11034850 Hs.22129 RefSeq, SEQID NO.: RefSeq Prot SEQID NO.: Chromosome Locus Link Regulation Probes mRina ID Nuc. ID A.A. 1 249 DOWN 215783 s at NM 000478 427 NP OOO469 11.78 1 23028 DOWN 212348 S. at NM O15013 428 NP 055828 1179 1 1870 DOWN 207042 at NM 004.091 429 NP 004082 118O 1 11313 DOWN 202292 X at NM 007260 430 NP O09191 1181 US 2009/0297536A1 Dec. 3, 2009 54

TABLE 4-continued

Markers of the invention which reside in MCRs of deletion and display decreased expression. 2S82 DOWN 202528 at NM OOO403 431 OOO394 82 3155 DOWN 202772 at NM OOO191 432 000182 83 11313 DOWN 215568 X at NM OO7260 433 O09191 84 2517 DOWN 202838 at NM OOO147 434 000138 85 1 O2SO DOWN 201225 s at NM OO5839 435 O0583.0 86 25932 DOWN 201559 s at NM O13943 436 O39234 87 57035 DOWN 209006 S. at NM 020317 437 064713 88 3925 DOWN 200783 s at NM 005563 438 OO5554 89 S1042 DOWN 204175 a NM O15871 439 056955 90 3151 DOWN 208668 x at NM OO5517 440 OO5508; 91 116138 6195 DOWN 203379 a NM OO2953 441 92 556.50 DOWN 2 9238 a NM O17837 442 060307 93 556.50 DOWN 5 146 at NM O17837 443 060307 94 63906 DOWN 2 8895 a NM O22078 444 071361 95 S4952 DOWN 218977 s at NM O17846 446 060316 97 6883 DOWN 2 at NM 005644 447 OO5635 98 10691 DOWN 220938 S. at NM OO6582; 448; 4.49 OO6573; 1199; 1200 NM 024482 O77808 51441 DOWN 7812 a NM O16258 450 O57342 2O1 6429 DOWN O1696 a NM OO5626 451 O05617 2O2 S1102 DOWN 8664 a NM 016011 452 057095 2O3 79570 DOWN 9438 a NM O24522 453 O78798 204 S7648 DOWN 2048 s at XM 036299 454 036299 205 127544 DOWN 3038 a NM 153341 455 699172 2O6 DOWN 2172 a 15O1 1912 DOWN O0919 a NM 004427; 456; 457 1207: 1208 NM 198040 79188 DOWN 7795 S NM 024334 459 210 7508 DOWN 09375 a. NMOO4628 460 211 27258 DOWN O2209 a NM O14463 461 212 S188 DOWN 04300 a NM 004564 462 OO4555 213 SS294 DOWN 8751 S. NM 018315; 463:464 060785; 121 4; 1215 NM 033632 361014 S1809 DOWN 8313 s NM O17423 465 059119 216 DOWN O918 a 503 9464 DOWN 20480 a NM O21973 466 068808 217 1635 DOWN O1571 S. NM OO1921 467 OO1912 218 556O2 DOWN 8929 a NM O17632 468 06.0102 219 3660 DOWN O3275 a. NM 002199 469 002190 220 55805 DOWN O7797 s. NM 018409 470 060879 221 553.25 DOWN 8449 a NM 018359 471 060829 222 51015 DOWN 8170 a NM 016048 472 057132 223 9474 DOWN O2511 s NM 004849 473 004840 224 55278 DOWN 8948 a NM 018292 474 O60762 225 57107 DOWN 219307 a NM O2O381 475 O65,114 226 11231 DOWN 201915 a. NM 007214 476 O09145 227 8724 DOWN 200067 x at NM 003795; 477; 478: 479 003786; 1228; 229; 1230 NM 152827; 690040; NM 152828 690041 8724 DOWN 213545 X at NM 003795; 480; 481; 482 003786; 1231; 232; 1233 NM 152827; 690040; NM 152828 690041 DOWN 217185 S at 8763 DOWN 208654 s at NM OO6016 483 OO6007 234 6610 DOWN 214206 a NM OO3O80 484 003071 235 6478O DOWN 218376 S. at NM O22765 485 O73602 236 984.1 DOWN 205340 a XM 376525 486 376525 237 98.96 DOWN 203656 a NM O14845 487 055660 238 8936 DOWN 204165 a NM OO3931 488 003922 239 S1362 DOWN 203377 S at NM O15891 489 O56975 240 23097 DOWN 212897 a NM O15076 490 O55891 241 23097 DOWN 212899 a NM O15076 491 O55891 242 262 DOWN 201196 s at NM OO1634 492 OO1625 243 598O DOWN 208070 S. at NM OO2912 493 OO2903 244 3066 DOWN 201833 a NM OO1527 494 OO1518 245 7259 DOWN 221493 a XM 371844 495 246 25842 DOWN 203427 a NM 014034 496 054753 247 10767 DOWN 209315 a. NM O06620 497 OO6611 248 S4806 DOWN 220841 s at NM O17651 498 06O121 249 4217 DOWN 203836 s at NM OO5923 499 005914 250 53832 DOWN 219115 s at NM O14432 500 055247 251 23593 DOWN 203430 a. NM O14320 5O1 O55135 252

US 2009/0297536A1 Dec. 3, 2009 56

TABLE 4-continued

Markers of the invention which reside in MCRs of deletion and display decreased expression. 377 DOWN 200011 s at NM OO1659 574 C OO1650 319 377; 377 DOWN 211622 S at NM OO1659 575 C OO1650 320 5571 DOWN 201805 at NM OO2733 576 C 002724 321 55.716 DOWN 22003.6 s at NM 0181.13 577 C O60583 322 65244 DOWN 218324 S at NM O23071 578 C 075559 323 104.45 DOWN 202556 S at NM OO6337 579 C OO6328 324 7009 DOWN 200803 s at NM O03217 C 003208 325 2.9127 DOWN 222077 s at NM 013277 581 C O37409 326 4891 DOWN 203123 s at NM 000617 582 C 000608 327 7024 DOWN 2O7627 s at NM OO5653 583 C 005644 328 98O2 DOWN 200794 x at NM 014764 C 055579 329 57228 DOWN 209679 s at NM 020467 585 C O65200 330 91 DOWN 205209 a NM 004302; 587; 588: 589 C 004293; 1332: 1333; 1334 NM 020327; C 064732; NM 020328 C 064733 91 DOWN 213198 a NM 004302; 590; 591; 592 C 004293; 1335; 1336; 1337 NM 020327; C 064732; NM 020328 C 064733 DOWN 222304 x at 1506 1975 DOWN 211937 a NM OO1417 593 C OO1408 338 S916 DOWN 217178 a NM OOO966 594 C OOO957 339 84975 DOWN 212861 a NM 032889 595 C 116278 340 S2O4 DOWN 207132 X at NM 002624; 596; 597; 598 C 002615; 1341; 1342; 1343 NM 145896; C 665903; NM 145897 C 665904 60314 DOWN 218220 a NM 021640 599 C 067653 344 3227 DOWN 206745 a. NM O 421.2 600 C O55O27 345 3223 DOWN 206194 a NM OO4503; 601: 602 C 004494; 1346; 1347 NM 153693 C 71.0160 23.583 DOWN 218685 sat NMO 4311 603 C O55126 348 DOWN 2 2126 a 1507 31.78 DOWN 200016 x at NM 002136; 604; 605 002127; 1349; 1350 NM 031157 112420 2281.8 DOWN 7726 a 6057 606 057141 351 2647 DOWN O2592 a NM OO1487 607 OO1478 352 967 DOWN O0663 a NM OO1780 608 OO1771 353 2909S DOWN 8556 a NM O 4182 609 054901 3S4 11260 DOWN 2160 a 614 O09166 359 291 10 DOWN 8520 a NM O 3254 615 O37386 360 2799 DOWN 2334 a 616 OO2O67 361 2799 DOWN O3676 a 617 OO2O67 362 23592 DOWN 8604 a NM O 4319 618 O55134 363 S1643 DOWN 2 9206 x at NM O 60S6 619 057140 364 55832 DOWN 207483 s at NM O 84.48 62O 060918 365 55832 DOWN 208838 a NM O 84.48 621 060918 366 8445 DOWN 202968 s at NM 003583; 622; 623 003574; 1367: 1368 NM OO6482 OO6473 5908 DOWN 200833 s at NM O S646 624 056461 369 55508 DOWN 218988 a NM O 8656 625 061126 370 8O89 DOWN 218911 a NM OO6530 626 OO6521 371 10576 DOWN 201947 s at NM OO6431 627 OO6422 372 4659 DOWN 201603 a NM 0024.80 628 OO2471 373 2908O DOWN 21893.6 s at NM O 41.67 629 054886 374 91298 DOWN 213701 a 1508 80184 DOWN 221683 s at NM O25114 630 O793.90 375 4254 DOWN 207029 a NM 000899; 631; 632 000890; 1376; 1377 NM OO3994 OO3985 2 1848 DOWN 208891 a NM OO1946; 633; 634 OO1937; 1378; 1379 NM 022652 073143 490 DOWN 212930 a. NM OO1682 635 OO1673 1380 490 DOWN 209281 is at NM OO1682 636 OO1673 1381 694 DOWN 200921 s at NM OO1731 637 OO1722 1382 527 DOWN 200954 a NM OO1694 638 OO1685 1383 527 DOWN 36994 at NM OO1694 639 OO1685 1384 51005 DOWN 219082 a NM O15944 640 057028 1385 5170 DOWN 204524 a NM 002613 641 002604 1386 5170 DOWN 32029 at NM 002613 642 002604 1387 23S24 DOWN 208.610 s at NM 016333 643 057417 1388 23S24 DOWN 213877 X at NM 016333 644 057417 1389 9570 DOWN 21.0009 s at NM 004287: 233; 234 004278: 984: 985 NM 054022 473363 8 S289 DOWN 204297 at NM 002647 645 002638 1390 8 498 DOWN 213738 s at NM 004.046 646 OO4037 1391 US 2009/0297536A1 Dec. 3, 2009 57

TABLE 4-continued

Markers of the invention which reside in MCRs of deletion and display decreased expression. 18 9063 DOWN 214593 at NM 004671; 647; 648 NP 004662; 1392: 1393 NM 1732O6 NP 775298 18 9063 DOWN 37433 at NM 004671; 649; 650 NP 004662; 1394; 1395 NM 1732O6 NP 775298 18 51124 DOWN 211406 at NM 016097 651 NP 057181; 1396 NP 060992 18 9306 DOWN 214462 at NM OO4232 652 NP OO4223 1397 18 4155 DOWN 207323 s at NM 002385 653 NP 002376 1398 18 4772 DOWN 210161 at NM OO6162; 654; 655; 666; 657; NP OO6153; 1399; 1400; 1401; NM 172387: 658 NP 765975; 1402: 1403 NM 172388; NP 765976; NM 172389; NP 765977; NM 172390 NP 765978 38 915.0 DOWN 205035 a. NM 004715; 659; 660 NP 004706; 1404: 1405 NM 0483.68 NP 430255 18 8O148 DOWN 218208 a NM O25078 661 NP 079354 1406 18 10907 DOWN 202835 a. NM OO6701 662 NP OO6692 1407 18 79863 DOWN 219419 a NM 024.805 663 NP 079081 1408 18 22850 DOWN 203321 s at XM 377498 664 XP 377498 1409 19 5536 DOWN 201979 s at NM OO6247 319 NP OO6238 1070 19 26121 DOWN 202408 S. at NM O15629 370 NP 056444 1121 19 3802 DOWN 210890 x at NM O14218 665 NP O55033 1410 19 9437 DOWN 207860 a NM 004.829 666 NP 004.820 1411 21 6612 DOWN 200740 S at NM OO6936 667 NP OO8867 1412 21 754 DOWN 200677 a NM 004339 668 NP 004330 1413 21 3275 DOWN 202098 s at NM OO1535 669 NP OO1526 1414 22 25812 DOWN 214570 x at NM O14348 670 NP 0551.63 1415 22 28831 DOWN 216846 a 1509 1510 22 9609 DOWN 211471 S. at NM 004914 671 NP 004.905 1416 22 613 DOWN 202315 s at NM 004327: 672; 673 NP 0043.18: 1417; 1418 NM 021574 NP 067585 22 3543 DOWN 206660 a NM 020070; 674; 675 NP 064455; 1419; 1420 NM 152855 NP 690594 22 375159 DOWN 215816 a 1511 1512 22 4320 DOWN 203876 s at NM OO5940 676 NP 005931 1421 22 6598 DOWN 206532 a. NM 003073 677 NP OO3064 1422 22 2952 DOWN 203815 a. NM OOO853 678 NP OOO844 1423 22 6634 DOWN 202567 a NM OO4175 679 NP 004166 1424 22 89781 DOWN 54037 at NM 022081; 680; 681; 682; 683; NP 071364; 1425; 1426; 1427: NM 152840; 684 NP 690053; 1428: 1429 NM 152841; NP 690054; NM 152842; NP 690055; NM 152843 NP 690056 22 89781 DOWN 218402 s at NM 022081; 685; 686; 687; 688; NP 071364; 1430; 1431; 1432: NM 152840; 689 NP 690053; 1433; 1434 NM 152841; NP 690054; NM 152842; NP 690055; NM 152843 NP 690056 22 24144 DOWN 202750 S at NM 012143 690 NP 036275 435 22 1413 DOWN 206843 at NM OO1886 691 NP OO1877 436 22 4330 DOWN 205330 at NM OO2430 692 NP OO2421 437 22 11200 DOWN 210416 s at NM 007194; 693; 694; NP O09125; 1438: 1439 NM 145862 NP 665861 22 8563 DOWN 209418 is at 1513 S14 22 84164 DOWN 215684 s at NM 032204 695 NP 115580 440 22 10291 DOWN 201357 s at NM OO5877 696 NP OO5868 441 22 23541 DOWN 204541 at NM 012429 697 NP 036561 442 22 23598 DOWN 20943.1 s at NM O14323; 698; 699; 700; 701 NP O55138; 1443; 1444; 1445; NM 032050; NP 114439; 446 NM 032051; NP 114440; NM 032052 NP 114441 22 9814 DOWN 213431 X at XM 038520 702 XP 038520 447 22 9814 DOWN 36545 s at XM 038520 703 XP 038520 448 22 23761 DOWN 202392 s at NM O14338 704 NP 055153 449 22 9814 DOWN 215699 x at XM 038520 705 XP 038520 450 22 9681 DOWN 205223 at XM 377498 706 XP 376007 451 22 7533 DOWN 201020 at NM OO3405 707 NP OO3396 452 22 DOWN 215762 at 1515 22 7078 DOWN 201149 s at NM OOO362 708 NP 000353 453 22 10043 DOWN 202807 s at NM OO5488 709 NP OO5479 454 22 80830 DOWN 219716 at NM 030641 710 NP 085144 455 22 23543 DOWN 212104 S at NM 014309 711 NP 055124 456 US 2009/0297536A1 Dec. 3, 2009 58

TABLE 4-continued

Markers of the invention which reside in MCRs of deletion and display decreased expression. 22 DOWN 221087 s at NM O14349; 712; 713; 714; 715 P O55164: 1457; 1458; 1459 NM 030644: 716; 717 P 085147; 1460; 1461: 1462 NM 145639; P 663614; NM 145640; P 663615; NM 145641; P 663616; NM 145642 P 663617 22 2378O DOWN 221653 x at NM 030882; 718; 719 P 112092; 1463; 1464 NM 145637 P 663612 22 4627 DOWN 211926 s at NM OO2473 720 P 002464 146S 22 2S828 DOWN 209077 a NM 012473 721 P 036605 1466 22 2S828 DOWN 209078 S. at NM 012473 722 P 036605 1467 22 8664 DOWN 200005 a NM 003753 723 P 003744 1468 22 7263 DOWN 209605 a. NM OO3312 724 P OO3303 1469 22 4357 DOWN 203524 s at NM 021126 725 P 066949 1470 22 79734 DOWN 205561 a NM O24681 726 P 078957 1471 22 4242 DOWN 213783 a NM OO2405 727 P 002396 1472 22 29775 DOWN 214207 s. at NM 014550 728 P O55365 1473 22 26088 DOWN 218114 a NM 013365 729 P 037497 1474 22 26088 DOWN 45572 s at NM 013365 730 P 037497 1475 22 23616 DOWN 213633 a NM 018957 731 P 061830 1476 22 23464 DOWN 205164 a NM O14291 732 P 055106 1477 22 5435 DOWN 209511 a NM O21974 733 P 068809 1478 22 1454 DOWN 222015 a. NM OO1894: 734; 735 P 001885; 1479; 1480 NM 152221 P 689407 22 1101S DOWN 204017 a NM OO6855; 736; 737 P OO6846; 1481; 1482 NM 016657 P 057839 22 11144 DOWN 208382 S at NM 007068 738 P OO8999 1483 22 25776 DOWN 203450 a NM O15373 739 P 056188 1484 22 56993 DOWN 217960 S at NM O2O243 740 P 064628 1485 22 9929 DOWN 201751 a NM O14876 741 P 055691 1486 22 25777 DOWN 212144 a NM O15374 742 P 056189 1487 22 9567 DOWN 205274 a NM 004286; 743 P OO4277; 1488 P 054746 22 9567 DOWN 219357 a NM 004286; 744 P OO4277; 1489 P 054746 22 5155 DOWN 216061 x at NM 002608; 745; 746 P 002599; 1490; 1491 NM 033016 P 148937 22 10454 DOWN 203901 a NM 006116; 747: 748 P OO6107; 1492; 1493 NM 153497 P 705717 22 4248 DOWN 208058 s at NM 0024.09 749 P 002400 1494 22 S4471 DOWN 221516 s at NM O19008 750 P 061881 1495 22 S4471 DOWN 204593 s at NM O19008 751 P 061881 1496 22 468 DOWN 200779 at NM OO1675; 752; 753 P OO1666: 1497; 1498 NM 1828.10 P 877962 22 4248 DOWN 209764 at NM 0024.09 754 P 002400 1499 22 27352 DOWN 20301.4 x at NM O15705 755 P 056520 1SOO

TABLE 5 Markers of the invention which reside in MCRs of amplification and display increased expression. POS Gene Minimum Chromosome (Mb) Weight p value Gene Description Gene Symbol GI UGIDhi 1 26.8 1.03 0.032 nuclear distribution gene Chomolog NUDC gi: 5729952 HS.263,812 (A. nidians) 116.9 2.41 0.038 transcription termination factor, RNA TTF2 gi: 573.3121 HS2O1774 polymerase II 117.8 0.029 WD repeat domain 3 gl: HS2O1375 11.3 0.035 hypothetical protein MGC33602 gi: 7328008 HS.274415 5 O.2 <.005 Succinate dehydrogenase complex, gi: 4759079 HS.440475 subunit A, flavoprotein (Fp) <.005 programmed cell death 6 PDCD6 gi: 7019484 HS.24087 <.005 Sec 6 (S. cerevisiae) homolog SEC6 gl:: 300S726 HS.44858O 0.005 hypothetical protein FLJ10565 FLJ10565 gi: 8922S2O HS1OO824 <.005 hypothetical protein FLJ13441 FLJ13441 gi: 1296.5190 HS.449178 0.014 hypothetical protein FLJ12443 FLJ12443 gi: 13376233 Hs.179882 US 2009/0297536A1 Dec. 3, 2009 59

TABLE 5-continued Markers of the invention which reside in MCRs of amplification and display increased expression. 1.8 O.96 NADH dehydrogenase (ubiquinone) Fe–S NDUFS6 gi: 475.8791 S.408257 protein 6, 13 kDa (NADH-coenzyme Q reductase) 5.5 0.72 KIAA0947 protein KIAAO947 gi: 134361.78 SSOTO 32 0.67 tenascin XB TNXB gi: 836.1667 S.411644 42.9 1.03 trinucleotide repeat containing 5 TNRCS gi: 133252O7 S.414099 43 O.25 protein phosphatase 2, regulatory Subunit PPP2RSD gi: 5453953 S.118244 B (B56), delta isoform O.8 O.47 G protein-coupled receptor 30 GPR30 gi: 1381668 S.1132O7 1.2 O.S6 G protein-coupled receptor 30 GPR30 gi: 26S612O S.1132O7 1.2 O.14 DKFZP586J0619 protein DKFZPS860619 gi: 10809392 S.112184 1.5 O.64 MICAL-like 2 FLU23471 gi: 13376030 S.3766.17 1.6 O.40 V-maf musculosponeurotic fibrosarcoma MAFK gi: 4505074 S.131953 oncogene homolog K (avian) 7 2.1 O.9S MAD1 mitotic arrest deficient-like 1 MAD1L1 gi: 4SOSO64 S.7345 (yeast) 30.7 O.47 glycyl-tRNA synthetase GARS gi: 577711 S.293.885 64.9 1.19 argininosuccinate lyase ASL gi: 4502256 S.442047 65 O.87 calcitonin gene-related peptide-receptor RCP9 gi: 7656976 S.3OO684 component protein 65.5 1.17 potassium channel tetramerisation domain KCTD7 gi:5596,067 S.119683 containing 7 65.6 1.04 RAB guanine nucleotide exchange factor RABGEF1 gi: 7657495 S.187660 (GEF) 1 65.8 1.42 hypothetical protein FLJ10099 FLJ10099 gi: 8922228 S.28795S 93.2 O.69 BET1 homolog (S. cerevisiae) BET1 gi: 12654.162 S.23.103 93.7 0.37 O-acetyltransferase CAS1 gi: 12597.638 S.324725 94.6 O.43 paraOXonase 3 PON3 gi: 1333.633 S.44O967 94.6 1.07 paraOXonase 2 PON2 gi: 2228776 S.165598 94.8 O.SO pyruvate dehydorgenase kinase, PDK4 gi: 450S692 S.8364 isoenzyme 4 95.4 0.57 solute carrier family 25, member 13 SLC25A13 gi: 765758O S.9599 (citrin) 77 95.9 1.01 SHFM1 S.33349S split hand foot malformation gi: 5453639 (ectrodactyly) type 1 96.3 O.42 ACN9 homolog (S. cerevisiae) ACN9 gi:9910179 S.42785 97.1 0.77 asparagine synthetase ASNS gi: 4SO2258 S.446546 97.2 1.23 Homo sapiens transcribed sequence with gi: 8008445 S.512431 weak similarity to protein pir: PC4369 (H. sapiens) PC4369 olfactory receptor. HT2 —human (fragment) 97.3 O.89 kinase phosphatase inhibitor 2 KPI2 gi: 7662475 S.122708 98.1 O.94 transformation/transcription domain TRRAP gi: 4507690 S.2O3952 associated protein 77 98.2 E3 ubiquitin ligase SMURF1 SMURF1 gi: 4738848 S.436249 98.2 transformation/transcription domain TRRAP gi: 3694.662 S.2O3952 associated protein 7 98.2 E3 ubiquitin ligase SMURF1 SMURF1 gi: 6446605 S.436249 98.2 Homo sapiens cDNA: FLJ21284 fis, gi: 10437358 S.288.218 clone COLO1911 98.5 O.49 Homo sapiens clone 24438 mRNA gi: 3283921 S.124126 Sequence 98.5 O.99 actin related protein 2/3 complex, Subunit ARPC1A gi: 5454.077 S.29.1981 1A, 41 kDa 98.5 1.01 actin related protein 2/3 complex, Subunit ARPC1B gi: SO316OO S.433506 1B, 41 kDa 98.7 O.76 Zinc finger protein 95 homolog (mouse) gi: 11036641 S.1108.39 98.8 O.60 cytochrome P450, family 3, Subfamily A, gi: 945OOS S.150276 polypeptide 5 98.8 0.67 cytochrome P450, family 3, Subfamily A, CYP3A5 gi: 4SO3230 S.150276 polypeptide 5 99.3 adaptor-related protein complex 4, mu1 gi: S.194703 Subunit 99.3 O.98 TAF6 RNA polymerase II, TATA box TAF6 gi: SO32146 S.2899SO binding protein (TBP)-associated factor, 80 kDa 99.5 O.38 postmelotic segregation increased 2-like 6 gi: 4175684 S.367667 99.8 O.94 O.OS guanine nucleotide binding protein (G GNB2 gi: 4.885282 S.185172 protein), beta polypeptide 2 1OO 0.67 O.OOS solute carrier family 12 SLC12A9 gi: 99.10385 S.437628 (potassium chloride transporters), member 9 US 2009/0297536A1 Dec. 3, 2009 60

TABLE 5-continued Markers of the invention which reside in MCRs of amplification and display increased expression. OO.4 O.94 procollagen-lysine, 2-oxoglutarate 5 PLOD3 gi: 4SOS890 S.153357 dioxygenase 3 OO.4 O.SO O.018 Zine finger, HIT domain containing 1 HIT1 gi: 5453616 S.211079 OO.4 O.36 O.044 etratricopeptide repeat domain 11 TTC11 gi: 77.05631 S423968 OO.8 O.36 O.O47 myosin light chain 2, precursor gi: 12803868 S.247831 ymphocyte-specific O16 O.35 chromosome 7 open reading frame 19 gi: 12357031 S.289053 O16 O.49 HSPC047 protein HS gi: 7661749 S.512142 O16 O.36 polymerase (RNA) II (DNA directed) PO gi: 5100.572 S.489461 polypeptide J, 13.3 kDa O1.7 O.38 DNA directed RNA polymerase II PO LR22 gi: S.406505 polypeptide J-related gene O1.7 O.48 DNA directed RNA polymerase II PO LR22 gi: 5901957 S.406505 polypeptide J-related gene 05 O.84 hypothetical protein MGC33190 MGC33190 gi: 3231718 S.211068 O.36 synaptophysin-like protein SY C gi: 5235354 S.80919 O.76 solute carrier family 26, member 4 gi: 450S696 S.512611 O.48 Cas-Br-M (murine) ecotropic retroviral LL1 gi: 133762O3 S.458382 tranforming sequence-like 1 dihydrolipoamide dehydrogenase (E3 DL gi: 181574 S.74635 component of pyruvate dehydrogenase complex, 2-oxo-glutarate complex, branched chain keto acid dehydrogenase complex) 111.1 O.SO dedicator of cytokinesis 4 DOCK4 gi: 7662263 S.118140 37.7 O.60 G protein-coupled receptor 124 GPR124 gi: 115946.13 S.17270 37.7 0.72 G protein-coupled receptor 124 GPR124 gi: 4739882 S.17270 37.7 1.12 BRF2, subunit of RNA polymerase III BR gi: 11096174 S274136 transcription initiation factor, BRF1-like 37.9 ash2 (absent, Small, or homeotic)-like ASH2L. gi: 44.17209 S.6856 (Drosophila) 38 O.OOS LSM1 homolog, U6 small nuclear RNA LSM1 gi: 7657312 S.4253.11 associated (S. cerevisiae) 38 BCL2-associated athanogene 4 BAG4 gi: 6631074 S.194726 38.1 KIAA0725 protein KIAAO725 gi: 38821.70 S.434966 38.2 Wolf-Hirschhorn syndrome candidate 1 WHSC1L1 gi: 13699812 S.415895 ike 1 20.7 O.43 TAP2 RNA polmerase II, TATA box TAF2 gi: 7022983 S.122752 binding protein (TBP)-associated factor, 50 kDa 21.3 O.38 mitochondrial ribosomal protein L13 MRPL13 gi: 7662495 S.3338.23 22.5 O.43 hysturonan synthase 2 HAS2 gi: 4.885390 S.159226 23.9 O.48 hypothetical protein MGC3067 MGC3067 gi: 8924181 S.241576 23.9 0.37 hypothetical protein MGC3067 MGC3067 gi: 132365.15 S.241576 24.1 0.44 unknown MGC21654 product MGC21654 gi: 3231900 S.95631 24.3 0.55 hypothetical protein FLJ10204 FLJ10204 gi: 892228O 24.6 O.S2 annexin A13 ANXA13 gi: 4757753 S.181107 25.4 1.03 ring finger 139 F139 gi: 339.5786 S.228285 25.9 O.66 KIAAO196 gene product KIAAO196 gi: 7661987 S.437991 28.7 O.62 v-myc myelocytomatosis viral oncogene MYC gi: 1296,2934 S.2O2453 homolog (avian) 8 28.9 Homo sapiens cDNA FLJ26234 fis, clone gi: 190753 S.459222 ADGO9627 30.8 O.S9 hypothetical protein BM-009 BM-009 gi: 7705303 S.369973 33.7 O.43 CGI-72 protein gi: 77.05782 S.441.59 34.2 O.47 N-myc downstream regulated gene 1 NDRG1 gi: S174656 S.318567 4.1.8 0.37 PTK2 protein tyrosine kinase 2 PT K2 gi: 5406748 S.434281 43.8 O41 mescochymal stem cell protein DSCD75 LOC51337 gi: 77O6199 S.25237 44.6 O.S9 KIAAO150 protein KIAAO150 gi: 1469881 S.370491 44.7 0.37 hypothetical protein FLJ12150 FL gi: 13376057 S.118983 44.7 0.37 eukaryotic translation elongation factor 1 EE gi: 46225SO S.334798 delta (guanine nucleotide exchange protein) 44.8 0.73 issue specific transplantation antigen TSTA3 gi: 6598326 S.404119 P35B 44.8 O.80 issue specific transplantation antigen TSTA3 gi: 1381.178 S.404119 P35B 44.8 O.61 KIAA.0628 gene product KIAAO628 gi: 7662213 S.431.33 44.9 O46 scribble SC RIB gi: 4331493 S.436329 45.2 O.61 5-oxoprolinase (ATP-hydrolysing) OP LAH gi: 5838792 S.305882 45.2 O.49 exosome complex exonuclease RRP41 P41 gi: 4S34672 S.343589 45.2 0.55 exosome complex exonuclease RRP41 P41 gi: 9506688 S.343589 45.2 O46 exosome complex exonuclease RRP41 P41 gi: 54085 17 S.343589 US 2009/0297536A1 Dec. 3, 2009 61

TABLE 5-continued Markers of the invention which reside in MCRs of amplification and display increased expression. 45.2 0.37 GPAA1P anchor attachment protein 1 GPAA1 gi: 6031166 S.4742 homolog (yeast) 45.2 GPAA1P anchor attachment protein 1 GPAA1 gi: 13623S46 S.4742: homolog (yeast): GPAA1P anchor S.4742 attachment protein 1 homolog (yeast) 45.2 O.45 GPAA1P anchor attachment protein 1 GPAA1 gi: 7018511 S.4742 homolog (yeast) 45.2 O.43 cytochrome c-1 gi: 4503184 S.289271 45.2 O.39 hypothetical protein DKFZp434N1923; gi: 13569949 s.295866; hypothetical protein DKFZp434N1923 S.295866 45.3 O.31 O.O14 brain protein 16 LOCS1236 gi: 13124772 S.3OO224 45.5 O.33 O.048 diacylglycerol O-acyltransferase homolog DGAT1 gi: 7382489 S.5128.10 (mouse) 45.5 0.67 putative G-protein coupled receptor FLJ11856 gi: 13375681 S.6459 GPCR41 45.6 O.43 cleavage and polyadenylation specific CPSF1 gi: 10O371.83 S.83727 actor 1, 160 kDa 45.6 solute carrier family 39 (zinc transporter), gi: 8923304 S.411274 member 4 45.6 O.43 vacuolar protein sorting 28 (yeast) VPS28 gi: 77.05884 S.418175 s 45.6 0.44 Homo sapiens mRNA, chromosome 1 gi: 7150895 S.4593.79 specific transcript KIAAO496. 21.9 methylthioadenosine phosphorylase M TAP gi: 6006O2S S.446152 35.6 tropomyosin 2 (beta) PM2 gi: 1219494 S.3OO772 35.7 talin LN1 gi: S4S4129 S.37SOO1 35.7 cAMP responsive element binding protein 3 REB3 gi: 2599559 S.287921 35.7 KIAAO258 AAO258 gi: 7662O29 S47313 35.7 natriuretic peptide receptor Biguanylate PR2 gi: 23373.54 S.785.18 cyclase B (atrionatriuretic peptide receptor B) 35.8 0.67 nasopharyngeal carcinoma related protein NGX6 gi: 7706546 S.44O953 36.1 1.33 clathrin, light polypeptide (Lca) CLTA gi: 600S992 S.207052 36.2 O.20 clathrin, light polypeptide (Lca) CLTA gi: 704460 S.207052 36.3 O.49 ring finger protein 38 RNF38 gi: 12232470 S.333 SO3 22.1 O.62 cytidine monophosphate N CMAS gi: 8923899 S.311346 acetylneuraminic acid synthetase 2 50 O.OO elastase 1, pancreatic ELA1 gi: S.348.395 2 54.8 0.77 myosin, light polypeptide 6, alkali, MYL6 gi: S. 77385 Smooth muscle and non-muscle 22.8 O.84 ADP-ribosyltransferase (NAD+: poly ADPRTL1 gi: 11496990 S. 437959 (ADP-ribose) polymerase)-like 1 23.6 0.67 myorubularin related protein 6 MTMR6 gi: 1669390 S. 79877 3 24.5 2.81 ring finger protein (C3H2C3 type) 6 gi: 126S 6362 S.136885 112.9 O.32 UPF3 regulator of nonsense transcripts U gi: 12620405 S.39974O homolog A (yeast) 29.1 chromosome 14 open reading frame 163 gi: 424O322 S.27023 29.5 adaptor-related protein complex 4, Sigma k gi: 12654832 S.496614 Subunit 30.1 O.80 chromosome 14 open reading frame 127 4orf127 gi: 13376746 S.288981 30.6 O46 Rho GTPase activating protein 5 RHGAPS gi: S90S160 S4O9546 31.8 O.S6 neuronal PAS domain protein 3 PAS3 gi: 11545846 S.243209 32.9 1.12 chromosome 14 open reading frame 11 s 4orf11 gi: 892.2092 S.433269 33 O.98 Sorting nexin 6 SNX6 gi: 13027619 S.283443 52.4 1.17 bone morphogenetic protein 4: bone BMP4 gi: 576934 s.68879; morphogenetic protein 4 S.68879 53.2 sterile alpha motif domain containing 4 SAMD4 gi: 56894.42 S.98259 53.4 WD repeat and HMG-box DNA binding WDHD1 gi: 7704203 S.385998 protein 1 :: 53.5 1.36 chromosome 14 open reading frame 32 gi: 10438139 S4O64O1 S3.6 O.S1 discs, large homolog7 (Drosophila) gi: 7661851 S.77695 53.8 O.42 F-box only protein 34 gi: 89236SO S.15467 S4 O.29 kinectin 1 (kinesin receptor) gi: 11681348 S. 368.212 55.6 O.62 SEC10-like 1 (S. cerevisiae) gi: 573.0036 S. 365863 55.7 O.68 chromosome 14 open reading frame 108 gi: 8922687 S.106,210 56.6 O.65 actin-related protein 10 homolog gi: 104336O4 S.24.8569 (S. cerevisiae) 58.5 O.65 chromosome 14 open reading frame 135 gi: 11968.054 S.413671 59.8 protein kinase C, eta PRKCH gi: 5453971 S.315366 60.1 hypoxia-inducible factor 1, alpha Subunit HIF1A gi: 4504384 S.412416 (basic helix-loop-helix transcription factor) 60.2 O.43 Small nuclear RNA activating complex, SNAPC1 gi: 45071OO S. 179312 polypeptide 1, 43 kDa US 2009/0297536A1 Dec. 3, 2009 62

TABLE 5-continued Markers of the invention which reside in MCRs of amp ification and display increased expression. 4 62.9 O.49 O.O21 Zinc finger and BTB domain containing 1 gi: 7662437 S. 511938 4 63.2 O.O3 O.O11 KIAAOS99 K AAO599 gi: 1327916O S. 198O37 4 39.1 O.66 &.OOS SWISNF related, matrix associated, actin SMARCE1 gi: 13045953 S.437546 dependent regulator of chromatin, Subfamily e, member 1 7 39.3 O6 &.OOS keratin 10 (epidermolytic hyperkeratosis; K RT10 gi: 4557696 S.99936 keratosis palmaris et plantaris) 7 40.4 .74 &.OOS ATP citrate lyase ACLY gi: 57681.07 S.387567 7 4.O.S 0.97 O.OO7 -kappa-B-interacting Ras-like protein 2 K BRAS2 gi: 89221SO SSO2910 7 40.6 17 O.O11 RAB5C, member RAS occogene family RABSC gi: 7672664 S.479 7 40.7 O.83 O.O33 signal transducer and activator of ST TATSB gi: 9970172 S.434992 transcription 5B 7 4 O1 &.OOS ATPase, H+ transporting, lysosomal VO gi: 4885.084 S.267871 Subunita isoform 1 7 4 O.69 O.OO7 transcription factor-like 4 gi: 1176 1691 S383,019 7 41.1 O.89 O.OO9 GT198, complete ORF UMGT198A gi: 11641S2 S.279.032 7 41.1 O6 O.OO9 hypothetical protein LOC162427 LOC162427 gi: 12779367 S.432850 7 41.2 30 O.OOS enhancer of Zeste homolog 1 (Drosophila) EZH1 gi: 2224716 S.194669 7 41.2 O8 &.OOS enhancer of Zeste homolog 1 (Drosophila) EZH1 gi: 2224716 S.194669 7 41.3 62 &.OOS HSPC009 protein HSPCOO9 gi: 7661731 S.16059 7 41.3 1S &.OOS beclin 1 (coiled-coil, myosin-like BCL2 BECN1 gi: 4502394 S.12272 interacting protein) 7 41.3 S6 &.OOS proteasome (prosome, macropain) PSME3 gi: SO31996 S.152978 activator subunit 3 (PA28 gamma; Ki) 7 41.3 09 &.OOS amine oxidase, copper containing 2 AOC2 gi: 6806881 S.143102 (retina-specific) 7 41.4 .74 &.OOS hypothetical protein MGC2744 M FC2744 gi: 34321.63 S.317403 7 41.5 86 &.OOS ribosomal protein L27 RPL27 gi: 4506622 S4OSS28 7 4.1.8 0.44 O.OS Homo sapiens cDNA FLJ35853 fis, clone gi: 5935.951 S.277721 TESTt2007078, highly similar to MEMBRANE COMPONENT, CHROMOSOME 17, SURFACE MARKER 2. 7 423 O.45 &.OOS dual specificity phosphatase 3 (vaccinia DUSP3 gi: 128O3692 S.181046 virus phosphatase VH1-related) 7 423 1.16 &.OOS membrane protein, palmitoylated 3 M PP3 gi: 4505238 S.396566 (MAGUK p55 subfamily member 3) 7 42.4 O.63 &.OOS membrane protein, palmitoylated 2 PP2 gi: 6991687 S.436326 (MAGUK p55 subfamily member 2) 7 42.6 1.57 &.OOS glucose-6-phosphatase catalytic subunit 3 gi: 12951784 S.294.005 7 42.6 1.39 &.OOS glucose-6-phosphatase catalytic subunit 3 gi: 4834429 S.294.005 7 42.7 1.55 &.OOS hypothetical protein MGC3123 GC3123 gi: 131291.17 S.181391 7 42.9 O.92 &.OOS granulin GRN gi: 4SO41SO S.180577 7 42.9 O.45 O.OO6 KIAA0553 protein KIAAOS53 gi: 11008117 S.396O47 7 43.6 0.55 O.018 N-myristoyltransferase 1 NMT1 gi: 2760893 S346743 7 43.7 O.34 O.O31 HMBA-inducible HIS1 gi: 7457641 S.15299 7 44 1.09 &.OOS hypothetical protein FLJ10120 FLJ1012O gi: 8922.238 S.378.860 7 44.8 O.S1 O.O12 ARF protein LOCS1326 gi: 7770214 SSOO496 7 44.8 1.09 &.OOS KIAA0563 gene product KIAAOS63 gi: 3647821 S38861 7 45.2 O.92 &.OOS N-ethylmaleimide-sensitive factor NSF gi: 11079227 S.431279 7 45.3 O.42 O.O41 wingless-type MMTV integration site WNT3 gi: 13S4O476 s.224667; family, member 3; wingless-type MMTV S.224667 integration site family, member 3 7 45.7 1.06 &.OOS Homo sapiens transcribed sequence with gi: 1126567 S.514263 weak similarity to protein sp: P30260 (H. sapiens) CC27 HUMAN Protein CD27Hs (Cell division cycle protein 27 homolog) (H-NUC) 7 46.1 0.55 O.O1 aminopeptidase poromycin sensitive NPEPPS gi: 421.0725 S.293.007 7 46.2 O.31 &.OOS karyopherin (importin) beta 1 KPNB2 gi: 13097743 S.439683 7 46.4 O.87 O.O1 pyridoxine-5'-phosphate oxidase PNPO gi: 8922497 S.327335 7 46.6 1.27 &.OOS chromobox homolog 1 (HP1 beta CBX1 gi: 5803075 S.772S4 homolog Drosophila) 7 46.6 O.81 O.OO8 Sorting nexin 1 SNX11 gi: 7019.538 S.15827 7 46.6 O.78 O.O1 Sorting nexin 1 SNX11 gi: 4827951 S.15827 7 47.4 O.86 &.OOS ATP synthase, H+ transporting, ATPSG1 gi: S.80986 mitochondrial PO complex, subunit c (subunit 9), isoform 1 7 47.4 O.60 O.O17 hypothetical protein FLJ13855 FLJ13855 gi: 12751494 S.36912O 7 47.4 1.17 &.OOS EAP30 subunit of ELL complex EAP30 gi: 6005754 S.127249 7 47.8 O.49 O.O21 KIAAO924 protein KIAAO924 gi: 7662383 S.190386 7 47.9 1.15 &.OOS prohibitin PHB gi: 6031190 S.7S323 7 48.1 1.06 &.OOS specide-type POZ protein SPOP gi: 4507182 S.129951 US 2009/0297536A1 Dec. 3, 2009 63

TABLE 5-continued Markers of the invention which reside in MCRs of amplification and display increased expression. 48.2 1.61 solute carrier family 35, member B1 SLC3SB1 gi: SO32212 s: 154O73 48.2 O.82 C/EBP-induced protein; C/EBP-induced LOC81558 gi: 13S40589 s.9851; protein S.9851 7 48.3 1.18 MYST histone acetyltransferase 2 MYST2 gi: S901961 S.21907 48.6 O.45 integrin, alpha 3 (antigen CD49C, alpha 3 TGA3 gi: 4504746 S.265829 subunit of VLA-3 receptor) 48.6 0.37 pyruvate dehydrogenase kinase, PDK2 gi: SS44583 S.922.61 isoenzyme 2 48.9 O.45 xylosyltransferase II XYLT2 gi: 11545913 S.32117 48.9 O.91 hypothetical protein PRO1855 PRO1855 gi: 10437822 S.370927 49 0.79 hypothetical protein FLJ20920 FLJ20920 gi: 13376740 S.288959 49 0.57 hypothetical protein FLJ11164 FLJ11164 gi: 8922910 S.8033 49 O.60 epsin 3 EPN3 gi: 8923677 S.165904 49.1 0.57 hypothetical protein FLJ21347 FL21347 gi: 12383,067 S.103147 49.2 O.93 hypothetical protein MGC15396; MGC15396 gi: 13543385 s.351247; hypothetical protein MGC15396 S.3S1247 49.2 O.42 cisplatin resistance-associated LUC7A gi: S.130293 overexpressed protein 77 49.3 O.40 cisplatin resistance-associated LUC7A gi: S174618 S.130293 overexpressed protein 7 49.5 O.69 sperm associated antigen 9 SPAG9 gi: 4504524 S.SOO367 49.7 1.32 non metastatic cells 1, protein (NM23A) NME1 gi: 4557796 S.118.638 expressed in 49.7 1.75 non metastatic cells 2, protein (NM23B) NME2 gi: 4SOS408 S.433416 expressed in 49.8 1.01 O.OOS CGI-48 protein CGI-48 gi: 3179644 S.441503 53.5 O.61 O.OOS COXII homolog, cytochrome c oxidase COXII gi: 4186577 S.436988 assembly protein (yeast) 53.8 O.23 hepatic leukemia factor HLF gi: 184223 S.250692 S4.3 O.64 phosphatidylcholine transfer protein PCTP gi: 10864026 S.285218 74 O.63 KIAAO195 gene product KIAAO195 gi: 7661985 S.301,132 74 1.07 CASK interacting protein 2 CASKIN2 gi: 5766922 S.2744.08 74 O.76 CASK interacting protein 2 CASKIN2 gi: S928.096 S.2744.08 19.3 O.S6 RIOkinase 3 (yeast) RIOK3 gi: 4507298 S.209061 19.3 0.79 RIOkinase 3 (yeast) RIOK3 gi: 585506.8 S.209061 19.3 1.02 colon cancer-associated protein Mic1 MIC gi: 7019454 S.287633 19.3 O.9S Niemann-Pick disease, type C1 NPC gi: 45578O2 S.404930 19.9 O.48 calcium-binding tyrosine-(Y)- CABYR gi: 6912377 S.51 1983 phosphorylation regulated (fibrousheathin 2) 8 20.1 O.48 oxysterol binding protein-like 1A: OSBPL1A gi: 13877169 s.415753; oxysterol binding protein-like 1A: S.415753 41.4 1.18 Zinc finger protein 146 ZNF146 gi: 6005965 42.9 O.71 hypothetical protein FLJ30921 FLJ30921 gi: 97.22561 S.290703 43.3 O.S3 D4, zinc and double PHD fingers family 1 DPF gi: 4758797 S.389.057 43.5 1.08 proteasome (prosome, macropain) 26S PSMD8 gi: 4506.232 S.78466 subunit, non-ATPase, 8 43.7 mitogen-activated protein kinase kinase MAP4K1 gi: 997O929 S.9S424 kinase kinase 1 43.8 1.41 eukaryotic translation initiation factor 3 eIF3k gi: S114OSO S.143773 Subunitk 43.8 1.43 eukaryotic translation initiation factor 3 eIF3k gi: 603828S S.143773 Subunitk O.43 heterogeneous nuclear ribonucleoprotein L. HNRPL gi: 5935.937 S.446623 1.01 sirtutin (silent mating type information STRT2 gi: 13775599 S.375214 regulation 2 homolog) 2 (S. cerevisiae) O.70 nuclear factor of kappa light polypeptide NFKBIB gi: 4SOS384 S.9731 gene enhancer in B-cells inhibitor, beta 44.1 O.86 seryl-tRNA synthetase 2 SARS2 gi: 892342O S.14220 44.1 O.12 mitochondrial ribosomal protein S12 MRPS12 gi: 2252149 S.411125 44.1 O.S6 F-box only protein 26 FBXO26 gi: 13376364 S.42S352 44.3 O.40 p21(CDKNIA)-activated kinase 4 PAK4 gi: 7382497 S.20447 44.3 O.83 p21(CDKNIA)-activated kinase 4 PAK4 gi: 41 O1586 S.20447 445 1.16 hypothetical protein F23149 1 PD2 gi: 9506582 S.152894 50.5 3.52 RelA-associated inhibitor RAI gi: 573OOOO S.324051 SO.6 1.27 CD3-epsilon-associated protein; antisense ASE-1 gi: 6912.245 S.446684 to ERCC-1 50.7 3.60 optic atrophy 3 (autosomal recessive, with OPA3 gi: 13376716 S.123473 chorea and spastic parsplegia) SO.8 3.04 echinoderm microtubulo associated EML2 gi: 4568182 S.241.78 protein like 2

US 2009/0297536A1 Dec. 3, 2009 65

TABLE 5-continued Markers of the invention which reside in MCRs of amplification and display increased expression. 19 61.3 0.55 0.039 zinc finger protein 444 ZNF444 gi: 8922893 Hs.24545 19 61.3 O.62 0.027 zinc finger protein 444 ZNF444 gi: 3916863 Hs.24545 19 624 O.69 0.022 zinc finger protein 264 ZNF264 gi: 4585.642 HS.426358 19 624 0.79 <.005 zinc finger protein 272 ZNF272 gi: 498733 HS.99971 19 62.5 0.44 0.043 Zinc finger protein 304 ZNF304 gi: 10190695 Hs.287374 19 626 O.66 0.008 hypothetical protein FLJ23233 FLU23233 gi: 13375967 Hs.98593 19 626 O.60 0.023 hypothetical protein FLJ23233 FLU23233 gi: 2112716 Hs.98593 19 62.8 O.60 0.022 zinc finger protein 134 (clone pHZ-15) ZNF134 gi: 4507982 HS.449971 19 62.8 1.08 <.005 zinc finger protein 211 ZNF211 gi: 5454175 Hs.449970 2O 30.9 O.62 0.008 inhibitor of DNA binding 1, dominant ID gi: 464181 HS.410900 negative helix-loop-helix protein 2O 58.2 O.70 <.005 ATP synthase, H+ transporting, ATP5E gi: 5901895 Hs.177530 mitochondrial F1 complex, epsilon Subunit 2O 58.2 O.65 <.005 chromosome 20 open reading frame 45 C20orf245 gi: 7705609 Hs.3945 2O 59.2 O.68 <.005 protein phosphatase 1, regulatory Subunit PPP1R3D gi: 6806895 Hs.504920 3D 2O 61.2 O.OO 0.025 TAF4 RNA polymerase II, TATA box TAF gi: 5112317 Hs.24644 binding protein (TBP)-associated factor. 135 kDa 2O 61.4 O.45 0.015 synovial sarcoma translocation gene on SS18L1 gi: 3327199 Hs.154429 chromosome 18-like 1 2O 61.4 O.38 0.048 proteasome (prosome, macropain) PSMA7 gi:9408092 Hs.233952 Subunit, alpha type, 7 2O 61.5 O.S9 <.005 oxysterol binding protein-like 2 OSBPL2 gi: 12653.062 Hs.15519 2O 61.5 0.37 0.049 laminin, alpha 5 LAMAS gi: 13097.167 Hs. 11669 2O 61.6 O.O6 <.005 ribosomal protein S21 RPS21 gi: 4506698 HS.372960 2O 62.2 O.49 0.018 transcription factor-like 5 (basic helix- TCFL5 gi: 5730O82 HS.30696 oop-helix) 2O 622 O.39 O.O3 open reading frame 11 C20orf11 gi: 8923556 Hs.103808 2O 62.5 O.82 <.005 chromosome 20 open reading frame 21 C20orf21 gi:9663381 Hs. 11747 2O 63 O46 0.018 ADP-ribosylation factor related protein 1 ARFRP1 gi: 82.46778 Hs.389277 2O 63 O41 O.O32 KIAA1847 KIAA1847 gi: 8246778 Hs. 11900 2O 63 0.37 O.O3 ADP-ribosylation factor related protein 1 ARFRP1 gi: 4507448 HS.389277 2O 63 O.48 O.O16 KIAA1847 KIAA1847 gi: 2279318 Hs. 11900 2O 63.2 0.77 <.005 tumor protein D52-like 2 TPD52L2 gi: 4507642 Hs.154718 2O 6.3.3 0.73 <.005 uridine kinase-like 1 URKL1 gi: 8923486 Hs.504998 2O 6.3.3 0.75 <.005 chromosome 20 open reading frame 14 C20orf14 gi: 13401215 Hs.31334 22 22.6 O.S9 0.031 D-dopachrome tautomerase DDT gi: 5453630 HS.433902 22 22.7 O.42 0.018 glutathione S-transferase theta 1 GSTT1 gi: 4504.184 Hs.268573 22 29.6 0.46 0.015 hypothetical protein FLJ20618 FLU20618 gi: 7021031 Hs.52184 22 29.6 O.65 <.005 hypothetical protein FLJ20618 FLU20618 gi: 4899032 Hs.52184 RefSeq, SEQID NO.: RefSeq, SEQID NO.: Chromosome Locus Link Regulation Probes mRina ID Nuc. Prot ID A.A. 1 10726 UP 201173 x at NM OO6600 445 NP OO6591 1196 1 8458 UP 204407 at NM 003594 1 NP 003585 756 1 10885 UP 218882 s at NM OO6784 2 NP OO6775 757 2 130814 UP 216458 at NM 152391 458 NP 689604 1209 5 6389 UP 201093 x at NM 004168 3 NP 004159 758 5 10016 UP 203415 at NM 013232 4 NP O37364 759 5 11336 UP 212630 at NM OO7277 5 NP OO9208 760 5 55722 UP 219531 at NM 018140 6 NP 060610 761 5 65980 UP 22O155 s at NM 023924 7 NP O76413 762 5 79888 UP 201818 at NM O24830 8 NP O79106 763 5 4726 UP 203606 at NM OO4553 9 NP 004544 764 5 23379 UP 209654 at XM 029101 10 XP 029101 765 6 7148 UP 213451 x at NM 019105; 11: 12 NP 061978; 766; 767 NM 032470 NP 115859 6 10695 UP 217931 at NM OO6586; 13; 14 NP 006577; 768; 769 NM 183010 NP 898828 6 5528 UP 202513 s at NM OO6245; 15; 16; 17 NP OO6236; 770; 771; 772 NM 180976; NP 851307; NM 180977 NP 85.1308 7 2852 UP 211829 s at NM OO1505 18 NP OO1496 773 7 2852 UP 210640 S at NM OO1505 19 NP OO1496 774 7 261.73 UP 212212 s at XM 291222 2O XP 2.91222 775 7 79778 UP 219332 at NM O24723; 21; 22 NP 078.999; 776; 777 NM 182924 NP 891554 7 7975 UP 206750 at NM 002360 23 NP 002351 778 7 8379 UP 204857 at NM 003550 24 NP 003541 779 7 2617 UP 208693 s at NM 002047 25 NP 002038 780

US 2009/0297536A1 Dec. 3, 2009 72

SEQUENCE LISTING The patent application contains a lengthy “Sequence Listing section. A copy of the “Sequence Listing is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090297536A1). An electronic copy of the “Sequence Listing will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

What is claimed is: 15. The method of claim 14, wherein said CGH is per 1. A method of assessing whether a subject is afflicted with formed on an array. cancer or at risk for developing cancer, the method compris 16. The method of claim 10, wherein the level of expres ing comparing the copy number of a minimal common region sion of the marker in the sample is assessed by detecting the (MCR) in a subject sample to the normal copy number of the presence in the sample of a protein corresponding to the MCR, wherein said MCR is selected from the group consist marker. ing of the MCRs listed in Table 1, and wherein an altered copy 17. The method of claim 16, wherein the presence of the number of the MCR in the sample indicates that the subject is protein is detected using a reagent which specifically binds afflicted with cancer or at risk for developing cancer. with the protein. 2. The method of claim 1, wherein the copy number is 18. The method of claim 17, wherein the reagent is selected assessed by fluorescent in situ hybridization (FISH). from the group consisting of an antibody, an antibody deriva 3. The method of claim 1, wherein the copy number is tive, and an antibody fragment. assessed by quantitative PCR (qPCR). 19. The method of claim 10, wherein the level of expres 4. The method of claim 1, wherein the normal copy number sion of the marker in the sample is assessed by detecting the is obtained from a control sample. presence in the sample of a transcribed polynucleotide or 5. A method of assessing whether a subject is afflicted with portion thereof, wherein the transcribed polynucleotide com cancer or at risk for developing cancer, the method compris prises the marker. ing comparing: 20. The method of claim 19, wherein the transcribed poly a) the amount, structure, and/or activity of a marker in a nucleotide is an mRNA. Subject sample, wherein the marker is a marker which 21. The method of claim 19, wherein the transcribed poly resides in an MCR listed in Table 1; and nucleotide is a cDNA. b) the normal amount, structure, and/or activity of the of the marker, 22. The method of claim 19, wherein the step of detecting wherein a significant difference between the amount, struc further comprises amplifying the transcribed polynucleotide. ture, and/or activity of the marker in the sample and the 23. The method of claim 10, wherein the level of expres normal amount, structure, and/or activity is an indica sion of the marker in the sample is assessed by detecting the tion that the subject is afflicted with cancer or at risk for presence in the sample of a transcribed polynucleotide which developing cancer. anneals with the marker or anneals with a portion of a poly 6. The method of claim 5, wherein the marker is selected nucleotide wherein the polynucleotide comprises the marker, from the group consisting of the markers listed in Tables 4 or under stringent hybridization conditions. 5. 24. A method for monitoring the progression of cancer in a 7. The method of claim 5, wherein the amount of a marker Subject, the method comprising: is compared. a) detecting in a subject sample at a first point in time, the 8. The method of claim 5, wherein the structure of a marker amount and/or activity of a marker, wherein the marker is compared. is a marker which resides in an MCR listed in Table 1: 9. The method of claim 5, wherein the activity of a marker b) repeating step a) at a Subsequent point in time; and is compared. c) comparing the amount and/or activity detected in steps 10. The method of claim 7, wherein amount of the marker a) and b), and therefrom monitoring the progression of is determined by determining the level of expression of the cancer in the Subject. marker. 25. The method of claim 24, wherein the marker is selected 11. The method of claim 5, wherein amount of the marker from the group consisting of the markers listed in Tables 4 or is determined by determining copy number of the marker. 5. 12. The method of claim 5, wherein the normal amount/ 26. The method of claim 24, wherein the sample is selected structure, and/or activity is obtained from a control sample. from the group consisting of tissue, whole blood, serum, 13. The method of claims 1 or 5, wherein the sample is plasma, buccal scrape, saliva, cerebrospinal fluid, urine, selected from the group consisting of tissue, whole blood, stool, bile, pancreatic juice, and pancreatic tissue. serum, plasma, buccal scrape, saliva, cerebrospinal fluid, 27. The method of claim 24, wherein the activity of a urine, stool, bile, pancreatic juice, and pancreatic tissue. marker is determined. 14. The method of claim 1 or 11, wherein the copy number 28. The method of claim 24, wherein the amount of a is assessed by comparative genomic hybridization (CGH). marker is determined. US 2009/0297536A1 Dec. 3, 2009

29. The method of claim 28, whereinamount of the marker 44. A method of assessing the efficacy of a therapy for is determined by determining the level of expression of the inhibiting cancer in a subject, the method comprising com marker. paring: 30. The method of claim 28, wherein the level of expres a) the amount and/or activity of a marker in the first sample sion of the marker in the sample is assessed by detecting the obtained from the Subject prior to providing at least a presence in the sample of a protein corresponding to the portion of the therapy to the subject, wherein the marker marker. is a marker which resides in an MCR listed in Table 1, 31. The method of claim 30, wherein the presence of the and protein is detected using a reagent which specifically binds b) the amount and/or activity of the marker in a second with the protein. sample obtained from the subject following provision of 32. The method of claim 31, wherein the reagent is selected the portion of the therapy, from the group consisting of an antibody, an antibody deriva wherein a significantly higher amount and/or activity of the tive, and an antibody fragment. marker in the first sample residing in an MCR which is 33. The method of claim 29, wherein the level of expres deleted in cancer, relative to the second sample, is an sion of the marker in the sample is assessed by detecting the indication that the test compound is efficacious for presence in the sample of a transcribed polynucleotide or inhibiting cancer and wherein a significantly lower portion thereof, wherein the transcribed polynucleotide com amount and/or activity of the marker in the first sample prises the marker. residing in an MCR which is amplified in cancer, relative 34. The method of claim 33, wherein the transcribed poly to the second sample, is an indication that the therapy is nucleotide is an mRNA. efficacious for inhibiting cancer in the Subject. 35. The method of claim 33, wherein the transcribed poly 45. The method of claim 44, wherein the marker is selected nucleotide is a cDNA. from the group consisting of the markers listed in Tables 4 or 36. The method of claim 33, wherein the step of detecting 5. further comprises amplifying the transcribed polynucleotide. 46. A method of selecting a composition capable of modu 37. The method of claim 29, wherein the level of expres lating cancer, the method comprising: sion of the marker in the sample is assessed by detecting the a) obtaining a sample comprising cancer cells; presence in the sample of a transcribed polynucleotide which b) contacting said cells with a test compound; and anneals with the marker or anneals with a portion of a poly c) determining the ability of the test compound to modulate nucleotide wherein the polynucleotide comprises the marker, the amount and/or activity of a marker, wherein the under stringent hybridization conditions. marker is a marker which resides in an MCR listed in 38. The method of claim 24, wherein the sample comprises Table 1, cells obtained from the subject. thereby identifying a modulator of cancer. 39. The method of claim 24, wherein between the first point 47. The method of claim 46, wherein the marker is selected in time and the Subsequent point in time, the Subject has from the group consisting of the markers listed in Tables 4 or undergone treatment for cancer, has completed treatment for 5. cancer, and/or is in remission. 48. The method of claim 46, wherein said cells are isolated 40. A method of assessing the efficacy of a test compound from an animal model of cancer. for inhibiting cancer in a subject, the method comprising 49. The method of claim 46, wherein said cells are from a comparing: cancer cell line. a) the amount and/or activity of a marker in a first sample obtained from the Subject and maintained in the pres 50. The method of claim 46, wherein said cells are from a ence of the test compound, wherein the marker is a Subject Suffering from cancer. marker which resides in an MCR listed in Table 1; and 51. The method of claim 49, wherein said cells are from a b) the amount and/or activity of the marker in a second pancreatic cancer cell line originating from a pancreatic sample obtained from the Subject and maintained in the tumor. absence of the test compound, 52. A method of selecting a composition capable of modu wherein a significantly higher amount and/or activity of a lating cancer, the method comprising: marker in the first sample residing in an MCR which is a) contacting a marker which resides in an MCR listed in deleted in cancer, relative to the second sample, is an Table iwith a test is compound; and indication that the test compound is efficacious for b) determining the ability of the test compound to modulate inhibiting cancer, and wherein a significantly lower the amount and/or activity of a marker which resides in amount and/or activity of a marker in the first sample an MCR listed in Table 1, residing in an MCR which is amplified in cancer, relative thereby identifying a composition capable of modulating to the second sample, is an indication that the test com CaCC. pound is efficacious for inhibiting cancer in the Subject. 53. The method of claim 52, wherein the marker is selected 41. The method of claim 40, wherein the marker is selected from the group consisting of the markers listed in Tables 4 or from the group consisting of the markers listed in Tables 4 or 5. 5. 54. The method of claim 46 or 52, further comprising 42. The method of claim 40, wherein the first and second administering the test compound to an animal model of can samples are portions of a single sample obtained from the C. Subject. 55. The method of claim 46 or 52, wherein the modulator 43. The method of claim 40, wherein the first and second inhibits amount and/or activity of a gene or protein corre samples are portions of pooled samples obtained from the sponding to a marker selected from the markers listed in Table Subject. 5. US 2009/0297536A1 Dec. 3, 2009 74

56. The method of claim 46 or 52, wherein the modulator 76. The method of claim 75, wherein said small molecule increases the amount and/or activity of a gene or protein inhibits a protein-protein interaction between a marker and a corresponding to a marker selected from the markers listed in target protein. Table 4. 77. The method of claim 66, wherein said compound is an 57. A kit for assessing the ability of a compound to inhibit aptamer which inhibits expression or activity of said marker. cancer, the kit comprising a reagent for assessing the amount, 78. The method of claim 66, wherein the marker is selected from the group consisting of the markers listed in Table 5. structure, and/or activity of a marker which resides in an 79. A method of treating a subject afflicted with cancer MCR listed in Table 1. comprising administering to the Subject a compound which 58. A kit for assessing whether a subject is afflicted with increases expression or activity of a gene or protein corre cancer, the kit comprising a reagent for assessing the copy sponding to a marker which resides in an MCR listed in Table number of an MCR selected from the group consisting of the 1 which is deleted in cancer, thereby treating a subject MCRS listed in Table 1. afflicted with cancer. 59. A kit for assessing whether a subject is afflicted with 80. A method of treating a subject afflicted with cancer cancer, the kit comprising a reagent for assessing the amount, comprising administering to the Subject a protein correspond structure, and/or activity of a markerwhich resides in an MCR ing to a marker which resides in an MCR listed in Table 1 listed in Table 1. which is deleted in cancer, thereby treating a subject afflicted 60. A kit for assessing the presence of human cancer cells, with cancer. 81. The method of any one of claims 79 or 80, wherein the the kit comprising an antibody or fragment thereof, wherein marker is selected from the group consisting of the markers the antibody or fragment thereof specifically binds with a listed in Table 4. protein corresponding to a marker which resides in an MCR 82. The method of claim 80, wherein the protein is pro listed in Table 1. vided to the cells of the Subject, by a vector comprising a 61. A kit for assessing the presence of cancer cells, the kit polynucleotide encoding the protein. comprising a nucleic acid probe wherein the probe specifi 83. The method of claim 79, wherein said compound is cally binds with a transcribed polynucleotide corresponding administered in a pharmaceutically acceptable formulation. to a marker which resides in an MCR listed in Table 1. 84. An isolated protein, or fragment thereof, corresponding to a marker selected from the markers listed in Table 4 or 62. The kit of claim 61, wherein the nucleic acid probe is a Table 5. molecular beacon probe. 85. An isolated nucleic acid molecule, or fragment thereof, 63. The kit of any one of claims 57, 59, 60, or 61, wherein corresponding to a marker selected from the markers listed in the marker is selected from the group consisting of the mark Table 4 or Table 5. ers listed in Tables 4 or 5. 86. An isolated antibody, or fragment thereof, which spe 64. A method of treating a subject afflicted with cancer cifically binds to a protein corresponding to a marker selected comprising administering to the Subject a modulator of from the markers listed in Table 4 or Table 5. amount and/or activity of a gene or protein corresponding to 87. An isolated nucleic acid molecule, or fragment thereof, a marker which resides in an MCR listed in Table 1, thereby contained within an MCR selected from the MCRs listed in treating a Subject afflicted with cancer. Table 1, wherein said nucleic acid molecule has an altered amount, structure, and/or activity in cancer. 65. The method of claim 64, wherein the marker is selected 88. An isolated polypeptide encoded by the nucleic acid from the group consisting of the markers listed in Tables 4 or molecule of claim 87. 5. 89. A method for identifying a marker associated with 66. A method of treating a subject afflicted with cancer cancer, said method comprising: comprising administering to the Subject a compound which a) performing profiling of the genome of cancer cells; inhibits the amount and/or activity of a gene or protein cor b) performing segmentation analysis of profiles identified responding to a marker which resides in an MCR listed in in step a); Table 1 which is amplified in cancer, thereby treating a sub c) identifying loci; ject afflicted with cancer. d) prioritizing said identified loci; and 67. The method of claim 66, wherein said compound is e) interrogating genes in the identified loci, administered in a pharmaceutically acceptable formulation. to thereby identify a marker associated with cancer. 68. The method of claim 66, wherein said compound is an 90. A method for identifying a locus associated with can antibody or an antigen binding fragment thereof, which spe cer, said method comprising the steps of cifically binds to a protein corresponding to said marker. a) performing profiling of the genome of cancer cells; 69. The method of claim 68, wherein said antibody is b) performing segmentation analysis of profiles identified conjugated to a toxin. in step a); 70. The method of claim 68, wherein said antibody is c) identifying loci; and conjugated to a chemotherapeutic agent. d) prioritizing said identified loci, to thereby identify a locus associated with cancer. 71. The method of claim 66, wherein said compound is an 91. The method of claim 89, wherein said interrogation of RNA interfering agent which inhibits expression of a gene genes in the identified loci is based on gene expression data. corresponding to said marker. 92. The method of claim 89, wherein said interrogation of 72. The method of claim 71, wherein said RNA interfering genes in the identified loci is based on in vitro Screening agent is an siRNA molecule oran shRNA molecule. assayS. 73. The method of claim 66, wherein said compound is an 93. The method of claim 89 or 90, wherein said profiling is antisense oligonucleotide complementary to a gene corre performed using comparative genomic hybridization (CGH). sponding to said marker. 94. The method of claim 89 or 90, wherein said cancer cells 74. The method of claim 66, wherein said compound is a are derived from a cancer cell line or a tumor. peptide or peptidomimetic. 95. A marker identified by the method of claim 89. 75. The method of claim 66, wherein said compound is a small molecule which inhibits activity of said marker. c c c c c