US20180334722A1

US20180334722A1 - Gene signatures for cancer prognosis

Info

Publication number: US20180334722A1
Application number: US15/921,416
Authority: US
Inventors: Steven Stone; Alexander Gutin; Susanne Wagner; Julia E. Reid
Original assignee: Inc Myriad Genetics; Myriad Genetics Inc
Current assignee: Inc Myriad Genetics; Myriad Genetics Inc
Priority date: 2010-07-07
Filing date: 2018-03-14
Publication date: 2018-11-22
Also published as: EP2591126A2; EP2591126B1; US20160355884A1; EP3812469A1; US20150167103A1; US20120053253A1; US10954568B2; EP2591126A4; WO2012006447A2; WO2012006447A3; US20220259675A1; CA2804391A1

Abstract

Biomarkers and methods using the biomarkers for the prediction of the recurrence risk of cancer in a patient are provided.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 15/060,090 (filed on Mar. 3, 2016), which is a continuation of U.S. application Ser. No. 14/632,888 (filed on Feb. 26, 2015), which is a continuation of U.S. application Ser. No. 13/178,380 (filed on Jul. 7, 2011), which claims the priority benefit of U.S. Provisional Application Ser. No. 61/362,209 (filed on Jul. 7, 2010), each of which are hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention generally relates to a molecular classification of disease and particularly to molecular markers for cancer prognosis and methods of use thereof.

BACKGROUND OF THE INVENTION

Cancer is a major public health problem, accounting for roughly 25% of all deaths in the United States. Though many treatments have been devised for various cancers, these treatments often vary in severity of side effects. It is useful for clinicians to know how aggressive a patient's cancer is in order to determine how aggressively to treat the cancer.
For example, most patients with early-stage asymptomatic prostate cancer are treated with radical prostatectomy or radiotherapy and optionally adjuvant therapy (e.g., hormone or chemotherapy), all of which have severe side effects. For many of these patients, however, these treatments and their associated side effects and costs are unnecessary because the cancer in these patients is not aggressive (i.e., grows slowly and is unlikely to cause mortality or significant morbidity during the patient's lifetime). In other patients the cancer is virulent (i.e., more likely to recur) and aggressive treatment is necessary to save the patient's life.
Some tools have been devised to help physicians in deciding which patients need aggressive treatment and which do not. In fact, several clinical parameters are currently in use for this purpose in various different cancers. In prostate cancer, for example, such clinical parameters include serum prostate-specific antigen (PSA), Gleason grade, pathologic stage, and surgical margins. In recent years clinical parameters have been made more helpful through their incorporation into continuous multivariable postoperative nomograms that calculate a patient's probability of having cancer progression/recurrence. See, e.g., Kattan et al., J. CLIN. ONCOL. (1999) 17:1499-1507; Stephenson et al., J. CLIN. ONCOL. (2005) 23:7005-7012. Despite these advances, however, many patients are given improper cancer treatments and there is still a serious need for novel and improved tools for predicting cancer recurrence.

SUMMARY OF THE INVENTION

The present invention is based in part on the surprising discovery that the expression of those genes whose expression closely tracks the cell cycle (“cell-cycle genes” or “CCGs” as further defined below) is particularly useful in classifying selected types of cancer and determining the prognosis of these cancers.
Accordingly, in a first aspect of the present invention, a method is provided for determining gene expression in a tumor sample from a patient identified as having prostate cancer, lung cancer, bladder cancer or brain cancer. Generally, the method includes at least the following steps: (1) obtaining a tumor sample from a patient identified as having prostate cancer, lung cancer, bladder cancer or brain cancer; (2) determining the expression of a panel of genes in said tumor sample including at least 4 cell-cycle genes; and (3) providing a test value by (a) weighting the determined expression of each of a plurality of test genes selected from said panel of genes with a predefined coefficient, and (b) combining the weighted expression to provide said test value, wherein at least 50%, at least 75% or at least 90% of said plurality of test genes are cell-cycle genes.
In preferred embodiments, the plurality of test genes includes at least 8 cell-cycle genes, or at least 10, 15, 20, 25 or 30 cell-cycle genes. Preferably, all of the test genes are cell-cycle genes.
Also in preferred embodiments, the step of determining the expression of the panel of genes in the tumor sample comprises measuring the amount of mRNA in the tumor sample transcribed from each of from 4 to about 200 cell-cycle genes; and measuring the amount of mRNA of one or more housekeeping genes in the tumor sample.
In another aspect of the present invention, a method is provided for determining the prognosis of prostate cancer, lung cancer, bladder cancer or brain cancer, which comprises determining in a tumor sample from a patient diagnosed of prostate cancer, lung cancer, bladder cancer or brain cancer, the expression of at least 6, 8 or 10 cell-cycle genes, wherein overexpression of said at least 6, 8 or 10 cell-cycle genes indicates a poor prognosis or an increased likelihood of recurrence of cancer in the patient.
In one embodiment, the prognosis method comprises (1) determining in a tumor sample from a patient diagnosed of prostate cancer, lung cancer, bladder cancer or brain cancer, the expression of a panel of genes in said tumor sample including at least 4 or at least 8 cell-cycle genes; and (2) providing a test value by (a) weighting the determined expression of each of a plurality of test genes selected from the panel of genes with a predefined coefficient, and (b) combining the weighted expression to provide the test value, wherein at least 50%, at least 75% or at least 85% of the plurality of test genes are cell-cycle genes, and wherein an increased level of overall expression of the plurality of test genes indicates a poor prognosis, whereas if there is no increase in the overall expression of the test genes, it would indicate a good prognosis or a low likelihood of recurrence of cancer in the patient.
In preferred embodiments, the prognosis method further includes a step of comparing the test value provided in step (2) above to one or more reference values, and correlating the test value to a risk of cancer progression or risk of cancer recurrence. Optionally an increased likelihood of poor prognosis is indicated if the test value is greater than the reference value.
In yet another aspect, the present invention also provide a method of treating cancer in a patient identified as having prostate cancer, lung cancer, bladder cancer or brain cancer, comprising: (1) determining in a tumor sample from a patient diagnosed of prostate cancer, lung cancer, bladder cancer or brain cancer, the expression of a panel of genes in the tumor sample including at least 4 or at least 8 cell-cycle genes; (2) providing a test value by (a) weighting the determined expression of each of a plurality of test genes selected from said panel of genes with a predefined coefficient, and (b) combining the weighted expression to provide said test value, wherein at least 50% or 75% or 85% of the plurality of test genes are cell-cycle genes, wherein an increased level of expression of the plurality of test genes indicates a poor prognosis, and an un-increased level of expression of the plurality of test genes indicates a good prognosis; and recommending, prescribing or administering a treatment regimen or watchful waiting based on the prognosis provided in step (2).
The present invention further provides a diagnostic kit for prognosing cancer in a patient diagnosed of prostate cancer, lung cancer, bladder cancer or brain cancer, comprising, in a compartmentalized container, a plurality of oligonucleotides hybridizing to at least 8 test genes, wherein less than 10%, 30% or less than 40% of all of the at least 8 test genes are non-cell-cycle genes; and one or more oligonucleotide hybridizing to at least one housekeeping gene. The oligonucleotides can be hybridizing probes for hybridization with the test genes under stringent conditions or primers suitable for PCR amplification of the test genes. In one embodiment, the kit consists essentially of, in a compartmentalized container, a first plurality of PCR reaction mixtures for PCR amplification of from 5 or 10 to about 300 test genes, wherein at least 50%, at least 60% or at least 80% of such test genes are cell-cycle genes, and wherein each reaction mixture comprises a PCR primer pair for PCR amplifying one of the test genes; and a second plurality of PCR reaction mixtures for PCR amplification of at least one housekeeping gene.
The present invention also provides the use of (1) a plurality of oligonucleotides hybridizing to at least 4 or at least 8 cell-cycle genes; and (2) one or more oligonucleotides hybridizing to at least one housekeeping gene, for the manufacture of a diagnostic product for determining the expression of the test genes in a tumor sample from a patient diagnosed of prostate cancer, lung cancer, bladder cancer or brain cancer, to predict the prognosis of cancer, wherein an increased level of the overall expression of the test genes indicates a poor prognosis or an increased likelihood of recurrence of cancer in the patient, whereas if there is no increase in the overall expression of the test genes, it would indicate a good prognosis or a low likelihood of recurrence of cancer in the patient. In some embodiments, the oligonucleotides are PCR primers suitable for PCR amplification of the test genes. In other embodiments, the oligonucleotides are probes hybridizing to the test genes under stringent conditions. In some embodiments, the plurality of oligonucleotides are probes for hybridization under stringent conditions to, or are suitable for PCR amplification of, from 4 to about 300 test genes, at least 50%, 70% or 80% or 90% of the test genes being cell-cycle genes. In some other embodiments, the plurality of oligonucleotides are hybridization probes for, or are suitable for PCR amplification of, from 20 to about 300 test genes, at least 30%, 40%, 50%, 70% or 80% or 90% of the test genes being cell-cycle genes.
The present invention further provides a system for determining gene expression in a tumor sample, comprising: (1) a sample analyzer for determining the expression levels of a panel of genes in a tumor sample including at least 4 cell-cycle genes, wherein the sample analyzer contains the tumor sample which is from a patient identified as having prostate cancer, lung cancer, bladder cancer or brain cancer, or cDNA molecules from mRNA expressed from the panel of genes; (2) a first computer program means for (a) receiving gene expression data on at least 4 test genes selected from the panel of genes, (b) weighting the determined expression of each of the test genes with a predefined coefficient, and (c) combining the weighted expression to provide a test value, wherein at least 50%, at least at least 75% of at least 4 test genes are cell-cycle genes; and optionally (3) a second computer program means for comparing the test value to one or more reference values each associated with a predetermined degree of risk of cancer recurrence or progression of the prostate cancer, lung cancer, bladder cancer or brain cancer. In some embodiments, the system further comprises a display module displaying the comparison between the test value to the one or more reference values, or displaying a result of the comparing step.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following Detailed Description, and from the Claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the predictive power over nomogram for CCG panels of different sizes.

FIG. 2 is an illustration of CCGs predicting time to recurrence.

FIG. 3 is an illustration of nomogram predicting time to recurrence.

FIG. 4 is an illustration of the non-overlapping recurrence predicted by nomogram and a CCG signature.

FIG. 5 is an illustration of time to recurrence for several patient populations defined by nomogram and/or CCG status.

FIG. 6 is an illustration of an example of a system useful in certain aspects and embodiments of the invention.

FIG. 7 is a flowchart illustrating an example of a computer-implemented method of the invention.

FIG. 8 a scatter plot comparing clinical parameters and CCG score as predictors of recurrence from Example 5.

FIG. 9 illustrates, from Example 5, the CCG threshold derived from analysis of the training cohort to the validation data set, with the CCG signature score effectively subdividing patients identified as low-risk using clinical parameters into patients with very low recurrence rates and a higher risk of recurrence.

FIG. 10 illustrates the predicted recurrence rate versus CCG score for patients in the validation cohort of Example 5.

FIG. 11 illustrates the predicted recurrence rate versus CCG score for patients in the validation cohort of Example 5.

FIG. 12 illustrates the distribution of clinical risk score in 443 patients studied in Example 5. The dark vertical line represents the threshold chosen by KM means to divide low- and high-risk patients and used throughout this study.

FIG. 13 illustrates the correlation between CCP score and survival in brain cancer.

FIG. 14 illustrates illustrates the correlation between CCP score and survival in bladder cancer.

FIG. 15 illustrates illustrates the correlation between CCP score and survival in breast cancer.

FIG. 16 illustrates the correlation between CCP score and survival in lung cancer.

FIG. 17 is an illustration of the predictive power over nomogram for CCG panels of different sizes.

DETAILED DESCRIPTION OF THE INVENTION

I. Determining Cell-Cycle Gene Expression

The present invention is based in part on the discovery that genes whose expression closely tracks the cell cycle (“cell-cycle genes” or “CCGs”) are particularly powerful genes for classifying selected cancers including prostate cancer, lung cancer, bladder cancer, brain cancer and breast cancer, but not other types of cancer such as colorectal cancer.
“Cell-cycle gene” and “CCG” herein refer to a gene whose expression level closely tracks the progression of the cell through the cell-cycle. See, e.g., Whitfield et al., MOL. BIOL. CELL (2002) 13:1977-2000. The term “cell-cycle progression” or “CCP” will also be used in this application and will generally be interchangeable with CCG (i.e., a CCP gene is a CCG; a CCP score is a CCG score). More specifically, CCGs show periodic increases and decreases in expression that coincide with certain phases of the cell cycle—e.g., STK15 and PLK show peak expression at G2/M. Id. Often CCGs have clear, recognized cell-cycle related function—e.g., in DNA synthesis or repair, in chromosome condensation, in cell-division, etc. However, some CCGs have expression levels that track the cell-cycle without having an obvious, direct role in the cell-cycle—e.g., UBE2S encodes a ubiquitin-conjugating enzyme, yet its expression closely tracks the cell-cycle. Thus a CCG according to the present invention need not have a recognized role in the cell-cycle. Exemplary CCGs are listed in Tables 1, 2, 3, and 4.
Whether a particular gene is a CCG may be determined by any technique known in the art, including that taught in Whitfield et al., MOL. BIOL. CELL (2002) 13:1977-2000. For example, a sample of cells, e.g., HeLa cells, can be synchronized such that they all progress through the different phases of the cell cycle at the same time. Generally this is done by arresting the cells in each phase—e.g., cells may be arrested in S phase by using a double thymidine block or in mitosis with a thymidine-nocodazole block. See, e.g., Whitfield et al., MOL. CELL. BIOL. (2000) 20:4188-4198. RNA is extracted from the cells after arrest in each phase and gene expression is quantitated using any suitable technique—e.g., expression microarray (genome-wide or specific genes of interest), real-time quantitative PCR™ (RTQ-PCR). Finally, statistical analysis (e.g., Fourier Transform) is applied to determine which genes show peak expression during particular cell-cycle phases. Genes may be ranked according to a periodicity score describing how closely the gene's expression tracks the cell-cycle—e.g., a high score indicates a gene very closely tracks the cell cycle. Finally, those genes whose periodicity score exceeds a defined threshold level (see Whitfield et al., MOL. BIOL. CELL (2002) 13:1977-2000) may be designated CCGs. A large, but not exhaustive, list of nucleic acids associated with CCGs (e.g., genes, ESTs, cDNA clones, etc.) is given in Table 1. See Whitfield et al., MOL. BIOL. CELL (2002) 13:1977-2000. All of the CCGs in Table 2 below form a panel of CCGs (“Panel A”) useful in the methods of the invention.

TABLE 2

	Entrez		RefSeq Accession
Gene Symbol	GeneID	ABI Assay ID	Nos.

APOBEC3B*	9582	Hs00358981_m1	NM_004900.3
ASF1B*	55723	Hs00216780_m1	NM_018154.2
ASPM*	259266	Hs00411505_m1	NM_018136.4
ATAD2*	29028	Hs00204205_m1	NM_014109.3
BIRC5*	332	Hs00153353_m1;	NM_001012271.1;
		Hs03043576_m1	NM_001012270.1;
			NM_001168.2
BLM*	641	Hs00172060_m1	NM_000057.2
BUB1	699	Hs00177821_m1	NM_004336.3
BUB1B*	701	Hs01084828_m1	NM_001211.5
C12orf48*	55010	Hs00215575_m1	NM_017915.2
C18orf24*	220134	Hs00536843_m1	NM_145060.3;
			NM_001039535.2
C1orf135*	79000	Hs00225211_m1	NM_024037.1
C21orf45*	54069	Hs00219050_m1	NM_018944.2
CCDC99*	54908	Hs00215019_m1	NM_017785.4
CCNA2*	890	Hs00153138_m1	NM_001237.3
CCNB1*	891	Hs00259126_m1	NM_031966.2
CCNB2*	9133	Hs00270424_m1	NM_004701.2
CCNE1*	898	Hs01026536_m1	NM_001238.1;
			NM_057182.1
CDC2*	983	Hs00364293_m1	NM_033379.3;
			NM_001130829.1;
			NM_001786.3
CDC20*	991	Hs03004916_g1	NM_001255.2
CDC45L*	8318	Hs00185895_m1	NM_003504.3
CDC6*	990	Hs00154374_m1	NM_001254.3
CDCA3*	83461	Hs00229905_m1	NM_031299.4
CDCA8*	55143	Hs00983655_m1	NM_018101.2
CDKN3*	1033	Hs00193192_m1	NM_001130851.1;
			NM_005192.3
CDT1*	81620	Hs00368864_m1	NM_030928.3
CENPA	1058	Hs00156455_m1	NM_001042426.1;
			NM_001809.3
CENPE*	1062	Hs00156507_m1	NM_001813.2
CENPF*	1063	Hs00193201_m1	NM_016343.3
CENPI*	2491	Hs00198791_m1	NM_006733.2
CENPM*	79019	Hs00608780_m1	NM_024053.3
CENPN*	55839	Hs00218401_m1	NM_018455.4;
			NM_001100624.1;
			NM_001100625.1
CEP55*	55165	Hs00216688_m1	NM_018131.4;
			NM_001127182.1
CHEK1*	1111	Hs00967506_m1	NM_001114121.1;
			NM_001114122.1;
			NM_001274.4
CKAP2*	26586	Hs00217068_m1	NM_018204.3;
			NM_001098525.1
CKS1B*	1163	Hs01029137_g1	NM_001826.2
CKS2*	1164	Hs01048812_g1	NM_001827.1
CTPS*	1503	Hs01041851_m1	NM_001905.2
CTSL2*	1515	Hs00952036_m1	NM_001333.2
DBF4*	10926	Hs00272696_m1	NM_006716.3
DDX39*	10212	Hs00271794_m1	NM_005804.2
DLGAP5/	9787	Hs00207323_m1	NM_014750.3
DLG7*
DONSON*	29980	Hs00375083_m1	NM_017613.2
DSN1*	79980	Hs00227760_m1	NM_024918.2
DTL*	51514	Hs00978565_m1	NM_016448.2
E2F8*	79733	Hs00226635_m1	NM_024680.2
ECT2*	1894	Hs00216455_m1	NM_018098.4
ESPL1*	9700	Hs00202246_m1	NM_012291.4
EXO1*	9156	Hs00243513_m1	NM_130398.2;
			NM_003686.3;
			NM_006027.3
EZH2*	2146	Hs00544830_m1	NM_152998.1;
			NM_004456.3
FANCI*	55215	Hs00289551_m1	NM_018193.2;
			NM_001113378.1
FBXO5*	26271	Hs03070834_m1	NM_001142522.1;
			NM_012177.3
FOXM1*	2305	Hs01073586_m1	NM_202003.1;
			NM_202002.1;
			NM_021953.2
GINS1*	9837	Hs00221421_m1	NM_021067.3
GMPS*	8833	Hs00269500_m1	NM_003875.2
GPSM2*	29899	Hs00203271_m1	NM_013296.4
GTSE1*	51512	Hs00212681_m1	NM_016426.5
H2AFX*	3014	Hs00266783_s1	NM_002105.2
HMMR*	3161	Hs00234864_m1	NM_001142556.1;
			NM_001142557.1;
			NM_012484.2;
			NM_012485.2
HN1*	51155	Hs00602957_m1	NM_001002033.1;
			NM_001002032.1;
			NM_016185.2
KIAA0101*	9768	Hs00207134_m1	NM_014736.4
KIF11*	3832	Hs00189698_m1	NM_004523.3
KIF15*	56992	Hs00173349_m1	NM_020242.2
KIF18A*	81930	Hs01015428_m1	NM_031217.3
KIF20A*	10112	Hs00993573_m1	NM_005733.2
KIF20B/	9585	Hs01027505_m1	NM_016195.2
MPHOSPH1*
KIF23*	9493	Hs00370852_m1	NM_138555.1;
			NM_004856.4
KIF2C*	11004	Hs00199232_m1	NM_006845.3
KIF4A*	24137	Hs01020169_m1	NM_012310.3
KIFC1*	3833	Hs00954801_m1	NM_002263.3
KPNA2	3838	Hs00818252_g1	NM_002266.2
LMNB2*	84823	Hs00383326_m1	NM_032737.2
MAD2L1	4085	Hs01554513_g1	NM_002358.3
MCAM*	4162	Hs00174838_m1	NM_006500.2
MCM10*	55388	Hs00960349_m1	NM_018518.3;
			NM_182751.1
MCM2*	4171	Hs00170472_m1	NM_004526.2
MCM4*	4173	Hs00381539_m1	NM_005914.2;
			NM_182746.1
MCM6*	4175	Hs00195504_m1	NM_005915.4
MCM7*	4176	Hs01097212_m1	NM_005916.3;
			NM_182776.1
MELK	9833	Hs00207681_m1	NM_014791.2
MKI67*	4288	Hs00606991_m1	NM_002417.3
MYBL2*	4605	Hs00231158_m1	NM_002466.2
NCAPD2*	9918	Hs00274505_m1	NM_014865.3
NCAPG*	64151	Hs00254617_m1	NM_022346.3
NCAPG2*	54892	Hs00375141_m1	NM_017760.5
NCAPH*	23397	Hs01010752_m1	NM_015341.3
NDC80*	10403	Hs00196101_m1	NM_006101.2
NEK2*	4751	Hs00601227_mH	NM_002497.2
NUSAP1*	51203	Hs01006195_m1	NM_018454.6;
			NM_001129897.1;
			NM_016359.3
OIP5*	11339	Hs00299079_m1	NM_007280.1
ORC6L*	23594	Hs00204876_m1	NM_014321.2
PAICS*	10606	Hs00272390_m1	NM_001079524.1;
			NM_001079525.1;
			NM_006452.3
PBK*	55872	Hs00218544_m1	NM_018492.2
PCNA*	5111	Hs00427214_g1	NM_182649.1;
			NM_002592.2
PDSS1*	23590	Hs00372008_m1	NM_014317.3
PLK1*	5347	Hs00153444_m1	NM_005030.3
PLK4*	10733	Hs00179514_m1	NM_014264.3
POLE2*	5427	Hs00160277_m1	NM_002692.2
PRC1*	9055	Hs00187740_m1	NM_199413.1;
			NM_199414.1;
			NM_003981.2
PSMA7*	5688	Hs00895424_m1	NM_002792.2
PSRC1*	84722	Hs00364137_m1	NM_032636.6;
			NM_001005290.2;
			NM_001032290.1;
			NM_001032291.1
PTTG1*	9232	Hs00851754_u1	NM_004219.2
RACGAP1*	29127	Hs00374747_m1	NM_013277.3
RAD51*	5888	Hs00153418_m1	NM_133487.2;
			NM_002875.3
RAD51AP1*	10635	Hs01548891_m1	NM_001130862.1;
			NM_006479.4
RAD54B*	25788	Hs00610716_m1	NM_012415.2
RAD54L*	8438	Hs00269177_m1	NM_001142548.1;
			NM_003579.3
RFC2*	5982	Hs00945948_m1	NM_181471.1;
			NM_002914.3
RFC4*	5984	Hs00427469_m1	NM_181573.2;
			NM_002916.3
RFC5*	5985	Hs00738859_m1	NM_181578.2;
			NM_001130112.1;
			NM_001130113.1;
			NM_007370.4
RNASEH2A*	10535	Hs00197370_m1	NM_006397.2
RRM2*	6241	Hs00357247_g1	NM_001034.2
SHCBP1*	79801	Hs00226915_m1	NM_024745.4
SMC2*	10592	Hs00197593_m1	NM_001042550.1;
			NM_001042551.1;
			NM_006444.2
SPAG5*	10615	Hs00197708_m1	NM_006461.3
SPC25*	57405	Hs00221100_m1	NM_020675.3
STIL*	6491	Hs00161700_m1	NM_001048166.1;
			NM_003035.2
STMN1*	3925	Hs00606370_m1;	NM_005563.3;
		Hs01033129_m1	NM_203399.1
TACC3*	10460	Hs00170751_m1	NM_006342.1
TIMELESS*	8914	Hs01086966_m1	NM_003920.2
TK1*	7083	Hs01062125_m1	NM_003258.4
TOP2A*	7153	Hs00172214_m1	NM_001067.2
TPX2*	22974	Hs00201616_m1	NM_012112.4
TRIP13*	9319	Hs01020073_m1	NM_004237.2
TTK*	7272	Hs00177412_m1	NM_003318.3
TUBA1C*	84790	Hs00733770_m1	NM_032704.3
TYMS*	7298	Hs00426591_m1	NM_001071.2
UBE2C	11065	Hs00964100_g1	NM_181799.1;
			NM_181800.1;
			NM_181801.1;
			NM_181802.1;
			NM_181803.1;
			NM_007019.2
UBE2S	27338	Hs00819350_m1	NM_014501.2
VRK1*	7443	Hs00177470_m1	NM_003384.2
ZWILCH*	55055	Hs01555249_m1	NM_017975.3;
			NR_003105.1
ZWINT*	11130	Hs00199952_m1	NM_032997.2;
			NM_001005413.1;
			NM_007057.3

*124-gene subset of CCGs useful in the invention (“Panel B”). ABI Assay ID means the catalogue ID number for the gene expression assay commercially available from Applied Biosystems Inc. (Foster City, CA) for the particular gene.

Accordingly, in a first aspect of the present invention, a method is provided for determining gene expression in a tumor sample from a patient identified as having prostate cancer, lung cancer, bladder cancer or brain cancer. Generally, the method includes at least the following steps: (1) obtaining a tumor sample from a patient identified as having prostate cancer, lung cancer, bladder cancer or brain cancer; (2) determining the expression of a panel of genes in the tumor sample including at least 2, 4, 6, 8 or 10 cell-cycle genes; and (3) providing a test value by (a) weighting the determined expression of each of a plurality of test genes selected from said panel of genes with a predefined coefficient, and (b) combining the weighted expression to provide said test value, wherein at least 20%, 50%, at least 75% or at least 90% of said plurality of test genes are cell-cycle genes.
Gene expression can be determined either at the RNA level (i.e., mRNA or noncoding RNA (ncRNA)) (e.g., miRNA, tRNA, rRNA, snoRNA, siRNA and piRNA) or at the protein level. Levels of proteins in a tumor sample can be determined by any known techniques in the art, e.g., HPLC, mass spectrometry, or using antibodies specific to selected proteins (e.g., IHC, ELISA, etc.).
In preferred embodiment, the amount of RNA transcribed from the panel of genes including test genes is measured in the tumor sample. In addition, the amount of RNA of one or more housekeeping genes in the tumor sample is also measured, and used to normalize or calibrate the expression of the test genes. The terms “normalizing genes” and “housekeeping genes” are defined herein below.
In some embodiments, the plurality of test genes includes at least 2, 3 or 4 cell-cycle genes, which constitute at least 50%, 75% or 80% of the plurality of test genes, and preferably 100% of the plurality of test genes. In some embodiments, the plurality of test genes includes at least 5, 6, 7, or at least 8 cell-cycle genes, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes. As will be clear from the context of this document, a panel of genes is a plurality of genes. Typically these genes are assayed together in one or more samples from a patient.
In some other embodiments, the plurality of test genes includes at least 8, 10, 12, 15, 20, 25 or 30 cell-cycle genes, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes.
As will be apparent to a skilled artisan apprised of the present invention and the disclosure herein, “tumor sample” means any biological sample containing one or more tumor cells, or one or more tumor derived RNA or protein, and obtained from a cancer patient. For example, a tissue sample obtained from a tumor tissue of a cancer patient is a useful tumor sample in the present invention. The tissue sample can be an FFPE sample, or fresh frozen sample, and preferably contain largely tumor cells. A single malignant cell from a cancer patient's tumor is also a useful tumor sample. Such a malignant cell can be obtained directly from the patient's tumor, or purified from the patient's bodily fluid such as blood and urine. In addition, a bodily fluid such as blood, urine, sputum and saliva containing one or tumor cells, or tumor-derived RNA or proteins, can also be useful as a tumor sample for purposes of practicing the present invention.
Those skilled in the art are familiar with various techniques for determining the status of a gene or protein in a tissue or cell sample including, but not limited to, microarray analysis (e.g., for assaying mRNA or microRNA expression, copy number, etc.), quantitative real-time PCR™ (“qRT-PCR™”, e.g., TaqMan™), immunoanalysis (e.g., ELISA, immunohistochemistry), etc. The activity level of a polypeptide encoded by a gene may be used in much the same way as the expression level of the gene or polypeptide. Often higher activity levels indicate higher expression levels and while lower activity levels indicate lower expression levels. Thus, in some embodiments, the invention provides any of the methods discussed above, wherein the activity level of a polypeptide encoded by the CCG is determined rather than or in addition to the expression level of the CCG. Those skilled in the art are familiar with techniques for measuring the activity of various such proteins, including those encoded by the genes listed in Tables 1, 2, 3, and 4. The methods of the invention may be practiced independent of the particular technique used.
In preferred embodiments, the expression of one or more normalizing genes is also obtained for use in normalizing the expression of test genes. As used herein, “normalizing genes” referred to the genes whose expression is used to calibrate or normalize the measured expression of the gene of interest (e.g., test genes). Importantly, the expression of normalizing genes should be independent of cancer outcome/prognosis, and the expression of the normalizing genes is very similar among all the tumor samples. The normalization ensures accurate comparison of expression of a test gene between different samples. For this purpose, housekeeping genes known in the art can be used. Housekeeping genes are well known in the art, with examples including, but are not limited to, GUSB (glucuronidase, beta), HMBS (hydroxymethylbilane synthase), SDHA (succinate dehydrogenase complex, subunit A, flavoprotein), UBC (ubiquitin C) and YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide). One or more housekeeping genes can be used. Preferably, at least 2, 5, 10 or 15 housekeeping genes are used to provide a combined normalizing gene set. The amount of gene expression of such normalizing genes can be averaged, combined together by straight additions or by a defined algorithm. Some examples of particularly useful housekeeper genes for use in the methods and compositions of the invention include those listed in Table A below.

TABLE A

	Entrez	Applied Biosystems	RefSeq
Gene Symbol	GeneID	Assay ID	Accession Nos.

CLTC*	1213	Hs00191535_m1	NM_004859.3
GUSB	2990	Hs99999908_m1	NM_000181.2
HMBS	3145	Hs00609297_m1	NM_000190.3
MMADHC*	27249	Hs00739517_g1	NM_015702.2
MRFAP1*	93621	Hs00738144_g1	NM_033296.1
PPP2CA*	5515	Hs00427259_m1	NM_002715.2
PSMA1*	5682	Hs00267631_m1
PSMC1*	5700	Hs02386942_g1	NM_002802.2
RPL13A*	23521	Hs03043885_g1	NM_012423.2
RPL37*	6167	Hs02340038_g1	NM_000997.4
RPL38*	6169	Hs00605263_g1	NM_000999.3
RPL4*	6124	Hs03044647_g1	NM_000968.2
RPL8*	6132	Hs00361285_g1	NM_033301.1;
			NM_000973.3
RPS29*	6235	Hs03004310_g1	NM_001030001.1;
			NM_001032.3
SDHA	6389	Hs00188166_m1	NM_004168.2
SLC25A3*	6515	Hs00358082_m1	NM_213611.1;
			NM_002635.2;
			NM_005888.2
TXNL1*	9352	Hs00355488_m1	NR_024546.1;
			NM_004786.2
UBA52*	7311	Hs03004332_g1	NM_001033930.1;
			NM_003333.3
UBC	7316	Hs00824723_m1	NM_021009.4
YWHAZ	7534	Hs00237047_m1	NM_003406.3

*Subset of housekeeping genes used in, e.g., Example 5.

In the case of measuring RNA levels for the genes, one convenient and sensitive approach is real-time quantitative PCR (qPCR) assay, following a reverse transcription reaction. Typically, a cycle threshold (C_t) is determined for each test gene and each normalizing gene, i.e., the number of cycle at which the fluorescence from a qPCR reaction above background is detectable.
The overall expression of the one or more normalizing genes can be represented by a “normalizing value” which can be generated by combining the expression of all normalizing genes, either weighted equally (straight addition or averaging) or by different predefined coefficients. For example, in a simplest manner, the normalizing value C_tHcan be the cycle threshold (C_t) of one single normalizing gene, or an average of the C_tvalues of 2 or more, preferably 10 or more, or 15 or more normalizing genes, in which case, the predefined coefficient is 1/N, where N is the total number of normalizing genes used. Thus, C_tH=(C_tH1+C_tH2+C_tHn)/N. As will be apparent to skilled artisans, depending on the normalizing genes used, and the weight desired to be given to each normalizing gene, any coefficients (from 0/N to N/N) can be given to the normalizing genes in weighting the expression of such normalizing genes. That is, C_tH=xC_tH1+yCtH₂+ . . . zC_tHn, wherein x+y+ . . . +z=1.
As discussed above, the methods of the invention generally involve determining the level of expression of a panel of CCGs. With modern high-throughput techniques, it is often possible to determine the expression level of tens, hundreds or thousands of genes. Indeed, it is possible to determine the level of expression of the entire transcriptome (i.e., each transcribed sequence in the genome). Once such a global assay has been performed, one may then informatically analyze one or more subsets of transcripts (i.e., panels or, as often used herein, pluralities of test genes). After measuring the expression of hundreds or thousands of transcripts in a sample, for example, one may analyze (e.g., informatically) the expression of a panel or plurality of test genes comprising primarily CCGs according to the present invention by combining the expression level values of the individual test genes to obtain a test value.
As will be apparent to a skilled artisan, the test value provided in the present invention represents the overall expression level of the plurality of test genes composed substantially of cell-cycle genes. In one embodiment, to provide a test value in the methods of the invention, the normalized expression for a test gene can be obtained by normalizing the measured C_tfor the test gene against the C_tH, i.e., ΔC_t1=(C_t1−C_tH). Thus, the test value representing the overall expression of the plurality of test genes can be provided by combining the normalized expression of all test genes, either by straight addition or averaging (i.e., weighted equally) or by a different predefined coefficient. For example, the simplest approach is averaging the normalized expression of all test genes: test value=(ΔC_t1+ΔC_t2+ΔC_tn)/n. As will be apparent to skilled artisans, depending on the test genes used, different weight can also be given to different test genes in the present invention.
It has been determined that, once the CCP phenomenon reported herein is appreciated, the choice of individual CCGs for a test panel can often be somewhat arbitrary. In other words, many CCGs have been found to be very good surrogates for each other. One way of assessing whether particular CCGs will serve well in the methods and compositions of the invention is by assessing their correlation with the mean expression of CCGs (e.g., all known CCGs, a specific set of CCGs, etc.). Those CCGs that correlate particularly well with the mean are expected to perform well in assays of the invention, e.g., because these will reduce noise in the assay. Rankings of select CCGs according to their correlation with the mean CCG expression as well as their ranking according to predictive value are given in Tables 9, 11 & 23 to 25.
Thus, in some embodiments of each of the various aspects of the invention the plurality of test genes comprises the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more CCGs listed in Tables 9, 11, 23, 24 or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 of the following genes: ASPM, BIRC5, BUB1B, CCNB2, CDC2, CDC20, CDCA8, CDKN3, CENPF, DLGAP5, FOXM1, KIAAO101, KIF11, KIF2C, KIF4A, MCM10, NUSAP1, PRC1, RACGAP1, and TPX2. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, or ten or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 of any of Tables 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, or nine or all of gene numbers 2 & 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, or 2 to 10 of any of Tables 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, or eight or all of gene numbers 3 & 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 of any of Tables 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, or seven or all of gene numbers 4 & 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, or 4 to 10 of any of Tables 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, or 15 or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, or 1 to 15 of any of Tables 9, 11, 23, 24, or 25.

II. Cancer Prognosis

It has been surprisingly discovered that in selected cancers such as prostate cancer, lung cancer, bladder cancer and brain cancer, but not other cancers including certain colon cancer, the expression of cell-cycle genes in tumor cells can accurately predict the degree of aggression of the cancer and risk of recurrence after treatment (e.g., surgical removal of cancer tissue, chemotherapy and radiation therapy, etc.). Thus, the above-described method of determining cell-cycle gene expression can be applied in the prognosis and treatment of such cancers.
Generally, a method is provided for prognosing cancer selected from prostate cancer, lung cancer, bladder cancer or brain cancer, which comprises determining in a tumor sample from a patient diagnosed of prostate cancer, lung cancer, bladder cancer or brain cancer, the expression of at least 2, 4, 5, 6, 7 or at least 8, 9, 10 or 12 cell-cycle genes, wherein overexpression of the at least 4 cell-cycle genes indicates a poor prognosis or an increased likelihood of recurrence of cancer in the patient. The expression can be determined in accordance with the method described above.
In some embodiments, the prognosis method includes (1) obtaining a tumor sample from a patient identified as having prostate cancer, lung cancer, bladder cancer or brain cancer; (2) determining the expression of a panel of genes in the tumor sample including at least 2, 4, 6, 8 or 10 cell-cycle genes; and (3) providing a test value by (a) weighting the determined expression of each of a plurality of test genes selected from the panel of genes with a predefined coefficient, and (b) combining the weighted expression to provide said test value, wherein at least 20%, 50%, at least 75% or at least 90% of said plurality of test genes are cell-cycle genes, and wherein an increased level of expression of the plurality of test genes indicates a poor prognosis or an increased likelihood of cancer recurrence.
In preferred embodiments, the test value representing the overall expression of the plurality of test genes is compared to one or more reference values (or index values), and optionally correlated to a risk of cancer progression or risk of cancer recurrence. Optionally an increased likelihood of poor prognosis is indicated if the test value is greater than the reference value.
For example, the index value may represent the gene expression levels found in a normal sample obtained from the patient of interest, in which case an expression level in the tumor sample significantly higher than this index value would indicate, e.g., a poor prognosis or increased likelihood of cancer recurrence or a need for aggressive treatment.
Alternatively, the index value may represent the average expression level of for a set of individuals from a diverse cancer population or a subset of the population. For example, one may determine the average expression level of a gene or gene panel in a random sampling of patients with cancer (e.g., prostate, bladder, brain, breast, or lung cancer). This average expression level may be termed the “threshold index value,” with patients having CCG expression higher than this value expected to have a poorer prognosis than those having expression lower than this value.
Alternatively the index value may represent the average expression level of a particular gene marker in a plurality of training patients (e.g., prostate cancer patients) with similar outcomes whose clinical and follow-up data are available and sufficient to define and categorize the patients by disease outcome, e.g., recurrence or prognosis. See, e.g., Examples, infra. For example, a “good prognosis index value” can be generated from a plurality of training cancer patients characterized as having “good outcome”, e.g., those who have not had cancer recurrence five years (or ten years or more) after initial treatment, or who have not had progression in their cancer five years (or ten years or more) after initial diagnosis. A “poor prognosis index value” can be generated from a plurality of training cancer patients defined as having “poor outcome”, e.g., those who have had cancer recurrence within five years (or ten years, etc.) after initial treatment, or who have had progression in their cancer within five years (or ten years, etc.) after initial diagnosis. Thus, a good prognosis index value of a particular gene may represent the average level of expression of the particular gene in patients having a “good outcome,” whereas a poor prognosis index value of a particular gene represents the average level of expression of the particular gene in patients having a “poor outcome.”
Thus one aspect of the invention provides a method of classifying cancer comprising determining the status of a panel of genes comprising at least two CCGs, in tissue or cell sample, particularly a tumor sample, from a patient, wherein an abnormal status indicates a negative cancer classification. As used herein, “determining the status” of a gene refers to determining the presence, absence, or extent/level of some physical, chemical, or genetic characteristic of the gene or its expression product(s). Such characteristics include, but are not limited to, expression levels, activity levels, mutations, copy number, methylation status, etc.
In the context of CCGs as used to determine risk of cancer recurrence or progression or need for aggressive treatment, particularly useful characteristics include expression levels (e.g., mRNA or protein levels) and activity levels. Characteristics may be assayed directly (e.g., by assaying a CCG's expression level) or determined indirectly (e.g., assaying the level of a gene or genes whose expression level is correlated to the expression level of the CCG). Thus some embodiments of the invention provide a method of classifying cancer comprising determining the expression level, particularly mRNA level of a panel of genes comprising at least two CCGs, in a tumor sample, wherein elevated expression indicates a negative cancer classification, or an increased risk of cancer recurrence or progression, or a need for aggressive treatment.
“Abnormal status” means a marker's status in a particular sample differs from the status generally found in average samples (e.g., healthy samples or average diseased samples). Examples include mutated, elevated, decreased, present, absent, etc. An “elevated status” means that one or more of the above characteristics (e.g., expression or mRNA level) is higher than normal levels. Generally this means an increase in the characteristic (e.g., expression or mRNA level) as compared to an index value. Conversely a “low status” means that one or more of the above characteristics (e.g., gene expression or mRNA level) is lower than normal levels. Generally this means a decrease in the characteristic (e.g., expression) as compared to an index value. In this context, a “negative status” generally means the characteristic is absent or undetectable. For example, PTEN status is negative if PTEN nucleic acid and/or protein is absent or undetectable in a sample. However, negative PTEN status also includes a mutation or copy number reduction in PTEN.
In some embodiments of the invention the methods comprise determining the expression of one or more CCGs and, if this expression is “increased,” the patient has a poor prognosis. In the context of the invention, “increased” expression of a CCG means the patient's expression level is either elevated over a normal index value or a threshold index (e.g., by at least some threshold amount) or closer to the “poor prognosis index value” than to the “good prognosis index value.”
Thus, when the determined level of expression of a relevant gene marker is closer to the good prognosis index value of the gene than to the poor prognosis index value of the gene, then it can be concluded that the patient is more likely to have a good prognosis, i.e., a low (or no increased) likelihood of cancer recurrence. On the other hand, if the determined level of expression of a relevant gene marker is closer to the poor prognosis index value of the gene than to the good prognosis index value of the gene, then it can be concluded that the patient is more likely to have a poor prognosis, i.e., an increased likelihood of cancer recurrence.
Alternatively index values may be determined thusly: In order to assign patients to risk groups, a threshold value will be set for the cell cycle mean. The optimal threshold value is selected based on the receiver operating characteristic (ROC) curve, which plots sensitivity vs (1−specificity). For each increment of the cell cycle mean, the sensitivity and specificity of the test is calculated using that value as a threshold. The actual threshold will be the value that optimizes these metrics according to the artisans requirements (e.g., what degree of sensitivity or specificity is desired, etc.). Example 5 demonstrates determination of a threshold value determined and validated experimentally.
Panels of CCGs (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more CCGs) can accurately predict prognosis, as shown in Example 3. Those skilled in the art are familiar with various ways of determining the expression of a panel of genes (i.e., a plurality of genes). One may determine the expression of a panel of genes by determining the average expression level (normalized or absolute) of all panel genes in a sample obtained from a particular patient (either throughout the sample or in a subset of cells from the sample or in a single cell). Increased expression in this context will mean the average expression is higher than the average expression level of these genes in normal patients (or higher than some index value that has been determined to represent the average expression level in a reference population such as patients with the same cancer). Alternatively, one may determine the expression of a panel of genes by determining the average expression level (normalized or absolute) of at least a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more) or at least a certain proportion (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%) of the genes in the panel. Alternatively, one may determine the expression of a panel of genes by determining the absolute copy number of the mRNA (or protein) of all the genes in the panel and either total or average these across the genes.
As used herein, “classifying a cancer” and “cancer classification” refer to determining one or more clinically-relevant features of a cancer and/or determining a particular prognosis of a patient having said cancer. Thus “classifying a cancer” includes, but is not limited to: (i) evaluating metastatic potential, potential to metastasize to specific organs, risk of recurrence, and/or course of the tumor; (ii) evaluating tumor stage; (iii) determining patient prognosis in the absence of treatment of the cancer; (iv) determining prognosis of patient response (e.g., tumor shrinkage or progression-free survival) to treatment (e.g., chemotherapy, radiation therapy, surgery to excise tumor, etc.); (v) diagnosis of actual patient response to current and/or past treatment; (vi) determining a preferred course of treatment for the patient; (vii) prognosis for patient relapse after treatment (either treatment in general or some particular treatment); (viii) prognosis of patient life expectancy (e.g., prognosis for overall survival), etc.
Thus, a “negative classification” means an unfavorable clinical feature of the cancer (e.g., a poor prognosis). Examples include (i) an increased metastatic potential, potential to metastasize to specific organs, and/or risk of recurrence; (ii) an advanced tumor stage; (iii) a poor patient prognosis in the absence of treatment of the cancer; (iv) a poor prognosis of patient response (e.g., tumor shrinkage or progression-free survival) to a particular treatment (e.g., chemotherapy, radiation therapy, surgery to excise tumor, etc.); (v) a poor prognosis for patient relapse after treatment (either treatment in general or some particular treatment); (vi) a poor prognosis of patient life expectancy (e.g., prognosis for overall survival), etc. In some embodiments a recurrence-associated clinical parameter (or a high nomogram score) and increased expression of a CCG indicate a negative classification in cancer (e.g., increased likelihood of recurrence or progression).
As discussed above, it is thought that elevated CCG expression accompanies rapidly proliferating (and thus more aggressive) cancer cells. Such a cancer in a patient will often mean the patient has an increased likelihood of recurrence after treatment (e.g., the cancer cells not killed or removed by the treatment will quickly grow back). Such a cancer can also mean the patient has an increased likelihood of cancer progression for more rapid progression (e.g., the rapidly proliferating cells will cause any tumor to grow quickly, gain in virulence, and/or metastasize). Such a cancer can also mean the patient may require a relatively more aggressive treatment. Thus, in some embodiments the invention provides a method of classifying cancer comprising determining the status of a panel of genes comprising at least two CCGs, wherein an abnormal status indicates an increased likelihood of recurrence or progression. As discussed above, in some embodiments the status to be determined is gene expression levels. Thus in some embodiments the invention provides a method of determining the prognosis of a patient's cancer comprising determining the expression level of a panel of genes comprising at least two CCGs, wherein elevated expression indicates an increased likelihood of recurrence or progression of the cancer.
“Recurrence” and “progression” are terms well-known in the art and are used herein according to their known meanings. As an example, the meaning of “progression” may be cancer-type dependent, with progression in lung cancer meaning something different from progression in prostate cancer. However, within each cancer-type and subtype “progression” is clearly understood to those skilled in the art. As used herein, a patient has an “increased likelihood” of some clinical feature or outcome (e.g., recurrence or progression) if the probability of the patient having the feature or outcome exceeds some reference probability or value. The reference probability may be the probability of the feature or outcome across the general relevant patient population. For example, if the probability of recurrence in the general prostate cancer population is X % and a particular patient has been determined by the methods of the present invention to have a probability of recurrence of Y %, and if Y>X, then the patient has an “increased likelihood” of recurrence. Alternatively, as discussed above, a threshold or reference value may be determined and a particular patient's probability of recurrence may be compared to that threshold or reference. Because predicting recurrence and predicting progression are prognostic endeavors, “predicting prognosis” will often be used herein to refer to either or both. In these cases, a “poor prognosis” will generally refer to an increased likelihood of recurrence, progression, or both.
As shown in Example 3, individual CCGs can predict prognosis quite well. Thus the invention provides a method of predicting prognosis comprising determining the expression of at least one CCG listed in Table 1 or Panels A through G.
Example 3 also shows that panels of CCGs (e.g., 2, 3, 4, 5, or 6 CCGs) can accurately predict prognosis. Thus in some aspects the invention provides a method of classifying a cancer comprising determining the status of a panel of genes (e.g., a plurality of test genes) comprising a plurality of CCGs. For example, increased expression in a panel of genes (or plurality of test genes) may refer to the average expression level of all panel or test genes in a particular patient being higher than the average expression level of these genes in normal patients (or higher than some index value that has been determined to represent the normal average expression level). Alternatively, increased expression in a panel of genes may refer to increased expression in at least a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more) or at least a certain proportion (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100%) of the genes in the panel as compared to the average normal expression level.
In some embodiments the panel comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 70, 80, 90, 100, 200, or more CCGs. In some embodiments the panel comprises at least 10, 15, 20, or more CCGs. In some embodiments the panel comprises between 5 and 100 CCGs, between 7 and 40 CCGs, between 5 and 25 CCGs, between 10 and 20 CCGs, or between 10 and 15 CCGs. In some embodiments CCGs comprise at least a certain proportion of the panel. Thus in some embodiments the panel comprises at least 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% CCGs. In some preferred embodiments the panel comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 70, 80, 90, 100, 200, or more CCGs, and such CCGs constitute of at least 50%, 60%, 70%, preferably at least 75%, 80%, 85%, more preferably at least 90%, 95%, 96%, 97%, 98%, or 99% or more of the total number of genes in the panel. In some embodiments the CCGs are chosen from the group consisting of the genes in Table 1 and Panels A through G. In some embodiments the panel comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or more of the genes in any of Table 1 and Panels A through G. In some embodiments the invention provides a method of predicting prognosis comprising determining the status of the CCGs in Panels A through G, wherein abnormal status indicates a poor prognosis.
In some of these embodiments elevated expression indicates an increased likelihood of recurrence or progression. Thus in a preferred embodiment the invention provides a method of predicting risk of cancer recurrence or progression in a patient comprising determining the status of a panel of genes, wherein the panel comprises between about 10 and about 15 CCGs, the CCGs constitute at least 90% of the panel, and an elevated status for the CCGs indicates an increased likelihood or recurrence or progression.
Several panels of CCGs (Table 2, supra, and Tables 3 & 4, infra) have been evaluated for their ability to predict prognosis in several different cancers. The results of these studies are described in Examples 1 through 6 below.

TABLE 3

“Panel C” Evaluated in Examples 1 through 4

	Gene Symbol	Entrez GeneID

	AURKA	6790
	BUB1*	699
	CCNB1*	891
	CCNB2*	9133
	CDC2*	983
	CDC20*	991
	CDC45L*	8318
	CDCA8*	55143
	CENPA	1058
	CKS2*	1164
	DLG7*	9787
	DTL*	51514
	FOXM1*	2305
	HMMR*	3161
	KIF23*	9493
	KPNA2	3838
	MAD2L1*	4085
	MELK	9833
	MYBL2*	4605
	NUSAP1*	51203
	PBK*	55872
	PRC1*	9055
	PTTG1*	9232
	RRM2*	6241
	TIMELESS*	8914
	TPX2*	22974
	TRIP13*	9319
	TTK*	7272
	UBE2C	11065
	UBE2S*	27338
	ZWINT*	11130

	*These genes were used as a 26-gene subset panel (“Panel D”) in the validation arm of the experiment described in Example 2.

TABLE 4

“Panel E”

	Name	GeneID

	ASF1B*	55723
	ASPM*	259266
	BIRC5*	332
	BUB1B*	701
	C18orf24*	220134
	CDC2*	983
	CDC20*	991
	CDCA3*	83461
	CDCA8*	55143
	CDKN3*	1033
	CENPF*	1063
	CENPM*	79019
	CEP55*	55165
	DLGAP5*	9787
	DTL*	51514
	FOXM1*	2305
	KIAA0101*	9768
	KIF11*	3832
	KIF20A*	10112
	KIF4A	24137
	MCM10*	55388
	NUSAP1*	51203
	ORC6L*	23594
	PBK*	55872
	PLK1*	5347
	PRC1*	9055
	PTTG1*	9232
	RAD51*	5888
	RAD54L*	8438
	RRM2*	6241
	TK1*	7083
	TOP2A*	7153

	*These genes were used as a 31-gene subset panel (“Panel F”) in the experiment described in Example 5.

It has been determined that the choice of individual CCGs for a panel can often be relatively arbitrary. In other words, most CCGs have been found to be very good surrogates for each other. One way of assessing whether particular CCGs will serve well in the methods and compositions of the invention is by assessing their correlation with the mean expression of CCGs (e.g., all known CCGs, a specific set of CCGs, etc.). Those CCGs that correlate particularly well with the mean are expected to perform well in assays of the invention, e.g., because these will reduce noise in the assay. A ranking of select CCGs according to their correlation with the mean CCG expression is given in Table 23.
In CCG signatures the particular CCGs assayed is often not as important as the total number of CCGs. The number of CCGs assayed can vary depending on many factors, e.g., technical constraints, cost considerations, the classification being made, the cancer being tested, the desired level of predictive power, etc. Increasing the number of CCGs assayed in a panel according to the invention is, as a general matter, advantageous because, e.g., a larger pool of mRNAs to be assayed means less “noise” caused by outliers and less chance of an assay error throwing off the overall predictive power of the test. However, cost and other considerations will generally limit this number and finding the optimal number of CCGs for a signature is desirable.
It has been discovered that the predictive power of a CCG signature often ceases to increase significantly beyond a certain number of CCGs (see FIG. 1; Example 1). More specifically, the optimal number of CCGs in a signature (no) can be found wherever the following is true
(P _n+1 −P _n)<C _O,
wherein P is the predictive power (i.e., P_nis the predictive power of a signature with n genes and P_n+1is the predictive power of a signature with n genes plus one) and C_Ois some optimization constant. Predictive power can be defined in many ways known to those skilled in the art including, but not limited to, the signature's p-value. C_Ocan be chosen by the artisan based on his or her specific constraints. For example, if cost is not a critical factor and extremely high levels of sensitivity and specificity are desired, C_Ocan be set very low such that only trivial increases in predictive power are disregarded. On the other hand, if cost is decisive and moderate levels of sensitivity and specificity are acceptable, C_Ocan be set higher such that only significant increases in predictive power warrant increasing the number of genes in the signature.
Alternatively, a graph of predictive power as a function of gene number may be plotted (as in FIG. 1) and the second derivative of this plot taken. The point at which the second derivative decreases to some predetermined value (C_O′) may be the optimal number of genes in the signature.
Examples 1 & 3 and FIGS. 1 & 17 illustrate the empirical determination of optimal numbers of CCGs in CCG panels of the invention. Randomly selected subsets of the 31 CCGs listed in Table 3 were tested as distinct CCG signatures and predictive power (i.e., p-value) was determined for each. As FIG. 1 shows, p-values ceased to improve significantly between about 10 and about 15 CCGs, thus indicating that an optimal number of CCGs in a prognostic panel is from about 10 to about 15. Thus some embodiments of the invention provide a method of predicting prognosis in a patient having prostate cancer comprising determining the status of a panel of genes, wherein the panel comprises between about 10 and about 15 CCGs and an elevated status for the CCGs indicates a poor prognosis. In some embodiments the panel comprises between about 10 and about 15 CCGs and the CCGs constitute at least 90% of the panel. In other embodiments the panel comprises CCGs plus one or more additional markers that significantly increase the predictive power of the panel (i.e., make the predictive power significantly better than if the panel consisted of only the CCGs). Any other combination of CCGs (including any of those listed in Table 1 or Panels A through G) can be used to practice the invention.
It has been discovered that CCGs are particularly predictive in certain cancers. For example, panels of CCGs have been determined to be accurate in predicting recurrence in prostate cancer (Examples 1 through 5). Further, CCGs can determine prognosis in bladder, brain, breast and lung cancers, as summarized in Example 6 and Tables 21 and 22 below.
Thus the invention provides a method comprising determining the status of a panel of genes comprising at least two CCGs, wherein an abnormal status indicates a poor prognosis. In some embodiments the panel comprises at least 2 genes chosen from the group of genes in at least one of Panels A through G. In some embodiments the panel comprises at least 10 genes chosen from the group of genes in at least one of Panels A through G. In some embodiments the panel comprises at least 15 genes chosen from the group of genes in at least one of Panels A through G. In some embodiments the panel comprises all of the genes in at least one of Panels A through G. The invention also provides a method of determining the prognosis of bladder cancer, comprising determining the status of a panel of genes comprising at least two CCGs (e.g., at least two of the genes in any of Panels B, C, & F), wherein an abnormal status indicates a poor prognosis. The invention also provides a method of determining the prognosis of brain cancer, comprising determining the status of a panel of genes comprising at least two CCGs (e.g., at least two of the genes in any of Panels B, C, & F), wherein an abnormal status indicates a poor prognosis. The invention further provides a method of determining the prognosis of breast cancer, comprising determining the status of a panel of genes comprising at least two CCGs (e.g., at least two of the genes in any of Panels B, C, & F), wherein an abnormal status indicates a poor prognosis. The invention also provides a method of determining the prognosis of lung cancer, comprising determining the status of a panel of genes comprising at least two CCGs (e.g., at least two of the genes in any of Panels B, C, & F), wherein an abnormal status indicates a poor prognosis.
In some embodiments the panel comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs. In some embodiments the panel comprises between 5 and 100 CCGs, between 7 and 40 CCGs, between 5 and 25 CCGs, between 10 and 20 CCGs, or between 10 and 15 CCGs. In some embodiments CCGs comprise at least a certain proportion of the panel. Thus in some embodiments the panel comprises at least 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% CCGs. In some embodiments the CCGs are chosen from the group consisting of the genes listed in Tables 1, 9 & 11 and Panels A through G. In some embodiments the panel comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more genes chosen from the group of genes in any of Tables 1, 9 or 11 or Panels A through G. In some embodiments the panel comprises all of the genes in any of Tables 1, 9, or 11 or Panels A through G.
As mentioned above, many of the CCGs of the invention have been analyzed to determine their correlation to the CCG mean and also, for the genes, to determine their relative predictive value within a panel (see Tables 9, 11, & 23 to 25). Thus in some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more CCGs listed in Table 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 of the following genes: ASPM, BIRC5, BUB1B, CCNB2, CDC2, CDC20, CDCA8, CDKN3, CENPF, DLGAP5, FOXM1, KIAA0101, KIF11, KIF2C, KIF4A, MCM10, NUSAP1, PRC1, RACGAP1, and TPX2. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, or ten or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 of any of Tables 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, or nine or all of gene numbers 2 & 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, or 2 to 10 of any of Tables 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, or eight or all of gene numbers 3 & 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 of any of Tables 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, or seven or all of gene numbers 4 & 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, or 4 to 10 of any of Tables 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, or 15 or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, or 1 to 15 of any of Tables 9, 11, 23, 24, or 25.
It has further been discovered that CCG status synergistically adds to clinical parameters in prognosing cancer. In the case of prostate cancer, for example, it has been discovered that a high level of gene expression of any one of the genes in Panels C through F is associated with an increased risk of prostate cancer recurrence or progression in patients whose clinical nomogram score indicates a relatively low risk of recurrence or progression. Because evaluating CCG expression levels can thus detect increased risk not detected using clinical parameters alone, the invention generally provides methods combining evaluating at least one clinical parameter with evaluating the status of at least one CCG.
As Example 3 shows, even individual CCGs add to clinical parameters in predicting cancer recurrence. Thus one aspect of the invention provides an in vitro diagnostic method comprising determining at least one clinical parameter for a cancer patient and determining the status of at least one CCG in a sample obtained from the patient. However, assessing the status of multiple CCGs improves predictive power even more (also shown in Example 1). Thus in some embodiments the status of a plurality of CCGs (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more) is determined. In some embodiments abnormal status indicates an increased likelihood of recurrence or progression. In some embodiments the patient has prostate cancer. In some embodiments the patient has lung cancer. Often the clinical parameter is at least somewhat independently predictive of recurrence or progression and the addition of CCG status improves the predictive power. As used herein, “clinical parameter” and “clinical measure” refer to disease or patient characteristics that are typically applied to assess disease course and/or predict outcome. Examples in cancer generally include tumor stage, tumor grade, lymph node status, histology, performance status, type of surgery, surgical margins, type of treatment, and age of onset. In prostate cancer clinicians often use pre-surgery blood PSA levels, stage (defined by size of tumor and evidence of metastasis), and Gleason score (similar to concept of grade). After surgical intervention, important clinical parameters in prostate cancer include margin and lymph node status. In breast cancer clinicians often use size of index lesion in cm, invasion, number of nodes involved, and grade.
Often certain clinical parameters are correlated with a particular disease character. For example, in cancer generally as well as in specific cancers, certain clinical parameters are correlated with, e.g., likelihood of recurrence or metastasis, prognosis for survival for a certain amount of time, likelihood of response to treatment generally or to a specific treatment, etc. In prostate cancer some clinical parameters are such that their status (presence, absence, level, etc.) is associated with increased likelihood of recurrence. Examples of such recurrence-associated parameters (some but not all of which are specific to prostate cancer) include high PSA levels (e.g., greater than 4 ng/ml), high Gleason score, large tumor size, evidence of metastasis, advanced tumor stage, nuclear grade, lymph node involvement, early age of onset. Other types of cancer may have different parameters correlated to likelihood of recurrence or progression, and CCG status, as a measure of proliferative activity, adds to these parameters in predicting prognosis in these cancers. As used herein, “recurrence-associated clinical parameter” has its conventional meaning for each specific cancer, with which those skilled in the art are quite familiar. In fact, those skilled in the art are familiar with various recurrence-associated clinical parameters beyond those listed here.
Often a physician will assess more than one clinical parameter in a patient and make a more comprehensive evaluation for the disease characters of interest. Example 5 shows how CCG status can add to one particular grouping of clinical parameters used to determine risk of recurrence in prostate cancer. Clinical parameters in Example 5 include binary variables for organ-confined disease and Gleason score less than or equal to 6, and a continuous variable for logarithmic PSA (Table 14). This model includes all of the clinical parameters incorporated in the post-RP nomogram (i.e., Kattan-Stephenson nomogram) except for Year of RP and the two components of the Gleason score. Thus in some embodiments at least two clinical parameters (e.g., two of the above listed parameters) are assessed along with the expression level of at least one CCG.
One way in which single, but more often multiple, clinical parameters are utilized by physicians is with the help of nomograms. In the clinical setting, nomograms are representations (often visual) of a correlation between one or more parameters and one or more patient or disease characters. An example of a prevalent clinical nomogram used in determining a prostate cancer patient's likelihood of recurrence is described in Kattan et al., J. CLIN. ONCOL. (1999) 17:1499-1507, and updated in Stephenson et al., J. CLIN. ONCOL. (2005) 23:7005-7012 (“Kattan-Stephenson nomogram”). This nomogram evaluates a patient by assigning a point value to each of several clinical parameters (year of RP, surgical margins, extracapsular extension, seminal vesicle invasion, lymph node involvement, primary Gleason score, secondary Gleason score, and preoperative PSA level), totaling the points for a patient into a nomogram score, and then predicting the patient's likelihood of being recurrence-free at varying time intervals (up to 10 years) based on this nomogram score. An example of a prevalent clinical nomogram used in determining a breast cancer patient's prognosis for survival is the Nottingham Prognostic Index (NPI). See, e.g., Galea et al., BREAST CANCER RES. & TREAT. (1992) 22:207-19.
It has been discovered that determining the status of a CCG in a sample obtained from a prostate cancer patient, along with the patient's Kattan-Stephenson nomogram score, is a better predictor of 10-year recurrence-free survival than the nomogram score alone. See, e.g., Examples 2 & 5, infra. Specifically, adding CCG status to the Kattan-Stephenson nomogram detects patients at significantly increased risk of recurrence that the nomogram alone does not. Table 3 above provides an exemplary panel of 31 CCGs (Panel C) and a subset panel of 26 CCGs (Panel D, shown with *) determined in Example 2 to show predictive synergy with the Kattan-Stephenson nomogram in prostate cancer prognosis. It has also been discovered that determining the status of a CCG in a sample obtained from a breast cancer patient, along with the patient's NPI score, is a better prognostic predictor than NPI score alone. See, e.g., Example 6, infra. Specifically, adding CCG status to the NPI nomogram detects patients at significantly increased risk of recurrence that the nomogram alone does not. Panels B, C and D were determined in Example 2 to show predictive synergy with the NPI nomogram in breast cancer prognosis.
Thus another aspect of the invention provides an in vitro method comprising determining a clinical nomogram score (e.g., Kattan-Stephenson or NPI nomogram score) for a cancer patient and determining the status of at least one CCG in a sample obtained from the patient. Example 3 illustrates the empirical determination of the predictive power of individual CCGs and of several CCG panels of varying size over the Kattan-Stephenson nomogram. Randomly selected subsets of the 31 CCGs listed in Table 3 were tested as distinct CCG signatures and predictive power (i.e., p-value) was determined for each. As FIG. 1 shows, CCG signatures of 2, 3, 4, 5, 6, 10, 15, 20, 25, and 26 genes each add predictive power to the nomogram. Thus the invention provides a method of determining whether a prostate cancer patient has an increased likelihood of recurrence comprising determining the status of a panel of genes comprising at least 2, 3, 4, 5, 6, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more CCGs, wherein an elevated status (e.g., increased expression) for the CCGs indicates an increased likelihood of recurrence. In some embodiments the method further comprises determining a clinical nomogram score of the patient. The invention further provides a method of determining whether a breast cancer patient has an increased likelihood of recurrence comprising determining the status of a panel of genes comprising at least 2, 3, 4, 5, 6, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more CCGs, wherein an elevated status (e.g., increased expression) for the CCGs indicates an increased likelihood of recurrence. In some embodiments the method further comprises determining a clinical nomogram score of the patient.
Often clinical nomograms for cancer are designed such that a particular value (e.g., high score) correlates with an increased risk of recurrence. Elevated CCG status (e.g., increased expression or activity) is also correlated with increased risk. Thus, in some embodiments the invention provides a method of determining whether a cancer patient has an increased likelihood of recurrence or progression comprising determining a clinical nomogram score for the patient and determining the status of at least one CCG in a sample obtained from the patient, wherein a high nomogram score and/or an elevated CCG status indicate the patient has an increased likelihood of recurrence or progression. In some embodiments the cancer is prostate cancer. In some embodiments the cancer is lung cancer.
In some embodiments this assessment is made before radical prostatectomy (e.g., using a prostate biopsy sample) while in some embodiments it is made after (e.g., using the resected prostate sample). In some embodiments, a sample of one or more cells are obtained from a prostate cancer patient before or after treatment for analysis according to the present invention. Prostate cancer treatment currently applied in the art includes, e.g., prostatectomy, radiotherapy, hormonal therapy (e.g., using GnRH antagonists, GnRH agonists, antiandrogens), chemotherapy, and high intensity focused ultrasound. In some embodiments, one or more prostate tumor cells from prostate cancer tissue are obtained from a prostate cancer patient during biopsy or prostatectomy and are used for analysis in the method of the present invention.
The present invention is also based on the discovery that PTEN status predicts aggressive prostate cancer. PTEN status adds to both clinical parameters (e.g., Kattan-Stephenson nomogram) and CCGs (e.g., the genes in Table 1 or Panels A through G). As described in more detail in Example 4 below, PTEN status was determined in 191 prostate cancer patient samples with accompanying clinical history data and CCG signature data. Negative PTEN status was found to be a significant predictor for risk of recurrence (p-value 0.031). PTEN remained a significant predictor of recurrence after adjusting for post-surgery clinical parameters and the CCG signature shown in Table 3 (p-value 0.026). In addition, and importantly, the combination of PTEN and the CCG signature seems to be a better predictor of recurrence than post-surgery clinical parameters (p-value 0.0002).
Because PTEN is an independent predictor of prostate cancer recurrence, one aspect of the invention provides a method of predicting a patient's likelihood of prostate cancer recurrence comprising determining PTEN status in a sample from the patient, wherein a low or negative PTEN status indicates the patient has a high likelihood of recurrence. PTEN status can be determined by any technique known in the art, including but not limited to those discussed herein.
Because PTEN adds to CCG status in predicting prostate cancer recurrence, another aspect of the invention provides an in vitro method comprising determining PTEN status and determining the status of a plurality of CCGs in a sample obtained from a patient. Different combinations of techniques can be used to determine the status the various markers. For example, in one embodiment PTEN status is determined by immunohistochemistry (IHC) while the status of the plurality of CCGs is determined by quantitative polymerase chain reaction (qPCR™), e.g., TaqMan™. Some embodiments of the invention provide a method of determining a prostate cancer patient's likelihood of recurrence comprising determining PTEN status in a sample obtained from the patient, determining the status of a plurality of CCGs in a sample obtained from the patient, wherein low or negative PTEN status and/or elevated CCG status indicate the patient has an increased likelihood of recurrence.
Because PTEN status adds predictive value to clinical parameters in predicting prostate recurrence, yet another aspect of the invention provides an in vitro method comprising determining PTEN status and determining at least one clinical parameter for a cancer patient. Often the clinical parameter is at least somewhat independently predictive of recurrence and the addition of PTEN status improves the predictive power. In some embodiments the invention provides a method of determining whether a cancer patient has an increased likelihood of recurrence comprising determining the status of PTEN in a sample obtained from the patient and determining a clinical nomogram score for the patient, wherein low or negative PTEN status and/or a high nomogram score indicate the patient has an increased likelihood of recurrence.
Because all three of the above markers are additive, some embodiments of the invention provide a method of determining whether a cancer patient has an increased likelihood of recurrence comprising determining the status of PTEN in a sample obtained from the patient, determining a clinical nomogram score for the patient and determining the status of at least one CCG in a sample obtained from the patient, wherein low or negative PTEN status, a high nomogram score and an elevated CCG status indicate the patient has an increased likelihood of recurrence.
The results of any analyses according to the invention will often be communicated to physicians, genetic counselors and/or patients (or other interested parties such as researchers) in a transmittable form that can be communicated or transmitted to any of the above parties. Such a form can vary and can be tangible or intangible. The results can be embodied in descriptive statements, diagrams, photographs, charts, images or any other visual forms. For example, graphs showing expression or activity level or sequence variation information for various genes can be used in explaining the results. Diagrams showing such information for additional target gene(s) are also useful in indicating some testing results. The statements and visual forms can be recorded on a tangible medium such as papers, computer readable media such as floppy disks, compact disks, etc., or on an intangible medium, e.g., an electronic medium in the form of email or website on internet or intranet. In addition, results can also be recorded in a sound form and transmitted through any suitable medium, e.g., analog or digital cable lines, fiber optic cables, etc., via telephone, facsimile, wireless mobile phone, internet phone and the like.
Thus, the information and data on a test result can be produced anywhere in the world and transmitted to a different location. As an illustrative example, when an expression level, activity level, or sequencing (or genotyping) assay is conducted outside the United States, the information and data on a test result may be generated, cast in a transmittable form as described above, and then imported into the United States. Accordingly, the present invention also encompasses a method for producing a transmittable form of information on at least one of (a) expression level or (b) activity level for at least one patient sample. The method comprises the steps of (1) determining at least one of (a) or (b) above according to methods of the present invention; and (2) embodying the result of the determining step in a transmittable form. The transmittable form is the product of such a method.
Techniques for analyzing such expression, activity, and/or sequence data (indeed any data obtained according to the invention) will often be implemented using hardware, software or a combination thereof in one or more computer systems or other processing systems capable of effectuating such analysis.
Thus, the present invention further provides a system for determining gene expression in a tumor sample, comprising: (1) a sample analyzer for determining the expression levels of a panel of genes in a tumor sample including at least 2, 4, 6, 8 or 10 cell-cycle genes, wherein the sample analyzer contains the tumor sample which is from a patient identified as having prostate cancer, lung cancer, bladder cancer or brain cancer, or cDNA molecules from mRNA expressed from the panel of genes; (2) a first computer program means for (a) receiving gene expression data on at least 4 test genes selected from the panel of genes, (b) weighting the determined expression of each of the test genes, and (c) combining the weighted expression to provide a test value, wherein at least 20%, 50%, at least 75% or at least 90% of the test genes are cell-cycle genes; and optionally (3) a second computer program means for comparing the test value to one or more reference values each associated with a predetermined degree of risk of cancer recurrence or progression of the prostate cancer, lung cancer, bladder cancer or brain cancer. In some embodiments, the system further comprises a display module displaying the comparison between the test value to the one or more reference values, or displaying a result of the comparing step.
In preferred embodiment, the amount of RNA transcribed from the panel of genes including test genes is measured in the tumor sample. In addition, the amount of RNA of one or more housekeeping genes in the tumor sample is also measured, and used to normalize or calibrate the expression of the test genes, as described above.
In some embodiments, the plurality of test genes includes at least 2, 3 or 4 cell-cycle genes, which constitute at least 50%, 75% or 80% of the plurality of test genes, and preferably 100% of the plurality of test genes. In some embodiments, the plurality of test genes includes at least 5, 6 or 7, or at least 8 cell-cycle genes, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes.
In some other embodiments, the plurality of test genes includes at least 8, 10, 12, 15, 20, 25 or 30 cell-cycle genes, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes.
The sample analyzer can be any instruments useful in determining gene expression, including, e.g., a sequencing machine, a real-time PCR machine, and a microarray instrument.
The computer-based analysis function can be implemented in any suitable language and/or browsers. For example, it may be implemented with C language and preferably using object-oriented high-level programming languages such as Visual Basic, SmallTalk, C++, and the like. The application can be written to suit environments such as the Microsoft Windows™ environment including Windows™ 98, Windows™ 2000, Windows™ NT, and the like. In addition, the application can also be written for the Macintosh™, SUN™, UNIX or LINUX environment. In addition, the functional steps can also be implemented using a universal or platform-independent programming language. Examples of such multi-platform programming languages include, but are not limited to, hypertext markup language (HTML), JAVA™, JavaScript™, Flash programming language, common gateway interface/structured query language (CGI/SQL), practical extraction report language (PERL), AppleScript™ and other system script languages, programming language/structured query language (PL/SQL), and the like. Java™- or JavaScript™-enabled browsers such as HotJava™, Microsoft™ Explorer™, or Netscape™ can be used. When active content web pages are used, they may include Java™ applets or ActiveX™ controls or other active content technologies.
The analysis function can also be embodied in computer program products and used in the systems described above or other computer- or internet-based systems. Accordingly, another aspect of the present invention relates to a computer program product comprising a computer-usable medium having computer-readable program codes or instructions embodied thereon for enabling a processor to carry out gene status analysis. These computer program instructions may be loaded onto a computer or other programmable apparatus to produce a machine, such that the instructions which execute on the computer or other programmable apparatus create means for implementing the functions or steps described above. These computer program instructions may also be stored in a computer-readable memory or medium that can direct a computer or other programmable apparatus to function in a particular manner, such that the instructions stored in the computer-readable memory or medium produce an article of manufacture including instruction means which implement the analysis. The computer program instructions may also be loaded onto a computer or other programmable apparatus to cause a series of operational steps to be performed on the computer or other programmable apparatus to produce a computer implemented process such that the instructions which execute on the computer or other programmable apparatus provide steps for implementing the functions or steps described above.
Thus one aspect of the present invention provides a system for determining whether a patient has increased likelihood of recurrence. Generally speaking, the system comprises (1) computer program means for receiving, storing, and/or retrieving a patient's gene status data (e.g., expression level, activity level, variants) and optionally clinical parameter data (e.g., Gleason score, nomogram score); (2) computer program means for querying this patient data; (3) computer program means for concluding whether there is an increased likelihood of recurrence based on this patient data; and optionally (4) computer program means for outputting/displaying this conclusion. In some embodiments this means for outputting the conclusion may comprise a computer program means for informing a health care professional of the conclusion.
One example of such a computer system is the computer system [600] illustrated in FIG. 6. Computer system [600] may include at least one input module [630] for entering patient data into the computer system [600]. The computer system [600] may include at least one output module [624] for indicating whether a patient has an increased or decreased likelihood of response and/or indicating suggested treatments determined by the computer system [600]. Computer system [600] may include at least one memory module [606] in communication with the at least one input module [630] and the at least one output module [624].
The at least one memory module [606] may include, e.g., a removable storage drive [608], which can be in various forms, including but not limited to, a magnetic tape drive, a floppy disk drive, a VCD drive, a DVD drive, an optical disk drive, etc. The removable storage drive [608] may be compatible with a removable storage unit [610] such that it can read from and/or write to the removable storage unit [610]. Removable storage unit [610] may include a computer usable storage medium having stored therein computer-readable program codes or instructions and/or computer readable data. For example, removable storage unit [610] may store patient data. Example of removable storage unit [610] are well known in the art, including, but not limited to, floppy disks, magnetic tapes, optical disks, and the like. The at least one memory module [606] may also include a hard disk drive [612], which can be used to store computer readable program codes or instructions, and/or computer readable data.
In addition, as shown in FIG. 1, the at least one memory module [606] may further include an interface [614] and a removable storage unit [616] that is compatible with interface [614] such that software, computer readable codes or instructions can be transferred from the removable storage unit [616] into computer system [600]. Examples of interface [614] and removable storage unit [616] pairs include, e.g., removable memory chips (e.g., EPROMs or PROMs) and sockets associated therewith, program cartridges and cartridge interface, and the like. Computer system [600] may also include a secondary memory module [618], such as random access memory (RAM).
Computer system [600] may include at least one processor module [602]. It should be understood that the at least one processor module [602] may consist of any number of devices. The at least one processor module [602] may include a data processing device, such as a microprocessor or microcontroller or a central processing unit. The at least one processor module [602] may include another logic device such as a DMA (Direct Memory Access) processor, an integrated communication processor device, a custom VLSI (Very Large Scale Integration) device or an ASIC (Application Specific Integrated Circuit) device. In addition, the at least one processor module [602] may include any other type of analog or digital circuitry that is designed to perform the processing functions described herein.
As shown in FIG. 6, in computer system [600], the at least one memory module [606], the at least one processor module [602], and secondary memory module [618] are all operably linked together through communication infrastructure [620], which may be a communications bus, system board, cross-bar, etc.). Through the communication infrastructure [620], computer program codes or instructions or computer readable data can be transferred and exchanged. Input interface [626] may operably connect the at least one input module [626] to the communication infrastructure [620]. Likewise, output interface [622] may operably connect the at least one output module [624] to the communication infrastructure [620].
The at least one input module [630] may include, for example, a keyboard, mouse, touch screen, scanner, and other input devices known in the art. The at least one output module [624] may include, for example, a display screen, such as a computer monitor, TV monitor, or the touch screen of the at least one input module [630]; a printer; and audio speakers. Computer system [600] may also include, modems, communication ports, network cards such as Ethernet cards, and newly developed devices for accessing intranets or the internet.
The at least one memory module [606] may be configured for storing patient data entered via the at least one input module [630] and processed via the at least one processor module [602]. Patient data relevant to the present invention may include expression level, activity level, copy number and/or sequence information for PTEN and/or a CCG. Patient data relevant to the present invention may also include clinical parameters relevant to the patient's disease. Any other patient data a physician might find useful in making treatment decisions/recommendations may also be entered into the system, including but not limited to age, gender, and race/ethnicity and lifestyle data such as diet information. Other possible types of patient data include symptoms currently or previously experienced, patient's history of illnesses, medications, and medical procedures.
The at least one memory module [606] may include a computer-implemented method stored therein. The at least one processor module [602] may be used to execute software or computer-readable instruction codes of the computer-implemented method. The computer-implemented method may be configured to, based upon the patient data, indicate whether the patient has an increased likelihood of recurrence, progression or response to any particular treatment, generate a list of possible treatments, etc.
In certain embodiments, the computer-implemented method may be configured to identify a patient as having or not having an increased likelihood of recurrence or progression. For example, the computer-implemented method may be configured to inform a physician that a particular patient has an increased likelihood of recurrence. Alternatively or additionally, the computer-implemented method may be configured to actually suggest a particular course of treatment based on the answers to/results for various queries.
FIG. 7 illustrates one embodiment of a computer-implemented method [700] of the invention that may be implemented with the computer system [600] of the invention. The method [700] begins with one of three queries ([710], [711], [712]), either sequentially or substantially simultaneously. If the answer to/result for any of these queries is “Yes” [720], the method concludes [730] that the patient has an increased likelihood of recurrence. If the answer to/result for all of these queries is “No” [721], the method concludes [731] that the patient does not have an increased likelihood of recurrence. The method [700] may then proceed with more queries, make a particular treatment recommendation ([740], [741]), or simply end.
When the queries are performed sequentially, they may be made in the order suggested by FIG. 7 or in any other order. Whether subsequent queries are made can also be dependent on the results/answers for preceding queries. In some embodiments of the method illustrated in FIG. 7, for example, the method asks about clinical parameters [712] first and, if the patient has one or more clinical parameters identifying the patient as at increased risk for recurrence then the method concludes such [730] or optionally confirms by querying CCG status, while if the patient has no such clinical parameters then the method proceeds to ask about CCG status [711]. Optionally, if CCG status is not elevated, then the method may continue to ask about PTEN status [710]. As mentioned above, the preceding order of queries may be modified. In some embodiments an answer of “yes” to one query (e.g., [712]) prompts one or more of the remaining queries to confirm that the patient has increased risk of recurrence.
In some embodiments, the computer-implemented method of the invention [700] is open-ended. In other words, the apparent first step [710, 711, and/or 712] in FIG. 7 may actually form part of a larger process and, within this larger process, need not be the first step/query. Additional steps may also be added onto the core methods discussed above. These additional steps include, but are not limited to, informing a health care professional (or the patient itself) of the conclusion reached; combining the conclusion reached by the illustrated method [700] with other facts or conclusions to reach some additional or refined conclusion regarding the patient's diagnosis, prognosis, treatment, etc.; making a recommendation for treatment (e.g., “patient should/should not undergo radical prostatectomy”); additional queries about additional biomarkers, clinical parameters, or other useful patient information (e.g., age at diagnosis, general patient health, etc.).
Regarding the above computer-implemented method [700], the answers to the queries may be determined by the method instituting a search of patient data for the answer. For example, to answer the respective queries [710, 711, 712], patient data may be searched for PTEN status (e.g., PTEN IHC or mutation screening), CCG status (e.g., CCG expression level data), or clinical parameters (e.g., Gleason score, nomogram score, etc.). If such a comparison has not already been performed, the method may compare these data to some reference in order to determine if the patient has an abnormal (e.g., elevated, low, negative) status. Additionally or alternatively, the method may present one or more of the queries [710, 711, 712] to a user (e.g., a physician) of the computer system [100]. For example, the questions [710, 711, 712] may be presented via an output module [624]. The user may then answer “Yes” or “No” via an input module [630]. The method may then proceed based upon the answer received. Likewise, the conclusions [730, 731] may be presented to a user of the computer-implemented method via an output module [624].
Thus in some embodiments the invention provides a method comprising: accessing information on a patient's CCG status, clinical parameters and/or PTEN status stored in a computer-readable medium; querying this information to determine at least one of whether a sample obtained from the patient shows increased expression of at least one CCG, whether the patient has a recurrence-associated clinical parameter, and/or whether the patient has a low/negative PTEN status, outputting [or displaying] the sample's CCG expression status, the patient's recurrence-associated clinical parameter status, and/or the sample's PTEN status. As used herein in the context of computer-implemented embodiments of the invention, “displaying” means communicating any information by any sensory means. Examples include, but are not limited to, visual displays, e.g., on a computer screen or on a sheet of paper printed at the command of the computer, and auditory displays, e.g., computer generated or recorded auditory expression of a patient's genotype.
As discussed at length above, recurrence-associated clinical parameters or PTEN status combined with elevated CCG status indicate a significantly increased likelihood of recurrence. Thus some embodiments provide a computer-implemented method of determining whether a patient has an increased likelihood of recurrence comprising accessing information on a patient's PTEN status (e.g., from a tumor sample obtained from the patient) or clinical parameters and CCG status (e.g., from a tumor sample obtained from the patient) stored in a computer-readable medium; querying this information to determine at least one of whether the patient has a low/negative PTEN status or whether the patient has a recurrence-associated clinical parameter; querying this information to determine whether a sample obtained from the patient shows increased expression of at least one CCG; outputting (or displaying) an indication that the patient has an increased likelihood of recurrence if the patient has a low/negative PTEN status or a recurrence-associated clinical parameter and the sample shows increased expression of at least one CCG. Some embodiments further comprise displaying PTEN, clinical parameters (or their values) and/or the CCGs and their status (including, e.g., expression levels), optionally together with an indication of whether the PTEN or CCG status and/or clinical parameter indicates increased likelihood of risk.
The practice of the present invention may also employ conventional biology methods, software and systems. Computer software products of the invention typically include computer readable media having computer-executable instructions for performing the logic steps of the method of the invention. Suitable computer readable medium include floppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM, magnetic tapes and etc. Basic computational biology methods are described in, for example, Setubal et al., INTRODUCTION TO COMPUTATIONAL BIOLOGY METHODS (PWS Publishing Company, Boston, 1997); Salzberg et al. (Ed.), COMPUTATIONAL METHODS IN MOLECULAR BIOLOGY, (Elsevier, Amsterdam, 1998); Rashidi & Buehler, BIOINFORMATICS BASICS: APPLICATION IN BIOLOGICAL SCIENCE AND MEDICINE (CRC Press, London, 2000); and Ouelette & Bzevanis, BIOINFORMATICS: A PRACTICAL GUIDE FOR ANALYSIS OF GENE AND PROTEINS (Wiley & Sons, Inc., 2^nded., 2001); see also, U.S. Pat. No. 6,420,108.
The present invention may also make use of various computer program products and software for a variety of purposes, such as probe design, management of data, analysis, and instrument operation. See U.S. Pat. Nos. 5,593,839; 5,795,716; 5,733,729; 5,974,164; 6,066,454; 6,090,555; 6,185,561; 6,188,783; 6,223,127; 6,229,911 and 6,308,170. Additionally, the present invention may have embodiments that include methods for providing genetic information over networks such as the Internet as shown in U.S. Ser. No. 10/197,621 (U.S. Pub. No. 20030097222); Ser. No. 10/063,559 (U.S. Pub. No. 20020183936), Ser. No. 10/065,856 (U.S. Pub. No. 20030100995); Ser. No. 10/065,868 (U.S. Pub. No. 20030120432); Ser. No. 10/423,403 (U.S. Pub. No. 20040049354).
Techniques for analyzing such expression, activity, and/or sequence data (indeed any data obtained according to the invention) will often be implemented using hardware, software or a combination thereof in one or more computer systems or other processing systems capable of effectuating such analysis.
Thus one aspect of the present invention provides systems related to the above methods of the invention. In one embodiment the invention provides a system for determining gene expression in a tumor sample, comprising:

- (1) a sample analyzer for determining the expression levels in a sample of a panel of genes including at least 4 CCGs, wherein the sample analyzer contains the sample, RNA from the sample and expressed from the panel of genes, or DNA synthesized from said RNA;
- (2) a first computer program for
  - (a) receiving gene expression data on at least 4 test genes selected from the panel of genes,
  - (b) weighting the determined expression of each of the test genes with a predefined coefficient, and
  - (c) combining the weighted expression to provide a test value, wherein the combined weight given to said at least 4 or 5 or 6 CCGs is at least 40% (or 50%, 60%, 70%, 80%, 90%, 95% or 100%) of the total weight given to the expression of all of said plurality of test genes; and optionally
- (3) a second computer program for comparing the test value to one or more reference values each associated with a predetermined degree of risk of cancer.
  In some embodiments at least 20%, 50%, 75%, or 90% of said plurality of test genes are CCGs. In some embodiments the sample analyzer contains reagents for determining the expression levels in the sample of said panel of genes including at least 4 CCGs. In some embodiments the sample analyzer contains CCG-specific reagents as described below.

In another embodiment the invention provides a system for determining gene expression in a tumor sample, comprising: (1) a sample analyzer for determining the expression levels of a panel of genes in a tumor sample including at least 4 CCGs, wherein the sample analyzer contains the tumor sample which is from a patient identified as having prostate cancer, breast cancer, brain cancer, bladder cancer, or lung cancer, RNA from the sample and expressed from the panel of genes, or DNA synthesized from said RNA; (2) a first computer program for (a) receiving gene expression data on at least 4 test genes selected from the panel of genes, (b) weighting the determined expression of each of the test genes with a predefined coefficient, and (c) combining the weighted expression to provide a test value, wherein the combined weight given to said at least 4 or 5 or 6 CCGs is at least 40% (or 50%, 60%, 70%, 80%, 90%, 95% or 100%) of the total weight given to the expression of all of said plurality of test genes; and optionally (3) a second computer program for comparing the test value to one or more reference values each associated with a predetermined degree of risk of cancer recurrence or progression of the prostate cancer, breast cancer, brain cancer, bladder cancer, or lung cancer. In some embodiments at least 20%, 50%, 75%, or 90% of said plurality of test genes are CCGs. In some embodiments the system comprises a computer program for determining the patient's prognosis and/or determining (including quantifying) the patient's degree of risk of cancer recurrence or progression based at least in part on the comparison of the test value with said one or more reference values.
In some embodiments, the system further comprises a display module displaying the comparison between the test value and the one or more reference values, or displaying a result of the comparing step, or displaying the patient's prognosis and/or degree of risk of cancer recurrence or progression.
In a preferred embodiment, the amount of RNA transcribed from the panel of genes including test genes (and/or DNA reverse transcribed therefrom) is measured in the sample. In addition, the amount of RNA of one or more housekeeping genes in the sample (and/or DNA reverse transcribed therefrom) is also measured, and used to normalize or calibrate the expression of the test genes, as described above.
In some embodiments, the plurality of test genes includes at least 2, 3 or 4 CCGs, which constitute at least 50%, 75% or 80% of the plurality of test genes, and preferably 100% of the plurality of test genes. In some embodiments, the plurality of test genes includes at least 5, 6 or 7, or at least 8 CCGs, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes. Thus in some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises the top 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more CCGs listed in Table 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 of the following genes: ASPM, BIRC5, BUB1B, CCNB2, CDC2, CDC20, CDCA8, CDKN3, CENPF, DLGAP5, FOXM1, KIAA0101, KIF11, KIF2C, KIF4A, MCM10, NUSAP1, PRC1, RACGAP1, and TPX2. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, or ten or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, or 1 to 10 of any of Tables 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, or nine or all of gene numbers 2 & 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, or 2 to 10 of any of Tables 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, or eight or all of gene numbers 3 & 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or 3 to 10 of any of Tables 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, or seven or all of gene numbers 4 & 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, or 4 to 10 of any of Tables 9, 11, 23, 24, or 25. In some embodiments the plurality of test genes comprises at least some number of CCGs (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more CCGs) and this plurality of CCGs comprises any one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, 14, or 15 or all of gene numbers 1 & 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 1 to 13, 1 to 14, or 1 to 15 of any of Tables 9, 11, 23, 24, or 25.
In some other embodiments, the plurality of test genes includes at least 8, 10, 12, 15, 20, 25 or 30 CCGs, which constitute at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80% or 90% of the plurality of test genes, and preferably 100% of the plurality of test genes.
The sample analyzer can be any instrument useful in determining gene expression, including, e.g., a sequencing machine (e.g., Illumina HiSeq™, Ion Torrent PGM, ABI SOLiD™ sequencer, PacBio RS, Helicos Heliscope™, etc.), a real-time PCR machine (e.g., ABI 7900, Fluidigm BioMark™, etc.), a microarray instrument, etc.
In one aspect, the present invention provides methods of treating a cancer patient comprising obtaining CCG status information (e.g., the CCGs in Table 1 or Panels A through G), and recommending, prescribing or administering a treatment for the cancer patient based on the CCG status. In some embodiments, the method further includes obtaining clinical parameter information, and/or obtaining PTEN status information from a sample from the patient and treating the patient with a particular treatment based on the CCG status, clinical parameter and/or PTEN status information. For example, the invention provides a method of treating a cancer patient comprising:

- (1) determining the status of at least one CCG;
- (2) determining the status of at least on clinical parameter;
- (3) determining the status of PTEN in a sample obtained from the patient; and
- (4) recommending, prescribing or administering either
  - (a) an active (including aggressive) treatment if the patient has at least one of increased expression of the CCG, recurrence-associated clinical parameter, or low/negative PTEN status, or
  - (b) a passive (or less aggressive) treatment if the patient has none of increased expression of the CCG, recurrence-associated clinical parameter, or low/negative PTEN status.

Whether a treatment is aggressive or not will generally depend on the cancer-type, the age of the patient, etc. For example, in breast cancer adjuvant chemotherapy is a common aggressive treatment given to complement the less aggressive standards of surgery and hormonal therapy. Those skilled in the art are familiar with various other aggressive and less aggressive treatments for each type of cancer. “Active treatment” in prostate cancer is well-understood by those skilled in the art and, as used herein, has the conventional meaning in the art. Generally speaking, active treatment in prostate cancer is anything other than “watchful waiting.” Active treatment currently applied in the art of prostate cancer treatment includes, e.g., prostatectomy, radiotherapy, hormonal therapy (e.g., GnRH antagonists, GnRH agonists, antiandrogens), chemotherapy, high intensity focused ultrasound (“HIFU”), etc. Each treatment option carries with it certain risks as well as side-effects of varying severity, e.g., impotence, urinary incontinence, etc. Thus it is common for doctors, depending on the age and general health of the man diagnosed with prostate cancer, to recommend a regime of “watchful-waiting.”
“Watchful-waiting,” also called “active surveillance,” also has its conventional meaning in the art. This generally means observation and regular monitoring without invasive treatment. Watchful-waiting is sometimes used, e.g., when an early stage, slow-growing prostate cancer is found in an older man. Watchful-waiting may also be suggested when the risks of surgery, radiation therapy, or hormonal therapy outweigh the possible benefits. Other treatments can be started if symptoms develop, or if there are signs that the cancer growth is accelerating (e.g., rapidly rising PSA, increase in Gleason score on repeat biopsy, etc.).
Although men who choose watchful-waiting avoid the risks of surgery and radiation, watchful-waiting carries its own risks, e.g., increased risk of metastasis. For younger men, a trial of active surveillance may not mean avoiding treatment altogether, but may reasonably allow a delay of a few years or more, during which time the quality of life impact of active treatment can be avoided. Published data to date suggest that carefully selected men will not miss a window for cure with this approach. Additional health problems that develop with advancing age during the observation period can also make it harder to undergo surgery and radiation therapy. Thus it is clinically important to carefully determine which prostate cancer patients are good candidates for watchful-waiting and which patients should receive active treatment.
Thus, the invention provides a method of treating a prostate cancer patient or providing guidance to the treatment of a patient. In this method, the status of at least one CCG (e.g., those in Table 1 or Panels A through G), at least one recurrence-associated clinical parameter, and/or the status of PTEN is determined, and (a) active treatment is recommended, initiated or continued if a sample from the patient has an elevated status for at least one CCG, the patient has at least one recurrence-associated clinical parameter, and/or low/negative PTEN status, or (b) watchful-waiting is recommended/initiated/continued if the patient has neither an elevated status for at least one CCG, a recurrence-associated clinical parameter, nor low/negative PTEN status. In certain embodiments, CCG status, the clinical parameter(s) and PTEN status may indicate not just that active treatment is recommended, but that a particular active treatment is preferable for the patient (including relatively aggressive treatments such as, e.g., RP and/or adjuvant therapy).
In general, adjuvant therapy (e.g., chemotherapy, radiotherapy, HIFU, hormonal therapy, etc. after prostatectomy or radiotherapy) is not the standard of care in prostate cancer. According to the present invention, however, physicians may be able to determine which prostate cancer patients have particularly aggressive disease and thus should receive adjuvant therapy. Thus in one embodiment, the invention provides a method of treating a patient (e.g., a prostate cancer patient) comprising determining the status of at least one CCG (e.g., those in Table 1 or Panels A through G), the status of at least one recurrence-associated clinical parameter, and/or the status of PTEN and initiating adjuvant therapy after prostatectomy or radiotherapy if a sample from the patient has an elevated status for at least one CCG, the patient has at least one recurrence-associated clinical parameter and/or the patient has low/negative PTEN status.
In one aspect, the invention provides compositions for use in the above methods. Such compositions include, but are not limited to, nucleic acid probes hybridizing to PTEN or a CCG (or to any nucleic acids encoded thereby or complementary thereto); nucleic acid primers and primer pairs suitable for amplifying all or a portion of PTEN or a CCG or any nucleic acids encoded thereby; antibodies binding immunologically to a polypeptide encoded by PTEN or a CCG; probe sets comprising a plurality of said nucleic acid probes, nucleic acid primers, antibodies, and/or polypeptides; microarrays comprising any of these; kits comprising any of these; etc. In some aspects, the invention provides computer methods, systems, software and/or modules for use in the above methods.
In some embodiments the invention provides a probe comprising an isolated oligonucleotide capable of selectively hybridizing to PTEN or at least one of the genes in Table 1 or Panels A through G. The terms “probe” and “oligonucleotide” (also “oligo”), when used in the context of nucleic acids, interchangeably refer to a relatively short nucleic acid fragment or sequence. The invention also provides primers useful in the methods of the invention. “Primers” are probes capable, under the right conditions and with the right companion reagents, of selectively amplifying a target nucleic acid (e.g., a target gene). In the context of nucleic acids, “probe” is used herein to encompass “primer” since primers can generally also serve as probes.
The probe can generally be of any suitable size/length. In some embodiments the probe has a length from about 8 to 200, 15 to 150, 15 to 100, 15 to 75, 15 to 60, or 20 to 55 bases in length. They can be labeled with detectable markers with any suitable detection marker including but not limited to, radioactive isotopes, fluorophores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies, etc. See Jablonski et al., NUCLEIC ACIDS RES. (1986) 14:6115-6128; Nguyen et al., BIOTECHNIQUES (1992) 13:116-123; Rigby et al., J. MOL. BIOL. (1977) 113:237-251. Indeed, probes may be modified in any conventional manner for various molecular biological applications. Techniques for producing and using such oligonucleotide probes are conventional in the art.
Probes according to the invention can be used in the hybridization/amplification/detection techniques discussed above. Thus, some embodiments of the invention comprise probe sets suitable for use in a microarray in detecting, amplifying and/or quantitating PTEN and/or a plurality of CCGs. In some embodiments the probe sets have a certain proportion of their probes directed to CCGs—e.g., a probe set consisting of 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% probes specific for CCGs. In some embodiments the probe set comprises probes directed to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 600, 700, or 800 or more, or all, of the genes in Table 1 or Panels A through G. Such probe sets can be incorporated into high-density arrays comprising 5,000, 10,000, 20,000, 50,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, or 1,000,000 or more different probes. In other embodiments the probe sets comprise primers (e.g., primer pairs) for amplifying nucleic acids comprising at least a portion of PTEN or of one or more of the CCGs in Table 1 or Panels A through G.
In another aspect of the present invention, a kit is provided for practicing the prognosis of the present invention. The kit may include a carrier for the various components of the kit. The carrier can be a container or support, in the form of, e.g., bag, box, tube, rack, and is optionally compartmentalized. The carrier may define an enclosed confinement for safety purposes during shipment and storage. The kit includes various components useful in determining the status of one or more CCGs and one or more housekeeping gene markers, using the above-discussed detection techniques. For example, the kit many include oligonucleotides specifically hybridizing under high stringency to mRNA or cDNA of the genes in Table 1 or Panels A through G. Such oligonucleotides can be used as PCR primers in RT-PCR reactions, or hybridization probes. In some embodiments the kit comprises reagents (e.g., probes, primers, and or antibodies) for determining the expression level of a panel of genes, where said panel comprises at least 25%, 30%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 99%, or 100% CCGs (e.g., CCGs in Table 1 or any of Panels A through G). In some embodiments the kit consists of reagents (e.g., probes, primers, and or antibodies) for determining the expression level of no more than 2500 genes, wherein at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 250, or more of these genes are CCGs (e.g., CCGs in Table 1 or any of Panels A through G).
The oligonucleotides in the detection kit can be labeled with any suitable detection marker including but not limited to, radioactive isotopes, fluorephores, biotin, enzymes (e.g., alkaline phosphatase), enzyme substrates, ligands and antibodies, etc. See Jablonski et al., Nucleic Acids Res., 14:6115-6128 (1986); Nguyen et al., Biotechniques, 13:116-123 (1992); Rigby et al., J. Mol. Biol., 113:237-251 (1977). Alternatively, the oligonucleotides included in the kit are not labeled, and instead, one or more markers are provided in the kit so that users may label the oligonucleotides at the time of use.
In another embodiment of the invention, the detection kit contains one or more antibodies selectively immunoreactive with one or more proteins encoded by PTEN or one or more CCGs or optionally any additional markers. Examples include antibodies that bind immunologically to PTEN or a protein encoded by a gene in Table 1 or Panels A through G. Methods for producing and using such antibodies have been described above in detail.
Various other components useful in the detection techniques may also be included in the detection kit of this invention. Examples of such components include, but are not limited to, Taq polymerase, deoxyribonucleotides, dideoxyribonucleotides, other primers suitable for the amplification of a target DNA sequence, RNase A, and the like. In addition, the detection kit preferably includes instructions on using the kit for practice the prognosis method of the present invention using human samples.
In a specific embodiment,

Example 1

The following cell cycle gene (CCG) signature was tested for predicting time to chemical recurrence after radical prostatectomy.


31-CCG Prostate Recurrence Signature

	AURKA
	BUB1
	CCNB1
	CCNB2
	CDC2
	CDC20
	CDC45L
	CDCA8
	CENPA
	CKS2
	DLG7
	DTL
	FOXM1
	HMMR
	KIF23
	KPNA2
	MAD2L1
	MELK
	MYBL2
	NUSAP1
	PBK
	PRC1
	PTTG1
	RRM2
	TIMELESS
	TPX2
	TRIP13
	TTK
	UBE2C
	UBE2S
	ZWINT

Mean mRNA expression for the above 31 CCGs was tested on 440 prostate tumor FFPE samples using a Cox Proportional Hazard model in Splus 7.1 (Insightful, Inc., Seattle Wash.). The p-value for the likelihood ratio test was 3.98×10⁻⁵.
The mean of CCG expression is robust to measurement error and individual variation between genes. In order to determine the optimal number of cell cycle genes for the signature, the predictive power of the mean was tested for randomly selected sets of from 1 to 30 of the CCGs listed above. This simulation showed that there is a threshold number of CCGs in a panel that provides significantly improved predictive power.

Example 2

In a univariate analysis a set of 31 CCGs (Table 3) was found to be a significant predictor of biochemical recurrence (p-value=1.8×10⁻⁹) after RP in prostate cancer patients. This signature was further evaluated to determine whether it added to an established clinical nomogram for prostate cancer recurrence (the Kattan-Stephenson nomogram). In summary, the nomogram was a highly significant predictor of recurrence (p-value 1.6×10⁻¹⁰) and, after adjusting for the nomogram, the CCG signature was a significant predictor of biochemical recurrence (p-value 4.8×10⁻⁵, Table 6).

Patients and Methods

Eight hundred four consecutive RP patients were followed for a median of 9.5 years. The patient characteristics and the treatment outcomes of the entire cohort have been previously reported (Swanson et al., UROL ONCOL. (2007) 25:110-114). Tissue blocks and/or slides from the final pathological evaluation with enough tissue for analysis were available for 430 patients. The cohort was divided randomly into 212 patients utilized for a training and 199 patient samples as a validation set.
Gene Expression (Statistical Methods):
Association between biochemical recurrence and CCG expression was evaluated using Cox PH models for time to recurrence. All of the p-values reported in this study were derived from a likelihood ratio test comparing the null model to the model containing the test variable. A set of 31 CCGs (Table 3, supra) was randomly selected. The assays were used to generate expression data from 212 patients in the training set. All of the expression data were generated in triplicate. The expression data were combined into a signature by calculating the mean expression level for 26 CCGs. Association between biochemical recurrence and CCG expression was evaluated using Cox PH models for time to recurrence.
Sample Preparation and Study Design:
RNA was isolated from FFPE tumor sections derived from 411 prostate cancer patients treated with RP. Representative 10 μm thick tumor sections were used to isolate RNA. When necessary, a pathologist guided macro- or micro-dissection of the sample was used to enrich for tumor tissue before RNA isolation. None of the samples in the validation cohort were micro-dissected. Prior to any analysis, the cohort was split into 212 patients for initial characterization of the signature (“training set”) and 199 patients for validation. The clinical characteristics of the training and validation cohort are listed on Table 5.

TABLE 5

Training	Validation	p-value	Statistic

Age in years at RP, mean (sd)

67.3

(5.9)

66.8

(5.8)

0.355

t-test

Ethnicity (% non-white)	2.80%	7.60%	0.042	Fisher's exact
Dissection method (% lcm)	24%	0%	NA	NA

Recurrence (%)

71/212

(33.5%)

72/199

(36.2%)

0.605

Fisher's exact

Days to recurrence, median	910	822	0.463	t-test
Days to follow-up, median	3373	3387	0.173	t-test
Pre-surgery PSA (median)	7.3	6.8	0.163	t-test of log

Seminal vesicle	23/212	(10.8%)	28/199	(14.1%)	0.37	Fisher's exact
Bladder	12/212	(5.7%)	17/199	(8.5%)	0.335	Fisher's exact
Lymph node	8/212	(3.8%)	10/199	(5.0%)	0.632	Fisher's exact
Capsular	100/212	(47.2%)	101/199	(50.8%)	0.49	Fisher's exact
Through capsule	59/212	(27.8%)	66/199	(33.2%)	0.283	Fisher's exact
Positive margins	43/212	(20.3%)	57/199	(28.6%)	0.051	Fisher's exact
Post-RP Gleason score > 6	80/212	(37.7%)	66/199	(33.2%)	0.354	Fisher's exact
Post-RP nomogram, mean (sd)	137	(19.5)	138	(23.0)	0.424	t-test

Results

The CCG expression signature (Table 3, supra) was predictive of disease recurrence in a univariate analysis (p-value=1.8×10⁻⁹, Table 6). The distribution of the signature score was skewed toward higher values (lower expression). The median value of signature score was used to divide the training cohort into two groups containing samples with either high or low CCG expression. The survival versus time for both groups is shown in FIG. 2.
Predictive power of the CCG signature after accounting for clinical variables typically included in a post-surgical nomogram (the Kattan-Stephenson nomogram) was also evaluated. The nomogram was a highly significant predictor of recurrence (p-value 1.6×10-10). After adjusting for the nomogram, the CCG signature was a significant predictor of biochemical recurrence (FIG. 3) in the discovery cohort (p-value 0.03) and in the clinical validation cohort (p-value 4.8×10⁻⁵).

TABLE 6

		CCG mean	Recurrence
N	Co-variates	p-value*	Hazard Ratio

TRAINING	212	none	0.00404	1.24
(31 CCGs)	204	post-surgery	0.03320	1.16
		nomogram
VALIDATION	199	none	1.8 × 10⁻⁹	2.68
(26-CCG subset)	197	post-surgery	4.8 × 10⁻⁵	1.94
		nomogram

*Mean of cell cycle gene expression with imputation of missing values, likelihood ratio test for Cox proportional hazards model.

To help understand the interaction between the nomogram and the CCG expression signature, a scatter plot comparing these predictors (FIG. 4) was generated (light gray stars represent patients whose cancer recurred while black stars represent patients whose cancer did not). Analysis of the scatter plot by KM means divided the samples into three clusters based on nomogram score only. Subsequently, it was discovered that the clusters were based on well-understood clinical parameters. The patients in the lowest scoring cluster (116/117) had organ-confined disease. Patients in the middle scoring cluster (48/60) had at least one post-surgical parameter known to be associated with poor outcome (i.e., disease through the capsule, disease positive lymph nodes, and/or disease positive seminal vesicles) and low pre-surgical PSA (<10 ng/ml). Patients in the highest scoring cluster had at least one unfavorable post-surgical parameter and high pre-surgery PSA. Next, the patients in the low and medium scoring clusters were divided by the mean of the CCG score. Outcomes for patients in the highest scoring cluster are adequately predicted by the nomogram and, therefore, were not divided further. As a result, the scatter plot defines five patient groups with disease recurrence rates of 2%, 40% (for two groups), 65%, and 80% (Table 7). The recurrence rate of all five groups versus time is shown in FIG. 5.

	TABLE 7

	Post-RP nomogram

CCG score	Low	Medium	High

Low
	1/62 (1.6%)	13/31 (41.9%)	16/20 (80%)
High	21/55 (38.2%)	19/29 (65.5%)

The scatter plot shown in FIG. 4 suggests that there is a non-linear interaction between the CCG signature and the post-surgical nomogram. That is, the CCG signature is a better predictor in patients with low nomogram scores. Therefore, the study tested for statistical evidence of an interaction between these variables in a multivariate model for predicting disease recurrence (Table 8). There was significant evidence for a favorable interaction in both training and validation studies. Including the interaction term in the model dramatically improved the prognostic significance of the CCG signature after adjusting for the nomogram (p-values of 0.0015 in training and 1.2×10⁻⁸in validation cohort).

TABLE 8

Statistical Summary

		Independent		Interaction p-	Variable p-	Recurrence
Cohort	N	variables	Co-variates	value	value	Hazard ratio

Training	204	nomogram	none	NA	1.6 × 10⁻¹⁰
	212	CCG signature	none	NA	0.004	1.24
	204	CCG signature	nomogram	0.021	0.0015
Validation	197	nomogram	none	NA	7.7 × 10⁻¹³
	199	CCG signature	none	NA	1.8 × 10⁻⁹	2.68
	197	CCG signature	nomogram	0.0001	1.28 × 10⁻⁸

Example 3

The following study aimed at determining the optimal number of CCGs to include in the signature. As mentioned above, CCG expression levels are correlated to each other so it was possible that measuring a small number of genes would be sufficient to predict disease outcome. In fact, single CCGs from the 31-gene set in Table 3 (Panel C) add significantly to the Kattan-Stephenson nomogram, as shown in Table 9 below (after adjustment for the nomogram and an interaction term between the nomogram and CCG expression):

TABLE 9

Gene #	Gene	p-value*

1	NUSAP1	2.8E−07
2	DLG7	5.9E−07
3	CDC2	6.0E−07
4	FOXM1	1.1E−06
5	MYBL2	1.1E−06
6	CDCA8	3.3E−06
7	CDC20	3.8E−06
8	RRM2	7.2E−06
9	PTTG1	1.8E−05
10	CCNB2	5.2E−05
11	HMMR	5.2E−05
12	BUB1	8.3E−05
13	PBK	1.2E−04
14	TTK	3.2E−04
15	CDC45L	7.7E−04
16	PRC1	1.2E−03
17	DTL	1.4E−03
18	CCNB1	1.5E−03
19	TPX2	1.9E−03
20	ZWINT	9.3E−03
21	KIF23	1.1E−02
22	TRIP13	1.7E−02
23	KPNA2	2.0E−02
24	UBE2C	2.2E−02
25	MELK	2.5E−02
26	CENPA	2.9E−02
27	CKS2	5.7E−02
28	MAD2L1	1.7E−01
29	UBE2S	2.0E−01
30	AURKA	4.8E−01
31	TIMELESS	4.8E−01

*p-value for likelihood ratio test of full (post-RP nomogram score + cell cycle expression + nomogram:cell cycle) vs reduced (post-RP nomogram score only) CoxPH model of time-to-recurrence.

To evaluate how smaller subsets of the larger CCG set (i.e., smaller CCG panels) performed, the study also compared how well the signature predicted outcome as a function of the number of CCGs included in the signature (FIG. 1). Time to chemical recurrence after prostate surgery was regressed on the CCG mean adjusted by the post-RP nomogram score. Data consist of TLDA assays expressed as deltaCT for 199 FFPE prostate tumor samples and 26 CCGs and were analyzed by a CoxPH multivariate model. P-values are for the likelihood ratio test of the full model (nomogram+cell cycle mean including interaction) vs the reduced model (nomogram only). As shown in Table 10 below and FIG. 1, small CCG signatures (e.g., 2, 3, 4, 5, 6 CCGS, etc.) add significantly to the Kattan-Stephenson nomogram:

	TABLE 10

	# of CCGs	Mean of log10(p-value)*

	1	−3.579
	2	−4.279
	3	−5.049
	4	−5.473
	5	−5.877
	6	−6.228

	*For 1000 randomly drawn subsets, size 1 through 6, of cell cycle genes.

Example 4

The aim of this experiment was to evaluate the association between PTEN mutations and biochemical recurrence in prostate cancer patients after radical prostatectomy. Somatic mutations in PTEN were found to be significantly associated with recurrence, and importantly, it added prognostic information beyond both the established clinical nomogram for prostate cancer recurrence (the Kattan-Stephenson nomogram) and the CCG signature score (described in Examples 1 & 2, supra).

Patients and Methods

Eight hundred four consecutive RP patients were followed for a median of 9.5 years. The patient characteristics and the treatment outcomes of the entire cohort have been previously reported (Swanson et al., UROL. ONCOL. (2007) 25:110-114). Tissue blocks and/or slides from the final pathological evaluation with enough tissue for analysis were available for 430 patients. Of these, 191 were selected for PTEN mutation screening based on the amount of available tumor.
Genomic DNA was isolated from the FFPE tumor samples for mutation screening of PTEN using the QIAamp DNA FFPE Tissue kit (Qiagen, Valencia, Calif.) according to the kit protocol. The FFPE slides were first stained with hematoxylin and eosin and examined by a pathologist to identify the tumor region. After deparaffinization, tumor tissue was cut out from the slides by a razor blade. For a few samples dissection was aided by laser capture microscopy (LCM), owing to the dispersion of the tumor cells
Mutations were detected by designing sequencing primers to interrogate the PTEN genomic sequence. The primers contained M13 forward and reverse tails to facilitate sequencing. After amplification, DNA sequence was determined on a Mega BASE 4500 (GE healthcare) using dye-primer chemistry as described in Frank et al., J. CLIN. ONCOL. (2002) 20:1480-1490. Due to the technical difficulties associated with sequencing DNA derived from FFPE material, each mutation was detected by at least two independent amplification and sequencing reactions.
Statistical Methods:
Unless otherwise specified, the association between biochemical recurrence and PTEN mutations was evaluated using Cox PH models for time to recurrence. The resultant p-values were derived from a likelihood ratio test comparing the null model to the model containing the test variable. In this example (Example 4), the CCG signature was derived from 26 CCGs (Panel D in Table 2, supra). All of the expression data were generated in triplicate. The expression data were combined into a signature by calculating the mean expression level for 26 CCGs. The clinical data were the variables included in the Kattan-Stephenson nomogram.

Results

PTEN mutations were found in 13 individuals (13/191). In this subset of 191 patients, PTEN was a significant predictor of biochemical recurrence (p-value=0.031). The recurrence rate in mutation carriers was 69% (9/13) compared to 36% (64/178) in non-mutant patients. The difference in recurrence rate is also significant using a Fisher's exact test (p-value=0.034). In the subset of patients with clinical parameter data, CCG signature score, and PTEN mutations, PTEN status was a significant predictor of biochemical recurrence after adjusting for both clinical parameters and CCG signature (p-value 0.024). Finally, the combination of PTEN mutation with CCG signature was a better predictor of outcome after adjusting for clinical parameters than using the CCG signature after adjusting for clinical parameters (p-value=0.0002 for the combination compared to 0.0028 for CCG only). These results show that PTEN mutations provide information about the likelihood of recurrence that is uncorrelated with either clinical parameters or CCG signature, and that using all three parameters to evaluate recurrence risk provides a more accurate estimate of recurrence probability than previously possible.

Example 5

This Example describes further studies to validate and refine some embodiments of the CCG signatures of the invention.

Patients and Methods

Eight hundred four consecutive radical prostatectomy patients were followed for a median of 9.5 years. The median age was 67 years. The clinical stage was T1 34%, T2 66% and T3<1%. The median preoperative PSA was 6.6 ng/ml with 72%<10 ng/ml and 28%>10 ng/ml. The specimens were inked and clinical parameters were recorded as to positive bladder neck or urethral margin, invasion into the capsule, extension through the capsule, positive margins and the involvement of the seminal vesicles. Biochemical recurrence was defined as a PSA>0.3 ng/ml. For this study we had access to clinical data on 690 patients. Tissue blocks and/or slides from the final pathological evaluation with enough tissue for analysis were available for 442 patients. The cohort was divided into 195 patients for a training cohort, and 247 patients for validation.

Selection of Genes

Assays of 126 CCGs and 47 HK (housekeeping) genes were run against 96 commercially obtained, anonymous prostate tumor FFPE samples without outcome or other clinical data. The working hypothesis was that the assays would measure with varying degrees of accuracy the same underlying phenomenon (cell cycle proliferation within the tumor for the CCGs, and sample concentration for the HK genes). Assays were ranked by the Pearson's correlation coefficient between the individual gene and the mean of all the candidate genes, that being the best available estimate of biological activity. Results for the correlation of each of the 126 CCGs to the mean are reported in Table 23. Not including CCGs with low average expression, or assays that produced sample failures, approximately half the CCGs had correlations less than 0.58, and a quarter of the HK genes had correlations less than 0.95. These assays were interpreted as not reflecting the underlying phenomenon and were eliminated, leaving a subset of 56 CCGs (Panel G) and 36 HK candidate genes (Tables 11 and 12). Correlation coefficients were recalculated on this subset, and the final selection was made from the ranked list.

TABLE 11

Complete list of evaluated CCGs (“Panel G”)

		Correl.
	Gene	w/ CCG
Gene #	Symbol	mean

1	FOXM1	0.908
2	CDC20	0.907
3	CDKN3	0.9
4	CDC2	0.899
5	KIF11	0.898
6	KIAA0101	0.89
7	NUSAP1	0.887
8	CENPF	0.882
9	ASPM	0.879
10	BUB1B	0.879
11	RRM2	0.876
12	DLGAP5	0.875
13	BIRC5	0.864
14	KIF20A	0.86
15	PLK1	0.86
16	TOP2A	0.851
17	TK1	0.837
18	PBK	0.831
19	ASF1B	0.827
20	C18orf24	0.817
21	RAD54L	0.816
22	PTTG1	0.814
23	KIF4A	0.814
24	CDCA3	0.811
25	MCM10	0.802
26	PRC1	0.79
27	DTL	0.788
28	CEP55	0.787
29	RAD51	0.783
30	CENPM	0.781
31	CDCA8	0.774
32	OIP5	0.773
33	SHCBP1	0.762
34	ORC6L	0.736
35	CCNB1	0.727
36	CHEK1	0.723
37	TACC3	0.722
38	MCM4	0.703
39	FANCI	0.702
40	KIF15	0.701
41	PLK4	0.688
42	APOBEC3B	0.67
43	NCAPG	0.667
44	TRIP13	0.653
45	KIF23	0.652
46	NCAPH	0.649
47	TYMS	0.648
48	GINS1	0.639
49	STMN1	0.63
50	ZWINT	0.621
51	BLM	0.62
52	TTK	0.62
53	CDC6	0.619
54	KIF2C	0.596
55	RAD51AP1	0.567
56	NCAPG2	0.535

TABLE 12

List of 15 housekeeping (HK) genes

	Gene Symbol	Correaltion with HK Mean

	RPL38	0.989
	UBA52	0.986
	PSMC1	0.985
	RPL4	0.984
	RPL37	0.983
	RPS29	0.983
	SLC25A3	0.982
	CLTC	0.981
	TXNL1	0.98
	PSMA1	0.98
	RPL8	0.98
	MMADHC	0.979
	RPL13A; LOC728658	0.979
	PPP2CA	0.978
	MRFAP1	0.978

Gene Expression

Total RNA was extracted from representative 5 μM thick FFPE tumor sections. The samples were de-paraffinized using a xylene bath and subsequently hydrated in graded series of ethanol baths. Afterward, the tumor region was dissected from the slide using a razor blade according to the pathologist instructions. Alternatively, the tumor region was dissected directly into an eppendorf tube and the paraffin was removed using xylene and washed with ethanol. After, samples were treated overnight with proteinase K digestion at 55° C. Total RNA was extracted using either RNeasy FFPE or miRNeasy (Qiagen) as described by the manufacturer (with the only exception being the extended proteinase K digestion described above). Isolated total RNA was treated with DNase I (Sigma) prior to cDNA synthesis. Subsequently, we employed the High-capacity cDNA Archive Kit (Applied Biosystems) to convert total RNA into single strand cDNA as described by the manufacturer. A minimum of 200 ng RNA was required for the RT reaction.
Prior to measuring expression levels, the cDNA was pre-amplified with a pooled reaction containing TaqMan™ assays. Pre-amplification reaction conditions were: 14 cycles of 95° C. for 15 sec and 60° C. for 4 minutes. The first cycle was modified to include a 10 minute incubation at 95° C. The amplification reaction was diluted 1:20 using the 1× TE buffer prior to loading on TaqMan™ Low Density Arrays (TLDA, Applied Biosystems) to measure gene expression.

CCG Score

The CCG score is calculated from RNA expression of 31 CCGs (Panel F) normalized by 15 housekeeper genes (HK). The relative numbers of CCGs (31) and HK genes (15) were optimized in order to minimize the variance of the CCG score. The CCG score is the unweighted mean of CT values for CCG expression, normalized by the unweighted mean of the HK genes so that higher values indicate higher expression. One unit is equivalent to a two-fold change in expression. Missing values were imputed using the mean expression for each gene determined in the training set using only good quality samples. The CCG scores were centered by the mean value, again determined in the training set.
A dilution experiment was performed on four of the commercial prostate samples to estimate the measurement error of the CCG score (se=0.10) and the effect of missing values. It was found that the CCG score remained stable as concentration decreased to the point of 5 failures out of the total 31 CCGs. Based on this result, samples with more than 4 missing values were not assigned a CCG score.
The CCG score threshold for determining low-risk was based on the lowest CCG score of recurrences in the training set. The threshold was then adjusted downward by 1 standard deviation in order to optimize the negative predictive value of the test.

Model of Clinical Risk

A Cox proportional hazards model was used to summarize the available clinical parameter data and estimate the prior clinical risk of biochemical recurrence for each patient. The data set consisted of 195 cases from the training set and 248 other cases with clinical parameter information but insufficient sample to measure RNA expression. Univariate tests were performed on clinical parameters known to be associated with outcome (see Table 13 below). Non-significant parameters were excluded from the model. A composite variable was created for organ-confined disease, with invasion defined as surgical margins, extracapsular extension, or involvement of any of seminal vesicles, bladder neck/urethral margins, or lymph nodes. The composite variable for organ-confined disease proved more significant in the model than any of its five components, some of which were inter-correlated or not prevalent. Model fitting was performed using the AIC criteria for post-operative covariates.

TABLE 13

Univariate analysis of clinical parameters
and association with biochemical recurrence

Cinical Variable	p-value*	# occurrences	Total	Frequency

BLADDER	0.0002	36	443	0.081
CAPSULAR	1.1 × 10⁻⁹	194	443	0.438
ETHNICITY	0.6741	416	439	0.948
(WHITE)
LYMPHNOD	0.0009	33	443	0.074
MARG.POS	6.1 × 10⁻¹¹	83	443	0.187
PATHGLEA	6.7 × 10⁻¹⁶	NA	443	NA
PATHGRAD	2.4 × 10⁻¹¹	NA	443	NA
PATHSTAG	3.1 × 10⁻¹⁵	NA	443	NA
PRE.PSA.LOG10	6.2 × 10⁻¹²	NA	443	NA
SEM.VES	3.0 × 10⁻⁸	56	443	0.126
SURGERY.YEAR	0.0803	NA	443	NA
THRU.CAP	1.3 × 10⁻¹⁰	114	443	0.257

*Cox PH p-value for likelihood ratio test

The final model (i.e., nomogram) has binary variables for organ-confined disease and Gleason score less than or equal to 6, and a continuous variable for logarithmic PSA (Table 14). This model includes all of the clinical parameters incorporated in the post-RP nomogram (i.e., Kattan-Stephenson nomogram) except for Year of RP and the two components of the Gleason score. The distribution of prior clinical risk shows three distinct nodes (FIG. 8). K-means clustering with 3 centers was used to set the threshold for the low-risk cluster, which comprises approximately 50% of the sample.

TABLE 14

Clinical Model

	Clinical Parameter	Coefficient	HR	p-value*

organ-confined disease	−0.827	0.44	3.4 × 10⁻⁶
Gleason score ≤ 6	−0.8734	0.42	4.2 × 10⁻⁷
log PSA	0.6678	1.95	2.0 × 10⁻⁴

*Cox PH p-value for likelihood ratio test

Statistical Analysis

Clinical parameters were compared between the training and validation sets using the Student's t-test for continuous parameters and Fisher's exact test for categorical parameters. The prior clinical risk of patients for biochemical recurrence after surgery was estimated by a post-RP nomogram score summarizing 7 covariates. K-means clustering of the nomogram score was used to categorize patients as low or high prior clinical risk. Expression data were expressed as the CT (the PCR cycle at which the fluorescence intensity exceeds a predetermined threshold) of each CCG normalized by the mean of the 15 housekeeper genes (Table 12 above).
Poor quality samples were excluded from analysis to eliminate poor quality samples or dubious readings without compromising the integrity of the signature by inadvertently excluding samples with low CCG expression. Accordingly, the thresholds for cleaning or filtering the data were set conservatively. Mean expression levels of the HK genes for each sample, which were higher than those of the CCGs, were used to identify poor quality samples. Technical metrics for the amplification efficiency and excessively high standard deviations of replicates were used to identify unreliable CT measurements. No failures of HK genes, and no more than 1 failure out of 3 replicates for CCGs, were allowed.
The association between biochemical recurrence and CCG expression after adjusting for clinical risk predicted by clinical parameters was evaluated using a Cox proportional hazards model for time-to-recurrence. The proportional hazards assumption of no time-dependence was tested for the full model of the CCG signature plus the binary clinical parameter score with an interaction term, and for the CCG signature only in the clinical risk subsets. It was not significant in either training or validation, indicating that there is no evidence for time-dependence. All of the p-values reported are from a likelihood ratio test comparing the reduced or null model to the model containing the test variable. Kaplan-Meier plots are used to show estimated survival probabilities for subsets of patients; however, p-values are from the Cox likelihood ratio test for the continuous values of the variable. All statistical analyses were performed in S+ Version 8.1.1 for Linux (TIBCO Spotfire) or R 2.9.0 (http://www.r-project.org).

Results

We isolated RNA from FFPE tumor sections derived from 442 prostate cancer patients treated with RP. The cohort was split into 195 patients for initial characterization of the signature (“training set”) and 247 patients for validation. The clinical parameters of the training and validation cohort are listed in Table 15. There were no significant differences after adjusting for multiple comparisons.

TABLE 15

Clinical parameters of training and validation patient cohorts

			p-	Statistical
Clinical Parameter	Training	Validation	value	Analysis

Age in years at RP,	67.5	(6.2)	66.8	(5.6)	0.204	t-test
mean (sd)

Ethnicity (% non-	3.10%	7.30%	0.058	Fisher's
white)	(2 Black, 3	(10 Black, 7		exact
	Hispanic, 1	Hispanic, 1
	other)	other)

Recurrence	73/195	(37.4%)	90/247	(36.4%)	0.843	Fisher's
						exact

Days to recurrence,	839	736	0.308	t-test
median
Days to follow-up,	3300	3332	0.556	t-test
median
Pre-RP surgery	7.4	6.4	0.022	t-test of
PSA, median				log

Seminal vesicles	23/195	(11.8%)	33/247	(13.4%)	0.668	Fisher's
						exact
Bladder neck/	12/195	(6.2%)	16/247	(6.5%)	1	Fisher's
urethral margin						exact
Lymph nodes	8/195	(4.1%)	12/247	(4.9%)	0.819	Fisher's
						exact
Capsular	104/195	(53.3%)	115/247	(46.6%)	0.18	Fisher's
penetration						exact
Through the	66/195	(33.8%)	73/247	(29.6%)	0.354	Fisher's
capsule						exact
Positive margins	51/195	(26.2%)	61/247	(24.7%)	0.742	Fisher's
						exact
Post-RP Gleason	114/195	(58.5%)	166/247	(67.2%)	0.06	Fisher's
score < 7						exact
Organ-confined	108/195	(55.4%)	156/247	(63.2%)	0.118	Fisher's
disease						exact
10-year PFP (95%	61%	(52%, 69%)	67%	(60%, 73%)	0.905	Log-rank
CI)						test

To analyze the CCG signature for this study, we tested 126 CCGs on RNA derived from 96 prostate tumors (Table 11). The tumor samples were anonymous and not associated with clinical data. From this set of genes, we selected 31 genes (Panel F) for inclusion in our signature (Table 16). The genes were selected based on their technical performance, and by how well each gene correlated with the mean expression level of the entire CCG set, in the 96 anonymous samples.

TABLE 16

CCG Signature from Training Set (Panel F)

	Symbol	GeneID

	ASF1B	55723
	ASPM	259266
	BIRC5	332
	BUB1B	701
	C18orf24	220134
	CDC2	983
	CDC20	991
	CDCA3	83461
	CDCA8	55143
	CDKN3	1033
	CENPF	1063
	CENPM	79019
	CEP55	55165
	DLGAP5	9787
	DTL	51514
	FOXM1	2305
	KIAA0101	9768
	KIF11	3832
	KIF20A	10112
	MCM10	55388
	NUSAP1	51203
	ORC6L	23594
	PBK	55872
	PLK1	5347
	PRC1	9055
	PTTG1	9232
	RAD51	5888
	RAD54L	8438
	RRM2	6241
	TK1	7083
	TOP2A	7153

To evaluate the prognostic utility of the CCG signature, we generated expression data on 195 patients in the training set. Since the individual gene expression levels were correlated, we combined them into a signature score by calculating the mean expression for the entire set of 31 genes (Panel F), normalized by 15 housekeepers (Table 12). The CCG score distribution was centered at zero, and each score unit corresponds to a 2-fold change in expression level. Poor quality samples were identified by observing either low expression of housekeeping genes or an unacceptable number of CCG failures, and excluded from the analysis. After applying our exclusion rules, there were 140 samples available for analysis. Association between biochemical recurrence and CCG expression was evaluated using Cox PH models for time to recurrence. A high CCG expression value was predictive of disease recurrence in a univariate analysis (p-value=0.01, Table 17).
Next, we evaluated the prognostic utility of the CCG signature after accounting for clinical parameters known to be associated with recurrence after RP. To account for clinical measures in our analysis, we created a model/nomogram that included preoperative PSA, Gleason score, and evidence of disease outside the prostate (i.e., any of either extracapsular extension, or positive post-surgical pathology on lymph nodes, margins, bladder neck, urethral margin or seminal vesicles). The model was optimized in 443 patients (Tables 13 & 14), including all patients for whom we had clinical data but were not in the validation set, and was a highly significant predictor of recurrence in the training cohort (p-value=2.5×10⁻¹¹). The distribution of the scores from the clinical model contained several modes (FIG. 8), separating high- and low-risk patient groups. Therefore, the score was used subsequently as a binary variable (high or low risk). The low-risk cluster correlated with a consistent set of clinical parameters. Specifically, the vast majority (215/218) had organ-confined disease and Gleason score<7. In addition, 80% had low pre-surgical PSA (<10 ng/ml). Patients in the high-risk cluster (N=225) were more heterogeneous, but tended to have clinical characteristics known to be associated with poor outcome (e.g., Gleason>6 and/or disease through the capsule).
Multivariate analysis of the training set incorporating our binary clinical model, showed evidence for a non-linear interaction between the expression signature and clinical parameters (Table 17). To help us understand the nature of this interaction, we generated a scatter plot comparing these predictors (FIG. 8). As evident from the figure, the CCG score proved useful for evaluating recurrence risk in patients defined as low risk by clinical parameters. In fact, even after adjusting for the clinical model within the low risk patients, the CCG signature was a strong predictor of biochemical recurrence (p-value=0.0071).

TABLE 17

Statistical Summary

Subset

31-gene training N = 19.5

31-gene validation N = 247

based	Main			Main
on clin.	effect p-	Interaction		effect p-	Interaction
model	value	p-value	n	value	p-value	n

CCG score		0.01		140	5.8 × 10⁻⁸		218
Binary clin.		5.1 × 10⁻⁶		133	1.1 × 10⁻¹⁰		215
risk (low vs
high)
CCG score		0.018	0.032	133	8.3 × 10⁻⁷	0.026	215
adjusted for
binary clin.
risk +
interaction
CCG score	low-risk	0.0038		54	7.5 × 10⁻⁵		112
only
Clin. risk	low-risk	0.22		54	0.044		112
score
CCG score	low-risk	0.0071		54	0.00019		112
adjusted for
clin, risk
(clin, risk vs
clin. risk +
CCG)
CCG score	high-risk	0.48		79	5.8 × 10⁻⁴		103
Clin. risk	high-risk	2.8 × 10⁻⁶		79	0.0076		103
score
CCG score	high-risk	0.51		79	0.0026		103
adjusted for
clin. risk
(clin, risk vs
clin. risk +
CCG)

We used our training data in the scatter plot to establish an optimized threshold score of −0.16 for the CCG signature (the mean CCG score is zero). FIG. 12 shows this threshold applied to the 443 patients studied in this example. Forty percent of low-risk patients fall below this threshold, and it was selected so that there were no recurrences 10-years after RP (i.e., negative predictive value (NPV) of 100%). As a result of establishing threshold values for both the clinical model and CCG score, the scatter plot was divided into four sections with recurrence rates of 0% (low CCG) and 26% (high CCG) for low-risk patients; and 60% (low CCG) and 50% for high-risk patients.
Next, we generated CCG expression data on 247 patients in our validation cohort. Thirty-two samples were eliminated from further analysis according to the exclusion rules developed on the training cohort. Panel F was a significant predictor of biochemical recurrence in a univariate analysis (p-value=5.8×10⁻⁸, Table 17). After adjusting for the binary clinical model, the CCG signature was highly predictive of recurrence in the validation cohort (p-value 8.3×10⁻⁷), and as in the training set, there was significant evidence for a non-linear interaction between variables. The CCG signature was informative across the entire spectrum of clinically defined risk (Table 17). In terms of validating the training results, the p-value for association between recurrence and CCG signature in low-risk patients was 1.9×10⁻⁴.
We applied the CCG threshold derived from our analysis of the training cohort to our validation data set (FIG. 9). Low risk patients with CCG scores below the threshold had a 10-year predicted recurrence rate of 5% (equivalent to validated NPV of 0.95). Overall, the combination of CCG score and clinical parameters divided the cohort into four groups with 10 year predicted recurrence rates of 5%, 22%, 36% and 70% (Table 18). The predicted recurrence rate versus CCG score for patients in the validation cohort is shown in FIGS. 10 & 11.

TABLE 18

Summary of recurrence rates in validation cohort
defined by clinical risk and CCG score

10-year recurrence rate

	Clinical risk	CCG score	Kaplan-Meier estimate	n

low	low	0.05	39
low	high	0.22	73
high	low	0.36	27
high	high	0.7	76

We tested our validated threshold versus various definitions of low-risk patients (Table 19). The signature score was a significant prognostic indicator in a variety of low-risk clinical definitions, and depending on definition, generated a 10-year predicted recurrence rate of 0.05 to 0.10.

TABLE 19

NPV of CCG signature in other definitions of low-risk patients

low CCP*

	10-yr predicted
Clinical definition of low risk	recurrence**	n	p-value***

Organ-confined disease and	0.05	39	9.4 × 10⁻⁴
Gleason score < 7 & PSA < 10
Organ-confined disease and	0.08	40	5.8 × 10⁻³
Gleason score < 7
Organ-confined disease and	0.07	42	8.7 × 10⁻⁴
Gleason score < 8 & PSA < 10
Organ-confined disease and	0.1	43	4.1 × 10⁻³
Gleason score < 8
Organ-confined disease only	0.1	44	2.4 × 10⁻³

*defined by validated threshold
**Kaplan-Meier estimates
***for difference between KM estimates for low and high risk adjusted by Greenwood variance.

Comment

We have developed and validated a prognostic molecular signature for prostate cancer. The signature is based on measuring mRNA expression levels of cell cycle genes (CCGs). By definition, expression of CCGs is regulated as a function of cell cycle stage. That is, they are turned on at specific cell cycle stages, so that actively growing cells have higher expression levels of CCG than quiescent cells. Presumably this fact underlies the signature's ability to predict cancer progression. Without wishing to be bound by theory, it is thought that by measuring the expression levels of CCG we are indirectly measuring the growth rate and inherent aggressiveness of the tumor, which ultimately impacts on the likelihood of prostate cancer recurrence after prostatectomy.
There is an important distinction between this study and many others that have attempted to generate prognostic molecular signatures. Often, similar studies begin with a very large number of candidate biomarkers (sometimes exceeding 1000's of genes) that are then evaluated for association with a clinical phenotype of interest. This approach may at times suffer from inherent multiple testing which can make the significance of the derived signature uncertain. Here we have tested a single hypothesis: CCG would be prognostic in prostate cancer (in fact we selected genes based on their correlation with CCG expression, not based on association with recurrence). And since CCG expression is correlated, we combined the expression data into a predictive signature by determining the mean expression value of all the genes in the signature. The simplicity of this approach, biologically and computationally, supports the view that the central claim of this study is likely to be highly robust, and replicated in subsequent studies.
The CCG signature (Panel F) is independently predictive and adds significantly to the predictive power of the clinical parameters typically employed to predict disease recurrence after surgery. This is true in both our training and validation cohorts.
The signature is immediately useful for defining the risk of patients who present with low-risk clinical parameters. Here, we essentially defined low-risk as Gleason<7, PSA<10 and organ-confined disease. The CCG signature score effectively subdivides the low-risk group into patients with very low recurrence rates (5%), and a higher risk of recurrence (22%) (FIG. 9 & Table 18). This is the most dramatic effect of the molecular signature—accurately redefining the risk of patients previously defined as low-risk based on clinical parameters. It is noteworthy that within this patient subpopulation (i.e., patients defined as low-risk based on clinical parameters) clinical parameters are not particularly prognostic (see Table 17). Therefore as a diagnostic test, the signature could be useful for a large number of patients. In this study, nearly 60% of the cohort was characterized as low-risk and 40% of those are expected to have low CCG scores. Therefore, the CCG signature can predict indolent disease in a quarter of the patients who have previously been identified as high-risk (and therefore identified as candidates for radical prostatectomy). Finally, the validation data in particular suggests that the CCG signature may be useful for defining risk in all patients. Specifically, it helped to divide patients defined as high-risk according to clinical parameters into those with 30% and 70% recurrence rates (Table 18).
The combination of clinical parameters and CCG signature enables physicians to more accurately predict risk of surgical failure, and therefore, identify the appropriate course of therapeutic intervention. As we have shown, the signature dramatically improves the recurrence prediction for patients who present with general clinical parameters of non-aggressive disease (Table 19). Within this clinical subgroup, patients with low CCG scores would benefit from the absolute reassurance that no further treatment is indicated. Conversely, the high CCG group may warrant immediate intervention. Patients with unfavorable post-surgical clinical parameters benefit from adjuvant radiation therapy. Therefore the CCG signature should predict the efficacy of adjuvant radiation for patients with low-risk clinical characteristics and high CCG scores. In the validation cohort, patients with high CCG scores and disease beyond the prostate have a recurrence rate of 70%, which should clearly identify patients who are good candidates for adjuvant radiation. Thus the combination of clinical parameters and CCG signature clearly leads to more accurately defined patient risk, which should enable a more intelligent assessment of the need for further treatment.

Example 6

Some of the CCGs panels described herein were further evaluated for their ability to prognose additional cancers. Panels C, D, and F were found to be prognostic to varying degrees in bladder, brain, breast, and lung cancer.

Methods

Gene expression and patient data was obtained from the following publicly available datasets: GSE7390 (Desmedt et al., CLIN. CANCER RES. (2007) 13:3207-14; PMID 17545524); GSE11121 (Schmidt et al., CANCER RES. (2008) 68:5405-13; PMID 18593943); GSE8894 (Son et al.; no publication); Shedden (Shedden et al., NATURE MED. (2008) 14:822; PMID 18641660); GSE4412 (Freije et al., CANCER RES. (2004) 64:6503-10; PMID 15374961); GSE4271 (Phillips et al., CANCER CELL (2006) 9:157-73; PMID 16530701); GSE5287 (Als et al., CLIN. CANCER RES. (2007) 13:4407-14; PMID 17671123). Each of these datasets has an associated detailed description of the experimental procedures used in gathering expression and patient data. The expression microarrays used to generate each dataset are summarized below in Table 20.

	TABLE 20

	Dataset	Array

	GSE7390	Affymetrix U133 A
	GSE11121	Affymetrix U133 A
	GSE8894	Affymetrix U133 plus 2.0
	Shedden	Affymetrix U133 A
	GSE4412	Affymetrix U133 A and B
	GSE4271	Affymetrix U133 A and B
	GSE5287	Affymetrix U133 A

Expression data for each of the genes in Panels C, D and F was gathered from these datasets and the mean expression level for each Panel was determined for each patient, whose clinical outcome was known (e.g., recurrence, progression, progression-free survival, overall survival, etc.). CCG score is an average expression of the genes in a panel. If a gene is represented by more than one probe set on the array, the gene expression is an average expression of all the probe sets representing the gene. The association between CCG score and survival or disease recurrence was tested using univariate and multivariate Cox proportional hazard model. Multivariate analysis was performed when relevant clinical parameters (grade in brain cancer, stage in lung cancer, NPI in breast cancer) were available.

Results

As shown in Table 21 below, each Panel, in univariate analysis, was a prognostic factor in each of the cancers analyzed.

	TABLE 21

	p-value

Cancer Type	Dataset	Panel C	Panel F	Panel B

ER positive breast cancer	GSE7390	2.4 × 10⁻³	2.3 × 10⁻³	4.3 × 10⁻³
ER positive breast cancer	GSE11121	1.2 × 10⁻⁵	8.7 × 10⁻⁶	1.5 × 10⁻⁵
Lung adenocarcinoma	GSE8894	2.0 × 10⁻³	2.5 × 10⁻³	5.6 × 10⁻³
Lung adenocarcinoma	Shedden	1.3 × 10⁻⁷	2.6 × 10⁻⁷	2.2 × 10⁻⁷
Brain cancer	GSE4412	3.2 × 10⁻⁵	2.2 × 10⁻⁵	9.0 × 10⁻⁵
Brain cancer	GSE4271	1.3 × 10⁻³	1.0 × 10⁻³	2.8 × 10⁻⁴
Bladder cancer	GSE5287	6.4 × 10⁻²	5.0 × 10⁻²	8.6 × 10⁻²

As shown in Table 22 below, each Panel was also prognostic in multivariate analysis when combined with at least one clinical parameter (or nomogram).

	TABLE 22

	p-value	Additional Clinical

Cancer Type	Dataset	Panel C	Panel F	Panel B	Variable/Nomogram

Brain cancer	GSE4271	0.022	0.017	0.0065	grade
Lung	Shedden
	1 × 10⁻⁶	2.1 × 10⁻⁶	1.4 × 10⁻⁶	stage
adenocarcinoma
ER positive breast	GSE7390	0.0077	0.0064	0.011	Nottingham Prognostic
cancer					Index (NPI)
ER positive breast	GSE11121	0.0041	0.0027	0.0045	NPI
cancer

Tables 23 & 24 below provide rankings of select CCGs according to their correlation with the mean CCG expression.

TABLE 23

		Correl.
	Gene	w/
Gene #	Symbol	Mean

1	TPX2	0.931
2	CCNB2	0.9287
3	KIF4A	0.9163
4	KIF2C	0.9147
5	BIRC5	0.9077
6	BIRC5	0.9077
7	RACGAP1	0.9073
8	CDC2	0.906
9	PRC1	0.9053
10	DLGAP5/	0.9033
	DLG7
11	CEP55	0.903
12	CCNB1	0.9
13	TOP2A	0.8967
14	CDC20	0.8953
15	KIF20A	0.8927
16	BUB1B	0.8927
17	CDKN3	0.8887
18	NUSAP1	0.8873
19	CCNA2	0.8853
20	KIF11	0.8723
21	CDCA8	0.8713
22	NCAPG	0.8707
23	ASPM	0.8703
24	FOXM1	0.87
25	NEK2	0.869
26	ZWINT	0.8683
27	PTTG1	0.8647
28	RRM2	0.8557
29	TTK	0.8483
30	TRIP13	0.841
31	GINS1	0.841
32	CENPF	0.8397
33	HMMR	0.8367
34	NCAPH	0.8353
35	NDC80	0.8313
36	KIF15	0.8307
37	CENPE	0.8287
38	TYMS	0.8283
39	KIAA0101	0.8203
40	FANCI	0.813
41	RAD51AP1	0.8107
42	CKS2	0.81
43	MCM2	0.8063
44	PBK	0.805
45	ESPL1	0.805
46	MKI67	0.7993
47	SPAG5	0.7993
48	MCM10	0.7963
49	MCM6	0.7957
50	OIP5	0.7943
51	CDC45L	0.7937
52	KIF23	0.7927
53	EZH2	0.789
54	SPC25	0.7887
55	STIL	0.7843
56	CENPN	0.783
57	GTSE1	0.7793
58	RAD51	0.779
59	CDCA3	0.7783
60	TACC3	0.778
61	PLK4	0.7753
62	ASF1B	0.7733
63	DTL	0.769
64	CHEK1	0.7673
65	NCAPG2	0.7667
66	PLK1	0.7657
67	TIMELESS	0.762
68	E2F8	0.7587
69	EXO1	0.758
70	ECT2	0.744
71	STMN1	0.737
72	STMN1	0.737
73	RFC4	0.737
74	CDC6	0.7363
75	CENPM	0.7267
76	MYBL2	0.725
77	SHCBP1	0.723
78	ATAD2	0.723
79	KIFC1	0.7183
80	DBF4	0.718
81	CKS1B	0.712
82	PCNA	0.7103
83	FBXO5	0.7053
84	C12orf48	0.7027
85	TK1	0.7017
86	BLM	0.701
87	KIF18A	0.6987
88	DONSON	0.688
89	MCM4	0.686
90	RAD54B	0.679
91	RNASEH2A	0.6733
92	TUBA1C	0.6697
93	C18orf24	0.6697
94	SMC2	0.6697
95	CENPI	0.6697
96	GMPS	0.6683
97	DDX39	0.6673
98	POLE2	0.6583
99	APOBEC3B	0.6513
100	RFC2	0.648
101	PSMA7	0.6473
102	MPHOSPH1/	0.6457
	kif20b
103	CDT1	0.645
104	H2AFX	0.6387
105	ORC6L	0.634
106	C1orf135	0.6333
107	PSRC1	0.633
108	VRK1	0.6323
109	CKAP2	0.6307
110	CCDC99	0.6303
111	CCNE1	0.6283
112	LMNB2	0.625
113	GPSM2	0.625
114	PAICS	0.6243
115	MCAM	0.6227
116	DSN1	0.622
117	NCAPD2	0.6213
118	RAD54L	0.6213
119	PDSS1	0.6203
120	HN1	0.62
121	C21orf45	0.6193
122	CTSL2	0.619
123	CTPS	0.6183
124	MCM7	0.618
125	ZWILCH	0.618
126	RFC5	0.6177

TABLE 24

	Gene	Correl. w/
Gene #	Symbol	CCG mean

1	DLGAP5	0.931
2	ASPM	0.931
3	KIF11	0.926
4	BIRC5	0.916
5	CDCA8	0.902
6	CDC20	0.9
7	MCM10	0.899
8	PRC1	0.895
9	BUB1B	0.892
10	FOXM1	0.889
11	NUSAP1	0.888
12	C18orf24	0.885
13	PLK1	0.879
14	CDKN3	0.874
15	RRM2	0.871
16	RAD51	0.864
17	CEP55	0.862
18	ORC6L	0.86
19	RAD54L	0.86
20	CDC2	0.858
21	CENPF	0.855
22	TOP2A	0.852
23	KIF20A	0.851
24	KIAA0101	0.839
25	CDCA3	0.835
26	ASF1B	0.797
27	CENPM	0.786
28	TK1	0.783
29	PBK	0.775
30	PTTG1	0.751
31	DTL	0.737

Table 25 below provides a ranking of the CCGs in Panel F according to their relative predictive value in Example 5 (analogous to Table 9).

TABLE 25

	Gene
Gene #	Symbol	p-value

1	MCM10	8.60E−10
2	ASPM	2.30E−09
3	DLGAP5	1.20E−08
4	CENPF	1.40E−08
5	CDC20	2.10E−08
6	FOXM1	3.40E−07
7	TOP2A	4.30E−07
8	NUSAP1	4.70E−07
9	CDKN3	5.50E−07
10	KIF11	6.30E−06
11	KIF20A	6.50E−06
12	BUB1B	1.10E−05
13	RAD54L	1.40E−05
14	CEP55	2.60E−05
15	CDCA8	3.10E−05
16	TK1	3.30E−05
17	DTL	3.60E−05
18	PRC1	3.90E−05
19	PTTG1	4.10E−05
20	CDC2	0.00013
21	ORC6L	0.00017
22	PLK1	0.0005
23	C18orf24	0.0011
24	BIRC5	0.00118
25	RRM2	0.00255
26	CENPM	0.0027
27	RAD51	0.0028
28	KIAA0101	0.00348
29	CDCA3	0.00863
30	PBK	0.00923
31	ASF1B	0.00936

Table 1 below provides a large, but not exhaustive, list of CCGs.

	TABLE 1

	Gene (Name and/or Symbol) or Number (EST, cDNA clone, or Accession)

1	STK15: serine/threonine kinase 15 Hs.48915 R11407
2	PLK: polo (Drosophia)-like kinase Hs.77597 AA629262
3	UBCH10: ubiquitin carrier protein E2-C Hs.93002 AA430504
4	MAPK13: mitogen-activated protein kinase 13 Hs.178695 AA157499 p38delta
	mRNA = stress-activated protein kinase 4
5	CDC2: cell division cycle 2, G1 to S and G2 to M Hs.184572 AA598974
6	TOP2A: topoisomerase (DNA) II alpha (170 kD) Hs.156346 AA504348
7	CENPE: centromere protein E (312 kD) Hs.75573 AA402431 CENP-E = putative
	kinetochore motor that accumulates just befo
8	TOP2A: topoisomerase (DNA) II alpha (170 kD) Hs.156346 AA026682
9	KPNA2: karyopherin alpha 2 (RAG cohort 1, importin alpha 1) Hs.159557 AA676460
10	FLJ10468: hypothetical protein FLJ10468 Hs.48855 N63744
11	CCNF: cyclin F Hs.1973 AA676797
12	DKFZp762E1312: hypothetical protein DKFZp762E1312 Hs.104859 T66935
13	CKS2: CDC2-Associated Protein CKS2 Hs.83758 AA292964
14	C20ORF1: chromosome 20 open reading frame 1 Hs.9329 H73329
15	BUB1: budding uninhibited by benzimidazoles 1 (yeast homolog) Hs.98658
	AA430092 BUB1 = putative mitotic checkpoint protein ser/thr kinase
16	TOP2A: **topoisomerase (DNA) II alpha (170 kD) Hs.156346 AI734240
17	CKS2: CDC2-Associated Protein CKS1 Hs.83758 AA010065 ckshs2 = homolog of
	Cks1 = p34Cdc28/Cdc2-associated protein
18	ARL6IP: ADP-ribosylation factor-like 6 interacting protein Hs.75249 H20558
19	L2DTL: L2DTL protein Hs.126774 R06900
20	STK15: **serine/threonine kinase 15 Hs.48915 H63492 aurora/IPL1-related kinase
21	E2-EPF: ubiquitin carrier protein Hs.174070 AA464019
22	UBCH10: ubiquitin carrier protein E2-C Hs.93002 R80790
23	KNSL5: kinesin-like 5 (mitotic kinesin-like protein 1) Hs.270845 AA452513 Mitotic
	kinesin-like protein-1
24	CENPF: centromere protein F (350/400 kD, mitosin) Hs.77204 AA701455
25	CCNA2: cyclin A2 Hs.85137 AA608568 Cyclin A
26	CDC2: cell division cycle 2, G1 to S and G2 to M Hs.184572 AA278152 CDC2 = Cell
	division control protein 2 homolog = P34 protein kin
27	HMMR: **hyaluronan-mediated motility receptor (RHAMM) Hs.72550 AA171715
28	KIAA0008: KIAA0008 gene product Hs.77695 AA262211
29	HSPC145: HSPC145 protein Hs.18349 R22949
30	FLJ20510: hypothetical protein FLJ20510 Hs.6844 N53214
31	Homo sapiens NUF2R mRNA, complete cds Hs.234545 AA421171:
32	HSPC216: hypothetical protein Hs.13525 T87341
33	P37NB: 37 kDa leucine-rich repeat (LRR) protein Hs.155545 AA423870
34	CDC20:
35	CCNE1: cyclin E1 Hs.9700 T54121
36	ESTs: Hs.221754 R84407
37	FLJ11252: hypothetical protein FLJ11252 Hs.23495 N30185
38	LOC51203: clone HQ0310 PRO0310p1 Hs.279905 AA620485
39	FLJ10491: hypothetical protein FLJ10491 Hs.274283 AA425404
40	KNSL1: kinesin-like 1 Hs.8878 AA504625
41	CENPA: centromere protein A (17 kD) Hs.1594 AI369629
42	Homo sapiens, clone IMAGE: 2823731, mRNA, partial cds Hs.70704 R96941:
43	CDC6: CDC6 (cell division cycle 6, S. cerevisiae) homolog Hs.69563 H59203
44	Homo sapiens DNA helicase homolog (PIF1) mRNA, partial cds Hs.112160
	AA464521:
45	ESTs: Hs.48480 AA135809
46	TSN: translin Hs.75066 AA460927
47	KPNA2: karyopherin alpha 2 (RAG cohort 1, importin alpha 1) Hs.159557 AA489087
48	RRM2: ribonucleotide reductase M2 polypeptide Hs.75319 AA187351
49	ESTs: Hs.14119 AA204830
50	CCNB1: cyclin B1 Hs.23960 R25788
51	GTSE1: G-2 and S-phase expressed 1 Hs.122552 AI369284
52	C20ORF1: chromosome 20 open reading frame 1 Hs.9329 AA936183
53	TACC3: transforming, acidic coiled-coil containing protein 3 Hs.104019 AA279990
	JkR1 mRNA downregulated upon T-cell activation
54	E2F1: E2F transcription factor 1 Hs.96055 H61303
55	BUB1B: budding uninhibited by benzimidazoles 1 (yeast homolog), beta Hs.36708
	AA488324
56	ESTs,: Weakly similar to CGHU7L collagen alpha 1(III) chain precursor [H. sapiens]
	Hs.19322 AA088457
57	KIAA0074: KIAA0074 protein Hs.1192 N54344
58	MPHOSPH1: M-phase phosphoprotein 1 Hs.240 AA282935
59	ANLN: anillin (Drosophila Scraps homolog), actin binding protein Hs.62180 R12261
60	BIRC5: baculoviral IAP repeat-containing 5 (survivin) Hs.1578 AA460685
	Survivin = apoptosis inhibitor = effector cell protease EPR-1
61	PTTG1: pituitary tumor-transforming 1 Hs.252587 AA430032
62	KIAA0159: chromosome condensation-related SMC-associated protein 1 Hs.5719
	AA668256
63	ESTs,: Weakly similar to OS-4 protein [H. sapiens] Hs.18714 W93120
64	HMMR: hyaluronan-mediated motility receptor (RHAMM) Hs.72550 R10284
65	DKFZp762E1312: hypothetical protein DKFZp762E1312 Hs.104859 AA936181
66	CKAP2: cytoskeleton associated protein 2 Hs.24641 T52152
67	RAMP: RA-regulated nuclear matrix-associated protein
68	SMAP: thyroid hormone receptor coactivating protein Hs.5464 AA481555
69	FLJ22624: hypothetical protein FLJ22624 Hs.166425 AA488791
70	CKS1: CDC2-Associated Protein CKS1 Hs.77550 N48162
71	NEK2: NIMA (never in mitosis gene a)-related kinase 2 Hs.153704 W93379
72	MKI67: antigen identified by monoclonal antibody Ki-67
73	TTK: TTK protein kinase Hs.169840 AI337292
74	VEGFC: vascular endothelial growth factor C Hs.79141 H07899 vascular endothelial
	growth factor related protein VRP
75	CDKN3: cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity
	phosphatase) Hs.84113 AA284072 CIP2 = Cdi1 = KAP1 phosphatase = G1/S cell cycle
	gene
76	Homo sapiens NUF2R mRNA, complete cds Hs.234545 R92435:
77	Homo sapiens cDNA FLJ10325 fis, clone NT2RM2000569 Hs.245342 AA235662:
78	HSPC145: HSPC145 protein Hs.18349 AA628867
79	HSU54999: LGN protein Hs.278338 W92010
80	FLJ20333: hypothetical protein FLJ20333 Hs.79828 R27552
81	KNSL2: kinesin-like 2 Hs.20830 N69491
82	ESTs: Hs.133294 AI053446
83	**ESTs: Hs.41294 H95819
84	SMTN: smoothelin Hs.149098 AA449234
85	FLJ23311: hypothetical protein FLJ23311 Hs.94292 N73916
86	USF1: upstream transcription factor 1 Hs.247842 AA719022
87	LOC51203: clone HQ0310 PRO0310p1 Hs.279905 AA779949
88	ADH4: alcohol dehydrogenase 4 (class II), pi polypeptide Hs.1219 AA007395
89	ESTs: Hs.186579 AA960844
90	CCNB2: cyclin B2 Hs.194698 AA774665
91	Homo sapiens, Similar to gene rich cluster, C8 gene, clone MGC: 2577, mRNA,
	complete cds Hs.30114 AA634371:
92	ESTs: Hs.99480 AA485454
93	Homo sapiens IRE1b mRNA for protein kinase/ribonuclease IRE1 beta, complete cds
	Hs.114905 AA088442:
94	PCNA: proliferating cell nuclear antigen Hs.78996 AA450264 PCNA = proliferating
	cell nuclear antigen
95	AA075920:
96	GTSE1: G-2 and S-phase expressed 1 Hs.122552 AA449474
97	CKS1: CDC2-Associated Protein CKS1 Hs.77550 AA278629
98	CDC25B: cell division cycle 25B Hs.153752 AA448659 cdc25B = M-phase inducer
	phosphatase 2
99	ESTs,: Weakly similar to unnamed protein product [H. sapiens] Hs.99807 AA489023
	Unknown UG Hs.99807 ESTs sc_id384
100	PCNA: proliferating cell nuclear antigen Hs.78996 H05891
101	LTBP3: **latent transforming growth factor beta binding protein 3 Hs.289019 R60197
102	Homo sapiens mRNA; cDNA DKFZp434D0818 (from clone DKFZp434D0818)
	Hs.5855 N95578:
103	ESTs: Hs.126714 AA919126
104	CIT: citron (rho-interacting, serine/threonine kinase 21) Hs.15767 H10788
105	LBR: lamin B receptor Hs.152931 AA099136
106	E2F1: E2F transcription factor 1 Hs.96055 AA424949
107	AA699928:
108	CDKN2C: cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) Hs.4854
	N72115 p18-INK6 = Cyclin-dependent kinase 6 inhibitor
109	STK12: serine/threonine kinase 12 Hs.180655 H81023 ARK2 = aurora-related kinase 2
110	ESTs: Hs.111471 AA682533
111	ESTs: Hs.44269 AA465090
112	MCM4: minichromosome maintenance deficient (S. cerevisiae) 4 Hs.154443
	AA485983
113	PMSCL1: **polymyositis/scleroderma autoantigen 1 (75 kD) Hs.91728 AA458994
	Cyclin A
114	MKI67: antigen identified by monoclonal antibody Ki-67 Hs.80976 AA425973 Ki67
	(long type)
115	ESTs: Hs.133294 AI144063
116	CDC25B: cell division cycle 25B Hs.153752 H14343 cdc25B = M-phase inducer
	phosphatase 2
117	FOXM1: forkhead box M1 Hs.239 AA129552 MPP2 = putative M phase
	phosphoprotein 2
118	FLJ11029: hypothetical protein FLJ11029 Hs.274448 AI124082
119	H2AFX: H2A histone family, member X Hs.147097 H95392
120	FLJ20333: hypothetical protein FLJ20333 Hs.79828 AA147792
121	SLC17A2: solute carrier family 17 (sodium phosphate), member 2 Hs.19710 H60423
122	Homo sapiens IRE1b mRNA for protein kinase/ribonuclease IRE1 beta, complete cds
	Hs.114905 AA102368:
123	ESTs: Hs.163921 AA573689
124	MCM5: minichromosome maintenance deficient (S. cerevisiae) 5 (cell division cycle
	46) Hs.77171 AA283961
125	CDKN1B: cyclin-dependent kinase inhibitor 1B (p27, Kip1) Hs.238990 AA630082
126	AA779865:
127	PTTG1: pituitary tumor-transforming 1 Hs.252587 AI362866
128	RAD21: RAD21 (S. pombe) homolog Hs.81848 AA683102
129	Homo sapiens cDNA FLJ10325 fis, clone NT2RM2000569 Hs.245342 AA430511:
130	NEK2: NIMA (never in mitosis gene a)-related kinase 2 Hs.153704 AA682321
131	FLJ20101: LIS1-interacting protein NUDE1, rat homolog Hs.263925 N79612
132	FZR1: Fzr1 protein Hs.268384 AA621026
133	ESTs: Hs.120605 AI220472
134	KIAA0855: golgin-67 Hs.182982 AA098902
135	SRD5A1: steroid-5-alpha-reductase, alpha polypeptide 1 (3-oxo-5 alpha-steroid delta
	4-dehydrogenase alpha 1) Hs.552 H16833
136	RAD51: RAD51 (S. cerevisiae) homolog (E coli RecA homolog) Hs.23044 N70010
137	KNSL2: kinesin-like 2 Hs.20830 R11542
138	KIAA0097: KIAA0097 gene product Hs.76989 AA598942
139	TUBB: tubulin, beta polypeptide Hs.179661 AA427899
140	HEC: highly expressed in cancer, rich in leucine heptad repeats Hs.58169 W72679
141	TROAP: trophinin associated protein (tastin) Hs.171955 H94949
142	ESTs: Hs.49047 N64737
143	ESTs: Hs.15091 AA678348
144	ESTs: Hs.133431 AI061169
145	KIAA0042: KIAA0042 gene product Hs.3104 AA477501
146	FZR1: Fzr1 protein Hs.268384 AA862886
147	FEN1: flap structure-specific endonuclease 1 Hs.4756 AA620553
148	CKS1: CDC2-Associated Protein CKS1 Hs.77550 AA459292 ckshs1 = homolog of
	Cks1 = p34Cdc28/Cdc2-associated protein
149	ESTs: Hs.193379 N57936
150	CASP8AP2: CASP8 associated protein 2 Hs.122843 H50582
151	BIRC2: baculoviral IAP repeat-containing 2 Hs.289107 R19628 c-IAP1 = MIHB = IAP
	homolog B
152	CKAP2: cytoskeleton associated protein 2 Hs.24641 AA504130
153	HLA-DRA: major histocompatibility complex, class II, DR alpha Hs.76807 R47979
154	HBP: Hairpin binding protein, histone Hs.75257 AA629558
155	FLJ10483: hypothetical protein FLJ10483 Hs.6877 H12254
156	CASP3: caspase 3, apoptosis-related cysteine protease Hs.74552 R14760 CASPASE-
	3 = CPP32 isoform alpha = yama = cysteine protease
157	**ESTs,: Weakly similar to protein that is immuno-reactive with anti-PTH polyclonal
	antibodies [H. sapiens] Hs.301486 AA088258
158	HMG2: high-mobility group (nonhistone chromosomal) protein 2 Hs.80684
	AA019203
159	PRO2000: PRO2000 protein Hs.46677 H58234
160	FLJ20333: hypothetical protein FLJ20333 Hs.79828 T48760
161	T56726:
162	TIMP1: tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity,
	collagenase inhibitor) Hs.5831 H80214
163	ESTs: Hs.102004 R94281
164	FLJ10858: hypothetical protein FLJ10858 Hs.134403 AA677552
165	Homo sapiens cDNA FLJ11883 fis, clone HEMBA1007178 Hs.157148 N62451:
166	RFC4: replication factor C (activator 1) 4 (37 kD) Hs.35120 N93924 replication factor C
167	PRO2000: PRO2000 protein Hs.46677 N47113
168	ECT2: epithelial cell transforming sequence 2 oncogene Hs.132808 AI031571
169	ESTs: Hs.165909 AA629538
170	PCF11: PCF11p homolog Hs.123654 AA053411
171	BIRC3: baculoviral IAP repeat-containing 3 Hs.127799 H48533 c-IAP2 = MIHC = IAP
	homolog C = TNFR2-TRAF signalling complex prot
172	EST,: Weakly similar to dJ45P21.2 [H. sapiens] Hs.326451 AA931528
173	KIAA0952: KIAA0952 protein Hs.7935 AA454989
174	KIF5B: kinesin family member 5B Hs.149436 AA608707
175	DKFZP566C134: DKFZP566C134 protein Hs.20237 N39306
176	ANLN: anillin (Drosophila Scraps homolog), actin binding protein Hs.62180 R17092
177	ORC1L: origin recognition complex, subunit 1 (yeast homolog)-like Hs.17908 H51719
178	ESTs: Hs.14139 T77757
179	IFIT1: interferon-induced protein with tetratricopeptide repeats 1 Hs.20315 AA074989
180	MGC5338: hypothetical protein MGC5338 Hs.99598 AA463627
181	COPEB: core promoter element binding protein Hs.285313 AA013481
182	UK114: translational inhibitor protein p14.5 Hs.18426 N72715
183	ESTs: Hs.265592 H67282
184	HMG4: high-mobility group (nonhistone chromosomal) protein 4 Hs.19114
	AA670197
185	MDS025: hypothetical protein MDS025 Hs.154938 AI225067
186	DKFZP564A122: DKFZP564A122 protein Hs.187991 N53236
187	TSC22: transforming growth factor beta-stimulated protein TSC-22 Hs.114360
	AA664389
188	AAAS: aladin Hs.125262 AA916726
189	PLAG1: **pleiomorphic adenoma gene 1 Hs.14968 AA418251
190	FLJ23293: **hypothetical protein FLJ23293 similar to ARL-6 interacting protein-2
	Hs.31236 R91583
191	H11: protein kinase H11; small stress protein-like protein HSP22 Hs.111676
	AA010110
192	POLD3: polymerase (DNA directed), delta 3 Hs.82502 AA504204
193	SERPINB3: serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 3
	Hs.227948 AA292860
194	DNAJB1: DnaJ (Hsp40) homolog, subfamily B, member 1 Hs.82646 AA435948
195	ESTs: Hs.99480 AA458886
196	BUB3: BUB3 (budding uninhibited by benzimidazoles 3, yeast) homolog Hs.40323
	AA405690
197	TUBB2: tubulin, beta, 2 Hs.251653 AI000256
198	Homo sapiens SNC73 protein (SNC73) mRNA, complete cds Hs.293441 H28469:
199	BUB3: BUB3 (budding uninhibited by benzimidazoles 3, yeast) homolog Hs.40323
	H38804
200	FLJ20699: hypothetical protein FLJ20699 Hs.15125 AA459420
201	KIAA0013: KIAA0013 gene product Hs.172652 N63575
202	ESTs: Hs.20575 N20305
203	CDC25C: cell division cycle 25C Hs.656 W95000 cdc25C = M-phase inducer
	phosphatase 3
204	FLJ11186: hypothetical protein FLJ11186 Hs.89278 AA394225
205	TOPK: PDZ-binding kinase; T-cell originated protein kinase Hs.104741 AA448898
206	KIAA0165: extra spindle poles, S. cerevisiae, homolog of Hs.153479 AA948058
207	LOC51659: HSPC037 protein Hs.108196 AA961752
208	ESTs: Hs.10338 AA436456
209	SUCLG2: succinate-CoA ligase, GDP-forming, beta subunit Hs.247309 AA465233
210	ZNF265: zinc finger protein 265 Hs.194718 AA452256
211	SKP2: S-phase kinase-associated protein 2 (p45) Hs.23348 R22188
212	NS1-BP: NS1-binding protein Hs.197298 AA486796
213	C21ORF50: chromosome 21 open reading frame 50 Hs.4055 AA416628
214	BIRC2: baculoviral IAP repeat-containing 2 Hs.289107 AA702174
215	BIRC3: baculoviral IAP repeat-containing 3 Hs.127799 AA002125 c-
	IAP2 = MIHC = IAP homolog C = TNFR2-TRAF signalling complex prot
216	INDO: indoleamine-pyrrole 2,3 dioxygenase Hs.840 AA478279
217	DEEPEST: mitotic spindle coiled-coil related protein Hs.16244 T97349
218	ESTs: Hs.105826 AA534321
219	C20ORF1: chromosome 20 open reading frame 1 Hs.9329 AI654707
220	Homo sapiens cDNA: FLJ21869 fis, clone HEP02442 Hs.28465 R63929:
221	RGS3: regulator of G-protein signalling 3 Hs.82294 AI369623
222	Homo sapiens DC29 mRNA, complete cds Hs.85573 AA186460:
223	MCM6: minichromosome maintenance deficient (mis5, S. pombe) 6 Hs.155462
	AA663995
224	NPAT: nuclear protein, ataxia-telangiectasia locus Hs.89385 AA284172
	NPAT = E14 = gene in ATM locus
225	KNSL6: kinesin-like 6 (mitotic centromere-associated kinesin) Hs.69360 AA400450
226	HN1: hematological and neurological expressed 1 Hs.109706 AA459865
227	TUBA3: Tubulin, alpha, brain-specific Hs.272897 AA865469
228	ESTs: Hs.221197 N55457
229	KIAA0175: KIAA0175 gene product Hs.184339 AA903137
230	CLASPIN: homolog of Xenopus Claspin Hs.175613 AA857804
231	CTNNA1: **catenin (cadherin-associated protein), alpha 1 (102 kD) Hs.178452
	AA026631
232	ESTs: Hs.221962 AA229644
233	SMC4L1: SMC4 (structural maintenance of chromosomes 4, yeast)-like 1 Hs.50758
	AA452095
234	ICBP90: transcription factor Hs.108106 AA026356
235	EXO1: exonuclease 1 Hs.47504 AA703000
236	Homo sapiens TRAF4 associated factor 1 mRNA, partial cds Hs.181466 T84975:
237	ESTs: Hs.186814 AA700879
238	FLJ11269: hypothetical protein FLJ11269 Hs.25245 R37817
239	SFPQ: splicing factor proline/glutamine rich (polypyrimidine tract-binding protein-
	associated) Hs.180610 AA425258
240	ZF: HCF-binding transcription factor Zhangfei Hs.29417 AA164474
241	TUBA2: tubulin, alpha 2 Hs.98102 AA626698
242	Homo sapiens mRNA; cDNA DKFZp434M0435 (from clone DKFZp434M0435)
	Hs.25700 N94435:
243	FLJ20530: **hypothetical protein FLJ20530 Hs.279521 AA425442
244	BTEB1: basic transcription element binding protein 1 Hs.150557 N80235
245	LOC51053: geminin Hs.234896 H51100
246	D21S2056E: DNA segment on chromosome 21 (unique) 2056 expressed sequence
	Hs.110757 AI362799
247	HDAC3: histone deacetylase 3 Hs.279789 H88540
248	USP1: ubiquitin specific protease 1 Hs.35086 AA099033
249	C21ORF50: chromosome 21 open reading frame 50 Hs.4055 AA135912
250	FLJ13046: **hypothetical protein FLJ13046 similar to exportin 4 Hs.117102 T95333
251	ESTs: Hs.181059 AA912032
252	FLJ22009: hypothetical protein FLJ22009 Hs.123253 AA401234
253	ESTs: Hs.62711 AA056377
254	RAD51C: RAD51 (S. cerevisiae) homolog C Hs.11393 R37145
	RAD51C = Recombination/repair Rad51-related protein
255	ESTs: Hs.268919 H53508
256	Homo sapiens cDNA FLJ11381 fis, clone HEMBA1000501 Hs.127797 AA885096:
257	SAP30: sin3-associated polypeptide, 30 kD Hs.20985 AA126982
258	H4FG: H4 histone family, member G Hs.46423 AA868008
259	TUBA1: tubulin, alpha 1 (testis specific) Hs.75318 AA180742 tubulin-alpha-4
260	DHFR: dihydrofolate reductase Hs.83765 R00884 DHFR = Dihydrofolate reductase
261	DHFR: dihydrofolate reductase Hs.83765 N52980
262	MGC5528: hypothetical protein MGC5528 Hs.315167 AA934904
263	NNMT: nicotinamide N-methyltransferase Hs.76669 T72089
264	TUBB: tubulin, beta polypeptide Hs.179661 AI672565
265	HSPA1L: heat shock 70 kD protein-like 1 Hs.80288 H17513 HSP70-HOM = Heat shock
	70 KD protein 1
266	TUBA1: **tubulin, alpha 1 (testis specific) Hs.75318 R36063
267	PRO1073: **PRO1073 protein Hs.6975 AA176999 CIP4 = Cdc42-interacting protein 4
268	POLD3: polymerase (DNA directed), delta 3 Hs.82502 AI017254
269	ESTs,: Moderately similar to T50635 hypothetical protein DKFZp762L0311.1
	[H. sapiens] Hs.47378 N38809
270	DKFZP564A122: DKFZP564A122 protein Hs.187991 N57723
271	LRRFIP1: **leucine rich repeat (in FLII) interacting protein 1 Hs.326159 T84633
272	ESTs: Hs.55468 AA165312
273	ESTs: Hs.31444 H16772
274	AFAP: actin filament associated protein Hs.80306 R69355
275	CXCR4: chemokine (C-X-C motif), receptor 4 (fusin) Hs.89414 T62491 CXC
	chemokine receptor 4 = fusin = neuropeptide Y receptor = L3
276	MSH2: *mutS (E. coli*) homolog 2 (colon cancer, nonpolyposis type 1) Hs.78934
	AA679697
277	ESTs: Hs.48474 N62074
278	AA677337:
279	ESTs,: Moderately similar to TBB2_HUMAN TUBULIN BETA-2 CHAIN
	[H. sapiens] Hs.23189 AA629908
280	HP1-BP74: HP1-BP74 Hs.142442 H79795
281	FLJ20101: LIS1-interacting protein NUDE1, rat homolog Hs.263925 AA459394
282	Homo sapiens mRNA; cDNA DKFZp434D1428 (from clone DKFZp434D1428);
	complete cds Hs.321775 AA431268:
283	ESTs: Hs.265592 AA992658
284	ESTs:
285	DDX11: DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 11 (S. cerevisiae CHL1-
	like helicase) Hs.27424 AA402879
286	CDC27: cell division cycle 27 Hs.172405 T81764
287	ARGBP2: Arg/Abl-interacting protein ArgBP2 Hs.278626 N89738
288	DKFZP564A122: DKFZP564A122 protein Hs.187991 AA025807
289	OPN3: opsin 3 (encephalopsin) Hs.279926 AA150060
290	DKFZP566C134: DKFZP566C134 protein Hs.20237 AA456319
291	KIAA0855: golgin-67 Hs.182982 H15101
292	PIN: dynein, cytoplasmic, light polypeptide Hs.5120 AA644679
293	ESTs,: Weakly similar to LIP1_HUMAN PANCREATIC LIPASE RELATED
	PROTEIN 1 PRECURSO [H. sapiens] Hs.68864 AA088857
294	HDAC3: histone deacetylase 3 Hs.279789 AA973283
295	DONSON: downstream neighbor of SON Hs.17834 AA417895
296	LOC51053: geminin Hs.234896 AA447662
297	FLJ10545: hypothetical protein FLJ10545 Hs.88663 AA460110
298	MAD2L1: MAD2 (mitotic arrest deficient, yeast, homolog)-like 1 Hs.79078
	AA481076 mitotic feedback control protein Madp2 homolog
299	TASR2: TLS-associated serine-arginine protein 2 Hs.3530 H11042
300	MCM6: minichromosome maintenance deficient (mis5, S. pombe) 6 Hs.155462
	N57722
301	CIT: citron (rho-interacting, serine/threonine kinase 21) Hs.15767 W69425
302	**ESTs: Hs.205066 AA284803
303	ICAM1: intercellular adhesion molecule 1 (CD54), human rhinovirus receptor
	Hs.168383 R77293 CD54 = ICAM-1
304	KIAA0855: golgin-67 Hs.182982 AA456818
305	ESTs,: Weakly similar to putative p150 [H. sapiens] Hs.300070 R10422
306	DEEPEST: mitotic spindle coiled-coil related protein Hs.16244 AI652290
307	MCM2: minichromosome maintenance deficient (S. cerevisiae) 2 (mitotin) Hs.57101
	AA454572
308	Homo sapiens cDNA: FLJ22272 fis, clone HRC03192 Hs.50740 AA495943:
309	WISP1: **WNT1 inducible signaling pathway protein 1 Hs.194680 T54850
310	KIAA0855: golgin-67 Hs.182982 AA280248
311	TEM8: tumor endothelial marker 8 Hs.8966 H58644
312	BITE: p10-binding protein Hs.42315 H96392
313	RAN: RAN, member RAS oncogene family Hs.10842 AA456636
314	EZH2: enhancer of zeste (Drosophila) homolog 2 Hs.77256 AA428252
315	MCM4: minichromosome maintenance deficient (S. cerevisiae) 4 Hs.154443 W74071
316	DKFZp434J0310: hypothetical protein Hs.278408 AA279657 Unknown UG Hs.23595
	ESTs sc_id6950
317	PPP1R10: protein phosphatase 1, regulatory subunit 10 Hs.106019 AA071526
318	H11: protein kinase H11; small stress protein-like protein HSP22 Hs.111676 H57493
319	ESTs,: Weakly similar to KIAA1074 protein [H. sapiens] Hs.200483 AA463220
320	ESTs,: Weakly similar to ALU8_HUMAN ALU SUBFAMILY SX SEQUENCE
	CONTAMINATION WARNING ENTRY [H. sapiens] Hs.226414 N72576
321	AA775033:
322	LOC51004: CGI-10 protein Hs.12239 AA677920
323	ESTs: Hs.150028 AI292036
324	MCM6: minichromosome maintenance deficient (mis5, S. pombe) 6 Hs.155462
	AA976533
325	ESTs,: Moderately similar to T50635 hypothetical protein DKFZp762L0311.1
	[H. sapiens] Hs.47378 AA406348
326	UCP4: uncoupling protein 4 Hs.40510 H60279
327	MSH5: mutS (E. coli) homolog 5 Hs.112193 AA621155
328	ROCK1: Rho-associated, coiled-coil containing protein kinase 1 Hs.17820 AA872143
329	KIAA0855: golgin-67 Hs.182982 AA694481
330	AA705332:
331	CDC27: cell division cycle 27 Hs.172405 N47994
332	DONSON: downstream neighbor of SON Hs.17834 AI732249
333	SH3GL2: SH3-domain GRB2-like 2 Hs.75149 R12817
334	PRC1: protein regulator of cytokinesis 1 Hs.5101 AA449336
335	ESTs,: Weakly similar to unnamed protein product [H. sapiens] Hs.99807 AA417744
	Unknown UG Hs.119424 ESTs sc_id2235
336	Human: clone 23719 mRNA sequence Hs.80305 AA425722
337	Homo sapiens mRNA; cDNA DKFZp564O2364 (from clone DKFZp564O2364)
	Hs.28893 W90240:
338	ESTs,: Weakly similar to LIP1_HUMAN PANCREATIC LIPASE RELATED
	PROTEIN 1 PRECURSO [H. sapiens] Hs.68864 AA132858
339	TUBA3: Tubulin, alpha, brain-specific Hs.272897 AA864642
340	AI283530:
341	ESTs: Hs.302878 R92512
342	PPP1R10: protein phosphatase 1, regulatory subunit 10 Hs.106019 T75485
343	SFRS5: splicing factor, arginine/serine-rich 5 Hs.166975 R73672
344	SFRS3: splicing factor, arginine/serine-rich 3 Hs.167460 AA598400
345	PRIM1: primase, polypeptide 1 (49 kD) Hs.82741 AA025937 DNA primase (subunit p48)
346	FLJ20333: hypothetical protein FLJ20333 Hs.79828 H66982
347	HSPA8: heat shock 70 kD protein 8 Hs.180414 AA620511
348	C4A: complement component 4A Hs.170250 AA664406
349	DKC1: dyskeratosis congenita 1, dyskerin Hs.4747 AA052960
350	HP1-BP74: HP1-BP74 Hs.142442 T84669
351	ETV4: ets variant gene 4 (E1A enhancer-binding protein, E1AF) Hs.77711 AA010400
	E1A-F = E1A enhancer binding protein = ETS translocation variant
352	Homo sapiens cDNA: FLJ23037 fis, clone LNG02036, highly similar to HSU68019
	Homo sapiens mad protein homolog (hMAD-3) mRNA Hs.288261 W42414
	Smad3 = hMAD-3 = Homologue of Mothers Against Decapentaplegic (M:
353	KIAA0952: KIAA0952 protein Hs.7935 AA679150
354	STK9: serine/threonine kinase 9 Hs.50905 N80713
355	NXF1: **nuclear RNA export factor 1 Hs.323502 R01238
356	FLJ12892: hypothetical protein FLJ12892 Hs.17731 AA449357
357	UNG: uracil-DNA glycosylase Hs.78853 H15111
358	STK17B: **serine/threonine kinase 17b (apoptosis-inducing) Hs.120996 AA419485
359	YWHAH: tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein,
	eta polypeptide Hs.75544 N69107
360	FLJ13154: hypothetical protein FLJ13154 Hs.25303 AA923560
361	LOC51116: CGI-91 protein Hs.20776 AA459419
362	SSXT: synovial sarcoma, translocated to X chromosome Hs.153221 N59206
363	KIAA0978: KIAA0978 protein Hs.3686 AA485878
364	EST: Hs.147907 AI223432
365	FLJ23468: hypothetical protein FLJ23468 Hs.38178 AA431741
366	FLJ10339: **hypothetical protein FLJ10339 Hs.203963 N95450
367	BMP2: bone morphogenetic protein 2 Hs.73853 AA011061
368	PIR51: RAD51-interacting protein Hs.24596 AI214426
369	FLJ20364: hypothetical protein FLJ20364 Hs.32471 AA676296
370	EIF4A2: **eukaryotic translation initiation factor 4A, isoform 2 Hs.173912 H54751
371	ESTs,: Weakly similar to MCAT_HUMAN MITOCHONDRIAL
	CARNITINE/ACYLCARNITINE CARRIER PROTEIN [H. sapiens] Hs.27769
	AA469975
372	FLJ11323: hypothetical protein FLJ11323 Hs.25625 AA775600
373	DKFZP564D0764: DKFZP564D0764 protein Hs.26799 AA460732
374	CTL2: CTL2 gene Hs.105509 AA454710
375	ESTs: Hs.293419 AA775845
376	IFIT1: interferon-induced protein with tetratricopeptide repeats 1 Hs.20315 AA489640
	Interferon-induced 56-KDa protein
377	RBBP8: retinoblastoma-binding protein 8 Hs.29287 H23021
378	**Homo sapiens clone 25061 mRNA sequence Hs.183475 R38944:
379	Human: DNA sequence from clone RP3-383J4 on chromosome 1q24.1-24.3 Contains
	part of a gene encoding a kelch motif containing protein, part of a novel gene encoding
	a protein similar to Aspartyl-TRNA sy Hs.117305 N29457
380	FLJ12888: hypothetical protein FLJ12888 Hs.284137 N68390
381	ESTs,: Weakly similar to IF38_HUMAN EUKARYOTIC TRANSLATION
	INITIATION FACTOR 3 SUBUNIT 8 [H. sapiens] Hs.222088 AI139629
382	ESTs: Hs.241101 AA133590
383	H4FI: H4 histone family, member I Hs.143080 AI218900
384	SP38: zona pellucida binding protein Hs.99875 AA400474
385	GABPB1: GA-binding protein transcription factor, beta subunit 1 (53 kD) Hs.78915
	H91651
386	LCHN: LCHN protein Hs.12461 AA029330
387	DKFZP564D0462: hypothetical protein DKFZp564D0462 Hs.44197 N32904
388	LENG8: leukocyte receptor cluster (LRC) encoded novel gene 8 Hs.306121
	AA464698
389	HIF1A: hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription
	factor) Hs.197540 AA598526
390	ESTs: Hs.93714 R09201
391	FLJ23468: hypothetical protein FLJ23468 Hs.38178 AA454949
392	DKFZP566C134: DKFZP566C134 protein Hs.20237 AA448164
393	PPP3CA: protein phosphatase 3 (formerly 2B), catalytic subunit, alpha isoform
	(calcineurin A alpha) Hs.272458 W60310
394	HMGE: GrpE-like protein cochaperone Hs.151903 H55907
395	CDK7: cyclin-dependent kinase 7 (homolog of Xenopus MO15 cdk-activating kinase)
	Hs.184298 R22624 CAK = cdk7 = NRTALRE = sdk = CDK activating kinase
396	ABCC5: **ATP-binding cassette, sub-family C (CFTR/MRP), member 5 Hs.108660
	AA186613
397	AA477707:
398	**ESTs: Hs.15607 R92899
399	LOC57209: Kruppel-type zinc finger protein Hs.25275 N50827
400	FLJ20101: LIS1-interacting protein NUDE1, rat homolog Hs.263925 R87716
401	KNSL4: kinesin-like 4 Hs.119324 AA430503
402	E2F5: E2F transcription factor 5, p130-binding Hs.2331 AA455521 E2F-5 = pRB-
	binding transcription factor
403	TMPO: thymopoietin Hs.11355 T63980
404	POLQ: polymerase (DNA directed), theta Hs.241517 AI057325
405	TGIF: TG-interacting factor (TALE family homeobox) Hs.90077 H51705
406	TRIP13: thyroid hormone receptor interactor 13 Hs.6566 AA630784
407	GAS6: growth arrest-specific 6 Hs.78501 AA461110
408	HN1: hematological and neurological expressed 1 Hs.109706 AA035429
409	BARD1: BRCA1 associated RING domain 1 Hs.54089 AA558464
410	DHFR: dihydrofolate reductase Hs.83765 AA424790
411	AA490946:
412	ESTs: Hs.130435 AA167114
413	HSPA8: heat shock 70 kD protein 8 Hs.180414 AA629567
414	RRM2: ribonucleotide reductase M2 polypeptide Hs.75319 AA826373
415	FLJ20036: hypothetical protein FLJ20036 Hs.32922 H59114
416	COPEB: core promoter element binding protein Hs.285313 AA055584
	CPBP = CBA1 = DNA-binding protein
417	FLJ10604: hypothetical protein FLJ10604 Hs.26516 N72697
418	ESTs,: Weakly similar to cDNA EST yk415c12.5 comes from this gene [C. elegans]
	Hs.108824 H97880
419	UBE2D3: **ubiquitin-conjugating enzyme E2D 3 (homologous to yeast UBC4/5)
	Hs.118797 AA017199
420	FLJ10890: **hypothetical protein FLJ10890 Hs.17283 AA004210
421	ESTs: Hs.214410 AA579336
422	OLR1: oxidised low density lipoprotein (lectin-like) receptor 1 Hs.77729 AA682386
423	FLJ13231: hypothetical protein FLJ13231 Hs.156148 W92787
424	EST: Hs.323101 W40398
425	ESTs,: Weakly similar to R06F6.5b [C. elegans] Hs.180591 N59330
426	Homo sapiens cDNA: FLJ23285 fis, clone HEP09071 Hs.90424 N26163:
427	Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 42408 Hs.284123
	AA211446:
428	NFKB1: nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (p105)
	Hs.83428 AA451716 NFkB1 = NF-kappaB p105 = p50
429	LOC58486: transposon-derived Buster1 transposase-like protein Hs.25726 AA630256
430	Homo sapiens cDNA FLJ10976 fis, clone PLACE1001399 Hs.296323 AA424756:
431	KIAA0182: KIAA0182 protein Hs.75909 AI023801
432	RANGAP1: Ran GTPase activating protein 1 Hs.183800 AA991855
433	PKMYT1: membrane-associated tyrosine- and threonine-specific cdc2-inhibitory
	kinase Hs.77783 AA478066 Myt1 kinase
434	HSPA8: heat shock 70 kD protein 8 Hs.180414 H64096
435	LUC7A: cisplatin resistance-associated overexpressed protein Hs.3688 AA411969
436	RRM1: ribonucleotide reductase M1 polypeptide Hs.2934 AA633549
437	SET07: PR/SET domain containing protein 7 Hs.111988 AA421470
438	**ESTs,: Weakly similar to ALU1_HUMAN ALU SUBFAMILY J SEQUENCE
	CONTAMINATION WARNING ENTRY [H. sapiens] Hs.193452 W96179
439	Homo sapiens clone 25058 mRNA sequence Hs.179397 R38894:
440	ESTs,: Weakly similar to KIAA0973 protein [H. sapiens] Hs.14014 AA780791
441	EST: Hs.105298 AA489813
442	CTCF: CCCTC-binding factor (zinc finger protein) Hs.57419 H89996
443	HRB: HIV-1 Rev binding protein Hs.171545 AA485958
444	**ESTs: Hs.294083 AA447679
445	KIAA0878: KIAA0878 protein Hs.188006 AA599094
446	ESTs,: Weakly similar to ALUB_HUMAN !!!! ALU CLASS B WARNING ENTRY
	!!! [H. sapiens] Hs.180552 AA481283
447	OGT: O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N-
	acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase) Hs.100293
	AA425229
448	Homo sapiens mRNA for KIAA1700 protein, partial cds Hs.20281 N40952:
449	Human: DNA sequence from clone RP1-187J11 on chromosome 6q11.1-22.33.
	Contains the gene for a novel protein similar to S. pombe and S. cerevisiae predicted
	proteins, the gene for a novel protein simila Hs.72325 AA159962
450	KIAA1265: KIAA1265 protein Hs.24936 AA479302
451	H1F0: H1 histone family, member 0 Hs.226117 H57830
452	ARGBP2: Arg/Abl-interacting protein ArgBP2 Hs.278626 H02525
453	ODF2: outer dense fibre of sperm tails 2 Hs.129055 AA149882
454	CD97: CD97 antigen Hs.3107 AI651871
455	BMI1: **murine leukemia viral (bmi-1) oncogene homolog Hs.431 AA193573
456	POLG: polymerase (DNA directed), gamma Hs.80961 AA188629
457	XPR1: xenotropic and polytropic retrovirus receptor Hs.227656 AA453474
458	ESTs: Hs.128096 AA971179
459	DNAJB1: DnaJ (Hsp40) homolog, subfamily B, member 1 Hs.82646 AA481022
460	ARL4: ADP-ribosylation factor-like 4 Hs.201672 AI142552
461	SFRS5: splicing factor, arginine/serine-rich 5 Hs.166975 AA598965
462	ESTs: Hs.25933 R11605
463	RIG-I: RNA helicase Hs.145612 AA126958
464	FLJ10339: hypothetical protein FLJ10339 Hs.203963 AA628231
465	DR1: down-regulator of transcription 1, TBP-binding (negative cofactor 2) Hs.16697
	AA043503
466	Homo sapiens, Similar to hypothetical protein FLJ20093, clone MGC: 1076, mRNA,
	complete cds Hs.298998 AA703249:
467	HSPC163: HSPC163 protein Hs.108854 H98963
468	DKFZP564A122: DKFZP564A122 protein Hs.187991 R27345
469	FLJ10128: uveal autoantigen with coiled coil domains and ankyrin repeats Hs.49753
	T47624
470	DSCR1: Down syndrome critical region gene 1 Hs.184222 AA629707
471	FLJ10342: hypothetical protein FLJ10342 Hs.101514 AA490935
472	Homo sapiens mRNA; cDNA DKFZp586N1323 (from clone DKFZp586N1323)
	Hs.24064 R26176:
473	ESTs: Hs.4983 H59921
474	ESTs,: Weakly similar to ALUB_HUMAN !!!! ALU CLASS B WARNING ENTRY
	!!! [H. sapiens] Hs.117949 H91167
475	CDC45L: CDC45 (cell division cycle 45, S. cerevisiae, homolog)-like Hs.114311
	AA700904
476	STAT5B: signal transducer and activator of transcription 5B Hs.244613 AA280647
	STAT5A/5B
477	Homo sapiens cDNA FLJ14028 fis, clone HEMBA1003838 Hs.281434 AA454682:
478	KIAA1524: KIAA1524 protein Hs.151343 AI248987
479	CTSD: cathepsin D (lysosomal aspartyl protease) Hs.79572 AA485373
480	Homo sapiens, Similar to hypothetical protein FLJ20093, clone MGC: 1076, mRNA,
	complete cds Hs.298998 AA682274:
481	GTPBP2: GTP binding protein 2 Hs.13011 T67069
482	LOC51003: CGI-125 protein Hs.27289 AA485945
483	VCL: vinculin Hs.75350 AA486727
484	KIF5B: kinesin family member 5B Hs.149436 AA046613
485	CDC25A: cell division cycle 25A Hs.1634 AA071514
486	LOC51141: insulin induced protein 2 Hs.7089 AA045308
487	*ESTs,: Moderately similar to CALD_HUMAN CALDESMON [H. sapiens*]
	Hs.117774 H48508
488	TBX3-iso: TBX3-iso protein Hs.267182 T48941
489	KIAA0176: KIAA0176 protein Hs.4935 R44371
490	PRKAR1A: protein kinase, cAMP-dependent, regulatory, type I, alpha (tissue specific
	extinguisher 1) Hs.183037 N25969 PKA-R1 alpha = cAMP-dependent protein kinase
	type I-alpha-cata
491	ESTs: Hs.268991 H77818
492	ESTs,: Weakly similar to A53028 isopentenyl-diphosphate Delta-isomerase
	[H. sapiens] Hs.9270 R17362
493	ESTs,: Weakly similar to B34087 hypothetical protein [H. sapiens] Hs.120946 H50656
494	TRN2: karyopherin beta 2b, transportin Hs.278378 R08897
495	LMNA: lamin A/C Hs.77886 AA489582
496	NFE2L2: nuclear factor (erythroid-derived 2)-like 2 Hs.155396 AA629687
497	DKFZp762L0311: hypothetical protein DKFZp762L0311 Hs.16520 AA486418
498	ESTs,: Weakly similar to S71752 giant protein p619 [H. sapiens] Hs.14870 T96829
499	Homo sapiens mRNA; cDNA DKFZp434A1315 (from clone DKFZp434A1315);
	complete cds Hs.298312 AA991355:
500	E2IG4: hypothetical protein, estradiol-induced Hs.8361 R13844
501	RANGAP1: Ran GTPase activating protein 1 Hs.183800 AA485734
502	H1F0: H1 histone family, member 0 Hs.226117 W69399
503	KIAA0239: KIAA0239 protein Hs.9729 AA454740
504	ESTs,: Weakly similar to ALU7_HUMAN ALU SUBFAMILY SQ SEQUENCE
	CONTAMINATION WARNING ENTRY [H. sapiens] Hs.68647 R96804
505	PRO0650: PRO0650 protein Hs.177258 N54333
506	DNAJB9: DnaJ (Hsp40) homolog, subfamily B, member 9 Hs.6790 AA045792
507	Homo sapiens cDNA: FLJ21971 fis, clone HEP05790 Hs.71331 AA774678:
508	LOC56996: **cation-chloride cotransporter-interacting protein Hs.119178 AA037466
509	AP3D1: adaptor-related protein complex 3, delta 1 subunit Hs.75056 AA630776
510	SGK: serum/glucocorticoid regulated kinase Hs.159640 AA486082 sgk = putative
	serine/threonine protein kinase transcriptional
511	HSPC148: hypothetical protein Hs.42743 R23666
512	MRPL19: mitochondrial ribosomal protein L19 Hs.75574 AA521243 KIAA0104
513	AA455102:
514	ESTs: Hs.150325 AI278813
515	**ESTs: Hs.40527 AA029844
516	HSPC145: HSPC145 protein Hs.18349 AI271431
517	KIAA0170: KIAA0170 gene product Hs.277585 H68789
518	FLJ11127: hypothetical protein Hs.91165 T98200
519	KIAA0182: KIAA0182 protein Hs.75909 H05099
520	FLJ23151: hypothetical protein FLJ23151 Hs.137260 AA284259
521	AMD1: S-adenosylmethionine decarboxylase 1 Hs.262476 AA425692
522	FLJ10342: **hypothetical protein FLJ10342 Hs.101514 AA934516
523	SPS: SELENOPHOSPHATE SYNTHETASE; Human selenium donor protein
	Hs.124027 AA486372
524	KIAA1586: KIAA1586 protein Hs.180663 AA779733
525	ICBP90: transcription factor Hs.108106 AA908902
526	Homo sapiens cDNA: FLJ21971 fis, clone HEP05790 Hs.71331 AI002036:
527	ABCC2: ATP-binding cassette, sub-family C (CFTR/MRP), member 2 Hs.193852
	R91502
528	ARHGDIB: Rho GDP dissociation inhibitor (GDI) beta Hs.83656 AA487426
	LyGDI = Rho GDP-dissociation inhibitor 2 = RHO GDI 2
529	RAD53: protein kinase Chk2 Hs.146329 AI653182
530	R96880:
531	TNFAIP3: tumor necrosis factor, alpha-induced protein 3 Hs.211600 AA433807
532	ESTs: Hs.26979 H23469
533	AOC2: amine oxidase, copper containing 2 (retina-specific) Hs.143102 N50959
534	Homo sapiens mRNA; cDNA DKFZp586N1323 (from clone DKFZp586N1323)
	Hs.24064 R30941:
535	AA452872:
536	ESTs: Hs.124169 R58970
537	ACYP1: acylphosphatase 1, erythrocyte (common) type Hs.18573 W78754
538	SIL: TAL1 (SCL) interrupting locus Hs.323032 AA704809
539	AA016234:
540	Homo sapiens mRNA; cDNA DKFZp566P1124 (from clone DKFZp566P1124)
	Hs.321022 N50895:
541	KIAA1067: KIAA1067 protein Hs.325530 AA099138
542	SMC4L1: SMC4 (structural maintenance of chromosomes 4, yeast)-like 1 Hs.50758
	AA283006
543	ESTs: Hs.29074 R70174
544	SNK: serum-inducible kinase Hs.3838 AA460152
545	FANCG: Fanconi anemia, complementation group G Hs.8047 AA427484
546	Homo sapiens cDNA: FLJ21531 fis, clone COL06036 Hs.102941 N95440:
547	Homo sapiens mRNA; cDNA DKFZp547B086 (from clone DKFZp547B086)
	Hs.36606 N48700:
548	C1ORF2: chromosome 1 open reading frame 2 Hs.19554 H11464 cote1 = ORF in
	glucocerebrosidase locus
549	HTF9C: HpaII tiny fragments locus 9C Hs.63609 H17888
550	ATF4: activating transcription factor 4 (tax-responsive enhancer element B67)
	Hs.181243 AA600217
551	ESTs: Hs.101014 AA194941
552	CDC25A: cell division cycle 25A Hs.1634 AA913262
553	TOPK: PDZ-binding kinase; T-cell originated protein kinase Hs.104741 AI002631
554	ASIP: agouti (mouse)-signaling protein Hs.37006 AI220203
555	DKFZP564F013: **hypothetical protein DKFZp564F013 Hs.128653 R14908
556	ZNF265: zinc finger protein 265 Hs.194718 N66014
557	SLC30A1: solute carrier family 30 (zinc transporter), member 1 Hs.55610 AA195463
558	ESTs: Hs.28462 R63922
559	ESTs: Hs.114055 R27431
560	IL6: interleukin 6 (interferon, beta 2) Hs.93913 N98591 IL-6
561	H3F3B: H3 histone, family 3B (H3.3B) Hs.180877 AA608514
562	ESTs: Hs.81263 W81524
563	Homo sapiens cDNA: FLJ23538 fis, clone LNG08010, highly similar to BETA2
	Human MEN1 region clone epsilon/beta mRNA Hs.240443 AA400234:
564	AMD1: S-adenosylmethionine decarboxylase 1 Hs.262476 R82299
565	MAP3K2: mitogen-activated protein kinase kinase kinase 2 Hs.28827 AA447971
566	NET1: neuroepithelial cell transforming gene 1 Hs.25155 R24543
567	CHAF1A: chromatin assembly factor 1, subunit A (p150) Hs.79018 AA704459
568	MGC5585: hypothetical protein MGC5585 Hs.5152 H50655
569	KIAA1598: KIAA1598 protein Hs.23740 H17868
570	PNN: pinin, desmosome associated protein Hs.44499 W86139
571	ESTs: Hs.238797 N70848
572	ESTs,: Weakly similar to ALUB_HUMAN !!!! ALU CLASS B WARNING ENTRY
	!!! [H. sapiens] Hs.180552 AA600192
573	PDGFA: platelet-derived growth factor alpha polypeptide Hs.37040 AA701502
574	Homo sapiens clone FLC0675 PRO2870 mRNA, complete cds Hs.306117 AA443127:
575	ESTs: Hs.143375 AA001841
576	TUBB: tubulin, beta polypeptide Hs.179661 H37989
577	MSH2: mutS (E. coli) homolog 2 (colon cancer, nonpolyposis type 1) Hs.78934
	AA219060 MSH2 = DNA mismatch repair mutS homologue
578	TOPBP1: topoisomerase (DNA) II binding protein Hs.91417 R97785
579	KIAA0869: KIAA0869 protein Hs.21543 R43798
580	H4FH: H4 histone family, member H Hs.93758 AA702781
581	FLJ23293: hypothetical protein FLJ23293 similar to ARL-6 interacting protein-2
	Hs.31236 AA629027
582	**Homo sapiens cDNA: FLJ23538 fis, clone LNG08010, highly similar to BETA2
	Human MEN1 region clone epsilon/beta mRNA Hs.240443 AA053165:
583	KIAA0978: KIAA0978 protein Hs.3686 N64780
584	KIAA1547: KIAA1547 protein Hs.31305 AA057737
585	DKFZP761C169: hypothetical protein DKFZp761C169 Hs.71252 AA608709
586	WS-3: novel RGD-containing protein Hs.39913 AA449975
587	FRZB: frizzled-related protein Hs.153684 H87275
588	BRCA1: breast cancer 1, early onset Hs.194143 H90415 BRCA1 = Mutated in breast
	and ovarian cancer
589	ESTs: Hs.4983 H22936
590	HSPC150: HSPC150 protein similar to ubiquitin-conjugating enzyme Hs.5199
	AA460431
591	Homo sapiens mRNA for KIAA1712 protein, partial cds Hs.29798 H54592:
592	FLJ11186: hypothetical protein FLJ11186 Hs.89278 AA504111 Unknown UG
	Hs.89278 ESTs
593	ESTs,: Weakly similar to unnamed protein product [H. sapiens] Hs.118338 R25481
594	APEXL2: apurinic/apyrimidinic endonuclease(APEX nuclease)-like 2 protein
	Hs.154149 AI674393
595	CDR2: cerebellar degeneration-related protein (62 kD) Hs.75124 AA074613
596	ESTs: Hs.69662 AA459724
597	PSCD2L: pleckstrin homology, Sec7 and coiled/coil domains 2-like Hs.8517
	AA464957
598	CRK: v-crk avian sarcoma virus CT10 oncogene homolog Hs.306088 H75530
599	CCNE2: cyclin E2 Hs.30464 AA520999 Unknown UG Hs.30464 cyclin E2
600	LOC51240: hypothetical protein Hs.7870 AA988037
601	FLJ11259: hypothetical protein FLJ11259 Hs.184465 AA485877
602	PTP4A1: protein tyrosine phosphatase type IVA, member 1 Hs.227777 AA482193
603	Homo sapiens cDNA: FLJ22355 fis, clone HRC06344 Hs.288283 AA026375:
604	Human: clone 23719 mRNA sequence Hs.80305 H43437
605	Homo sapiens clone FLC0675 PRO2870 mRNA, complete cds Hs.306117 AA485453:
606	MSE55: serum constituent protein Hs.148101 H73234
607	CFLAR: CASP8 and FADD-like apoptosis regulator Hs.195175 AA453766
608	Homo sapiens cDNA: FLJ22844 fis, clone KAIA5181 Hs.296322 AA975103:
609	Human: DNA sequence from clone RP11-371L19 on chromosome 20 Contains two
	novel genes, the gene for a novel protein similar to 40S ribosomal protein S10
	(RPS10), ESTs, STSs, GSSs and five CpG islands Hs.19002 R00846
610	ESTs: Hs.60054 R26390
611	ESTs,: Weakly similar to ALU7_HUMAN ALU SUBFAMILY SQ SEQUENCE
	CONTAMINATION WARNING ENTRY [H. sapiens] Hs.325158 AA032084
612	FLJ10980: hypothetical protein FLJ10980 Hs.29716 N45467
613	IFIT1: **interferon-induced protein with tetratricopeptide repeats 1 Hs.20315
	AA157787
614	ESTs: Hs.21734 AA429809
615	DKFZP434C245: DKFZP434C245 protein Hs.59461 AA705518
616	RNPS1: RNA-binding protein S1, serine-rich domain Hs.75104 AA496837
617	FLJ13639: hypothetical protein FLJ13639 Hs.101821 AA131681
618	PCF11: PCF11p homolog Hs.123654 W73749
619	EIF4G3: eukaryotic translation initiation factor 4 gamma, 3 Hs.25732 N92469
620	Homo sapiens cDNA: FLJ21971 fis, clone HEP05790 Hs.71331 AA130595:
621	STAT1: signal transducer and activator of transcription 1, 91 kD Hs.21486 AA079495
622	BIRC3: baculoviral IAP repeat-containing 3 Hs.127799 R07870
623	HP1-BP74: HP1-BP74 Hs.142442 N20589
624	HSPC228: hypothetical protein Hs.267288 AI734268
625	KIAA0675: KIAA0675 gene product Hs.165662 AA454867
626	AMD1: S-adenosylmethionine decarboxylase 1 Hs.262476 AA504772
627	EST: Hs.149338 AI249089
628	PWP1: nuclear phosphoprotein similar to S. cerevisiae PWP1 Hs.172589 AA485992
629	AI336973:
630	DUSP4: dual specificity phosphatase 4 Hs.2359 AA444049
631	FLJ12788: hypothetical protein FLJ12788 Hs.20242 AA497041
632	HSPC150: HSPC150 protein similar to ubiquitin-conjugating enzyme Hs.5199
	AA985450
633	FLJ11729: hypothetical protein FLJ11729 Hs.286212 W15533
634	KLF4: Kruppel-like factor 4 (gut) Hs.7934 H45668
635	FLJ11058: hypothetical protein FLJ11058 Hs.180817 N63911
636	FLJ23468: hypothetical protein FLJ23468 Hs.38178 AA460299
637	ESTs: Hs.115315 AI278336
638	EBI3: Epstein-Barr virus induced gene 3 Hs.185705 AA425028 EBI3 = cytokine
	receptor
639	ESTs: Hs.293797 N63988
640	MGAT2: mannosyl (alpha-1,6-)-glycoprotein beta-1,2-N-
	acetylglucosaminyltransferase Hs.172195 AA485653
641	H2BFQ: H2B histone family, member Q Hs.2178 AA456298
642	NMB: neuromedin B Hs.83321 AI650675
643	SSR3: signal sequence receptor, gamma (translocon-associated protein gamma)
	Hs.28707 AA453486
644	HSPC196: hypothetical protein Hs.239938 R78498
645	EST: Hs.44522 N33610
646	BRF1: butyrate response factor 1 (EGF-response factor 1) Hs.85155 AA723035
647	MAN1A2: mannosidase, alpha, class 1A, member 2 Hs.239114 H97940
648	KIAA1201: KIAA1201 protein Hs.251278 AA427719
649	NUCKS: similar to rat nuclear ubiquitous casein kinase 2 Hs.118064 AA158345
650	MAGEF1: MAGEF1 protein Hs.306123 AA425302
651	Human: Chromosome 16 BAC clone CIT987SK-A-362G6 Hs.6349 N75498
652	R40377:
653	AP3M2: adaptor-related protein complex 3, mu 2 subunit Hs.77770 R14443
654	ESTs,: Weakly similar to 1207289A reverse transcriptase related protein [H. sapiens]
	Hs.272135 AA705010
655	Homo sapiens mRNA for FLJ00116 protein, partial cds Hs.72363 AA159893:
656	EIF4E: eukaryotic translation initiation factor 4E Hs.79306 AA193254
657	Homo sapiens mRNA for hypothetical protein (TR2/D15 gene) Hs.180545 N47285:
658	ESTs: Hs.99542 AA461474
659	CTNND1: catenin (cadherin-associated protein), delta 1 Hs.166011 AA024656
660	ESTs: Hs.188554 R75884
661	ZNF217: zinc finger protein 217 Hs.155040 R81830
662	FLJ12892: hypothetical protein FLJ12892 Hs.17731 AI243595
663	ETV5: ets variant gene 5 (ets-related molecule) Hs.43697 AA460265
664	EST: Hs.251574 T54821
665	RPS25: ribosomal protein S25 Hs.113029 T98662
666	CNN2: calponin 2 Hs.169718 AA284568
667	ESTs,: Weakly similar to plakophilin 2b [H. sapiens] Hs.12705 AA485365
668	PAPPA: pregnancy-associated plasma protein A Hs.75874 AA609463
669	TFF3: trefoil factor 3 (intestinal) Hs.82961 N74131
670	AI204264:
671	DJ328E19.C1.1: hypothetical protein Hs.218329 AA486041
672	ME3: malic enzyme 3, NADP(+)-dependent, mitochondrial Hs.2838 AA779401
673	ESTs,: Weakly similar to IEFS_HUMAN TRANSFORMATION-SENSITIVE
	PROTEIN IEF SSP 3521 [H. sapiens] Hs.43213 AA490554
674	FLJ13181: hypothetical protein FLJ13181 Hs.301526 AA057266
675	KIAA1547: KIAA1547 protein Hs.31305 AA136692
676	ZNF281: zinc finger protein 281 Hs.59757 N47468
677	Homo sapiens cDNA: FLJ23260 fis, clone COL05804, highly similar to HSU90911
	Human clone 23652 mRNA sequence Hs.13996 AA463961:
678	ESTs: Hs.25933 AA411392
679	NCBP1: nuclear cap binding protein subunit 1, 80 kD Hs.89563 AA278749 nuclear cap
	binding protein
680	H2BFL: H2B histone family, member L Hs.239884 H70774
681	DKFZP564A122: DKFZP564A122 protein Hs.187991 H66150
682	NASP: nuclear autoantigenic sperm protein (histone-binding) Hs.243886 AA644128
683	*ESTs,: Weakly similar to KIAA0822 protein [H. sapiens*] Hs.98368 AA422008
684	MAP2K6: mitogen-activated protein kinase kinase 6 Hs.118825 H07920
685	ESTs: Hs.158357 AA865842
686	GADD45A: growth arrest and DNA-damage-inducible, alpha Hs.80409 AA147214
	GADD45 alpha = growth arrest and DNA-damage-inducible protein
687	DHFR: dihydrofolate reductase Hs.83765 AA488803
688	AA151930:
689	Homo sapiens mRNA; cDNA DKFZp434P116 (from clone DKFZp434P116);
	complete cds Hs.103378 AA431133:
690	Homo sapiens mRNA; cDNA DKFZp564D156 (from clone DKFZp564D156)
	Hs.9927 T55704:
691	ESTs: Hs.32204 R93719
692	PRPSAP1: phosphoribosyl pyrophosphate synthetase-associated protein 1 Hs.77498
	R20005
693	ZNF42: zinc finger protein 42 (myeloid-specific retinoic acid-responsive) Hs.169832
	AA987906
694	**ESTs: Hs.43712 N25936
695	RUNX1: runt-related transcription factor 1 (acute myeloid leukemia 1; aml1 oncogene)
	Hs.129914 AA146826
696	Homo sapiens mRNA; cDNA DKFZp547C244 (from clone DKFZp547C244) Hs.9460
	T64452:
697	TYMS: thymidylate synthetase Hs.82962 AA663310
698	MGC5528: hypothetical protein MGC5528 Hs.315167 AA843451
699	ESTs: Hs.268685 R22952
700	SFPQ: splicing factor proline/glutamine rich (polypyrimidine tract-binding protein-
	associated) Hs.180610 AA418910
701	ESTs: Hs.155105 AI221390
702	FLJ10624: hypothetical protein FLJ10624 Hs.306000 AA489592
703	TRIP8: thyroid hormone receptor interactor 8 Hs.6685 AA425205
704	DNAJB6: DnaJ (Hsp40) homolog, subfamily B, member 6 Hs.181195 AA496105
705	ESTs: Hs.18331 T98244
706	RBM14: RNA binding motif protein 14 Hs.11170 AA421233
707	SCYA2: small inducible cytokine A2 (monocyte chemotactic protein 1, homologous to
	mouse Sig-je) Hs.303649 AA425102 MCP-1 = MCAF = small inducible cytokine
	A2 = JE = chemokine
708	MGC4161: hypothetical protein MGC4161 Hs.177688 AI224867
709	TUBB2: tubulin, beta, 2 Hs.251653 AA888148
710	FLJ20280: hypothetical protein FLJ20280 Hs.270134 N74086
711	TERA: TERA protein Hs.180780 AA465096
712	CPS1: **carbamoyl-phosphate synthetase 1, mitochondrial Hs.50966 N68399
713	KIAA0802: KIAA0802 protein Hs.27657 W55875
714	FYN: FYN oncogene related to SRC, FGR, YES Hs.169370 N22980
715	Homo sapiens PRO2751 mRNA, complete cds Hs.283978 H12784:
716	CLTH: Clathrin assembly lymphoid-myeloid leukemia gene Hs.7885 AA441930
717	CHMP1.5: CHMP1.5 protein Hs.42733 W85875
718	SMARCB1: SWI/SNF related, matrix associated, actin dependent regulator of
	chromatin, subfamily b, member 1 Hs.159971 AA446018
719	AA487823: SRF = c-fos serum response element-binding transcription facto
720	**ESTs: Hs.130741 AA608725
721	Homo sapiens cDNA FLJ10976 fis, clone PLACE1001399 Hs.296323 R36085:
722	FLJ20036: hypothetical protein FLJ20036 Hs.32922 N91145
723	C11ORF5: chromosome 11 open reading frame 5 Hs.121025 AA776702
724	AF3P21: SH3 protein Hs.102929 N94372
725	LOC54104: hypothetical protein Hs.12871 H05934
726	DF: D component of complement (adipsin) Hs.155597 AA233549
727	CEP4: Cdc42 effector protein 4; binder of Rho GTPases 4 Hs.3903 AA449061
728	KIF5B: kinesin family member 5B Hs.149436 AA644218
729	MGC5627: hypothetical protein MGC5627 Hs.237971 H02336
730	G3BP: Ras-GTPase-activating protein SH3-domain-binding protein Hs.220689
	AA449834
731	ESTs: Hs.293987 AA229758
732	ESTs: Hs.36828 AA194796
733	Homo sapiens mRNA for FLJ00101 protein, partial cds Hs.221600 W92262:
734	Homo sapiens cDNA: FLJ21288 fis, clone COL01927 Hs.6019 R07184:
735	ESTs,: Weakly similar to 1207289A reverse transcriptase related protein [H. sapiens]
	Hs.250594 H86813
736	Homo sapiens cDNA FLJ11941 fis, clone HEMBB1000649 Hs.124106 AI301573:
737	ESTs: Hs.24908 H77726
738	TOB2: transducer of ERBB2, 2 Hs.4994 AA486088
739	ESTs: Hs.143900 AI193212
740	Homo sapiens clone FLC0675 PRO2870 mRNA, complete cds Hs.306117 H16589:
741	ESTs,: Weakly similar to KIAA0638 protein [H. sapiens] Hs.296288 T83657
742	FLJ20039: hypothetical protein FLJ20039 Hs.267448 AA448268
743	RPA2: replication protein A2 (32 kD) Hs.79411 R13557
744	GAS1: growth arrest-specific 1 Hs.65029 AA025819
745	Human: DNA sequence from clone 967N21 on chromosome 20p12.3-13. Contains the
	CHGB gene for chromogranin B (secretogranin 1, SCG1), a pseudogene similar to part
	of KIAA0172, the gene for a novel protein Hs.88959 R56678
746	ESTs: Hs.21175 AI341642
747	LBC: lymphoid blast crisis oncogene Hs.301946 AA135716
748	ESTs: Hs.194595 R06761
749	MGC4707: hypothetical protein MGC4707 Hs.291003 R14653
750	ZNF183: zinc finger protein 183 (RING finger, C3HC4 type) Hs.64794 AA132766
751	RAD18: postreplication repair protein hRAD18p Hs.21320 R59197
752	EIF4EBP2: **eukaryotic translation initiation factor 4E binding protein 2 Hs.278712
	H15159
753	**Homo sapiens mRNA; cDNA DKFZp586M0723 (from clone DKFZp586M0723)
	Hs.27860 AA446650:
754	ORC3L: origin recognition complex, subunit 3 (yeast homolog)-like Hs.74420 H99257
755	CDK7: cyclin-dependent kinase 7 (homolog of Xenopus MO15 cdk-activating kinase)
	Hs.184298 AI311067
756	USP10: ubiquitin specific protease 10 Hs.78829 AA455233
757	KIAA0733: TAK1-binding protein 2; KIAA0733 protein Hs.109727 AA931658
758	R89286:
759	ALDH4: aldehyde dehydrogenase 4 (glutamate gamma-semialdehyde dehydrogenase;
	pyrroline-5-carboxylate dehydrogenase) Hs.77448 AA181378
760	IDN3: IDN3 protein Hs.225767 N62911
761	ESTs: Hs.50180 H48143
762	MIG2: mitogen inducible 2 Hs.75260 H29252
763	KIAA0856: KIAA0856 protein Hs.13264 R12847
764	EST: Hs.47763 N54162
765	Homo sapiens mRNA; cDNA DKFZp547C244 (from clone DKFZp547C244) Hs.9460
	AA447553:
766	KIAA0855: golgin-67 Hs.182982 AA775625
767	ESTs,: Weakly similar to JH0148 nucleolin - rat [R. norvegicus] Hs.30120 R54659
768	FLJ22313: hypothetical protein FLJ22313 Hs.30211 H52061
769	ESTs: Hs.71818 AI028074
770	KIAA0618: KIAA0618 gene product Hs.295112 AA455506
771	ESTs: Hs.59413 W93056
772	ESTs: Hs.165607 AA992090
773	UBAP: ubiquitin associated protein Hs.75425 AA446016
774	HAN11: WD-repeat protein Hs.176600 AA725641
775	USP16: ubiquitin specific protease 16 Hs.99819 AA489619
776	ESTs: Hs.67776 AA464963
777	SM-20: similar to rat smooth muscle protein SM-20 Hs.6523 H56028
778	CCNG2: cyclin G2 Hs.79069 AA489647
779	Homo sapiens mRNA; cDNA DKFZp566P1124 (from clone DKFZp566P1124)
	Hs.321022 N62953:
780	FLJ20094: hypothetical protein FLJ20094 Hs.29700 N95490
781	LOC51174: delta-tubulin Hs.270847 W33133
782	Homo sapiens mRNA; cDNA DKFZp434I1820 (from clone DKFZp434I1820); partial
	cds Hs.14235 N52394:
783	FANCA: Fanconi anemia, complementation group A Hs.284153 AA644129
784	P5-1: MHC class I region ORF Hs.1845 T58146
785	DNA2L: DNA2 (DNA replication helicase, yeast, homolog)-like Hs.194665
	AA974495 KIAA0083
786	LOC51578: **adrenal gland protein AD-004 Hs.279586 AA150301
787	ESTs: Hs.326417 AA913304
788	CDKN2D: cyclin-dependent kinase inhibitor 2D (p19, inhibits CDK4) Hs.29656
	R77517 p19-INK4D = Cyclin-dependent kinase 4 inhibitor D
789	FABP1: fatty acid binding protein 1, liver Hs.5241 AA682392
790	TERA: TERA protein Hs.180780 AA906997
791	ESTs: Hs.145383 AI253072
792	SLC7A5: solute carrier family 7 (cationic amino acid transporter, y+ system), member
	5 Hs.184601 AA419176
793	AXL: AXL receptor tyrosine kinase Hs.83341 H15336 axl = ufo = tyrosine kinase
	receptor
794	LOC57190: selenoprotein N Hs.8518 AA284276
795	ESTs: Hs.99037 AA443948
796	STCH: stress 70 protein chaperone, microsome-associated, 60 kD Hs.288799 H85311
797	ESTs: Hs.88523 AA278591 Unknown UG Hs.88523 ESTs
798	ESD: **esterase D/formylglutathione hydrolase Hs.82193 AA250931
799	ESTs: Hs.122444 R31021
800	ESTs: Hs.283127 AI291262
801	KIAA0480: **KIAA0480 gene product Hs.92200 H91332
802	HP1-BP74: HP1-BP74 Hs.142442 AA598791
803	**ESTs,: Moderately similar to ALU1_HUMAN ALU SUBFAMILY J SEQUENCE
	CONTAMINATION WARNING ENTRY [H. sapiens] Hs.144662 AA987667
804	TTF2: transcription termination factor, RNA polymerase II Hs.142157 AI023603
805	ESTs: Hs.13740 T70541
806	DJ37E16.5: hypothetical protein dJ37E16.5 Hs.5790 AA400021
807	CDH24: cadherin-like 24 Hs.155912 AI732266
808	DJ465N24.2.1: **hypothetical protein dJ465N24.2.1 Hs.8084 AA932375
809	ESTs,: Weakly similar to S57447 HPBRII-7 protein [H. sapiens] Hs.16346 AA410490
810	Homo sapiens cDNA: FLJ23285 fis, clone HEP09071 Hs.90424 AI005038:
811	KRAS2: v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog Hs.184050 N95249
812	FLJ20038: hypothetical protein FLJ20038 Hs.72071 H96090
813	ESTs,: Weakly similar to ALU4_HUMAN ALU SUBFAMILY SB2 SEQUENCE
	CONTAMINATION WARNING ENTRY [H. sapiens] Hs.28848 AA486607
814	H2AFN: H2A histone family, member N Hs.134999 AI095013
815	RERE: arginine-glutamic acid dipeptide (RE) repeats Hs.194369 AA490249
816	USP1: ubiquitin specific protease 1 Hs.35086 T55607
817	TIP47: cargo selection protein (mannose 6 phosphate receptor binding protein)
	Hs.140452 AA416787
818	KIAA0135: KIAA0135 protein Hs.79337 AA427740 KIAA0135 = related to pim-1
	kinase
819	ESTs: Hs.214410 T95273
820	PPP1R2: protein phosphatase 1, regulatory (inhibitor) subunit 2 Hs.267819 N52605
821	Homo sapiens cDNA: FLJ21210 fis, clone COL00479 Hs.325093 AA978323:
822	CSNK2A2: casein kinase 2, alpha prime polypeptide Hs.82201 AA054996
823	HSRTSBETA: rTS beta protein Hs.180433 N66132
824	FLJ13110: hypothetical protein FLJ13110 Hs.7358 AA431233
825	ESTs: Hs.238797 N30704
826	FYN: FYN oncogene related to SRC, FGR, YES Hs.169370 N35086
827	RBM8A: RNA binding motif protein 8A Hs.65648 AA448402
828	ESTs: Hs.21906 AA608546
829	ESTs: Hs.128081 AA971042
830	PP591: hypothetical protein PP591 Hs.118666 AA626336
831	N63866:
832	HM74: putative chemokine receptor; GTP-binding protein Hs.137555 R02739
833	MID1: midline 1 (Opitz/BBB syndrome) Hs.27695 AA598640
834	KIAA1586: KIAA1586 protein Hs.180663 AA938639
835	Homo sapiens clone CDABP0014 mRNA sequence Hs.92679 AA443139:
836	HSU79274: protein predicted by clone 23733 Hs.150555 AA451900
837	AOC3: amine oxidase, copper containing 3 (vascular adhesion protein 1) Hs.198241
	AA036974
838	AA548037:
839	FLJ10154: hypothetical protein FLJ10154 Hs.179972 AA457133
840	THBS1: thrombospondin 1 Hs.87409 AA464532
841	DNAJB6: DnaJ (Hsp40) homolog, subfamily B, member 6 Hs.181195 AA431203
842	KIAA1547: KIAA1547 protein Hs.31305 AI216623
843	GATA2: GATA-binding protein 2 Hs.760 R32405
844	ESTs: Hs.176950 R82522
845	KIAA1018: KIAA1018 protein Hs.5400 AA156859
846	B4GALT1: **UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 1
	Hs.198248 AA043795
847	HMGCR: 3-hydroxy-3-methylglutaryl-Coenzyme A reductase Hs.11899 AA779417
848	ESTs,: Weakly similar to 1819485A CENP-E protein [H. sapiens] Hs.167652 H94466
849	ESTs: Hs.294088 AA971073
850	KIAA1637: coactivator independent of AF-2 (CIA); KIAA1637 protein Hs.288140
	AA918007
851	HSPC196: hypothetical protein Hs.239938 H66023
852	DR1: down-regulator of transcription 1, TBP-binding (negative cofactor 2) Hs.16697
	AA132007
853	CG1I: putative cyclin G1 interacting protein Hs.10028 AA486444
854	IGSF4: immunoglobulin superfamily, member 4 Hs.70337 AA487505
855	ESTs: Hs.179309 AA664350
856	HSPC163: HSPC163 protein Hs.108854 AA053139
857	FLJ12788: hypothetical protein FLJ12788 Hs.20242 AI061317
858	FEM1B: FEM-1 (C. elegans) homolog b Hs.6048 H82273
859	FXR1: fragile X mental retardation, autosomal homolog 1 Hs.82712 N62761
860	NCOA3: nuclear receptor coactivator 3 Hs.225977 AA156793
861	H2BFB: H2B histone family, member B Hs.180779 N33927
862	ESTs: Hs.23830 AA460601
863	CDK7: cyclin-dependent kinase 7 (homolog of Xenopus MO15 cdk-activating kinase)
	Hs.184298 AA031961 CAK = cdk7 = NRTALRE = sdk = CDK activating kinase
864	FLJ20259: hypothetical protein FLJ20259 Hs.9956 T55949
865	Homo sapiens cDNA FLJ20678 fis, clone KAIA4163 Hs.143601 T95823:
866	RPS19: ribosomal protein S19 Hs.298262 T72208
867	Homo sapiens mRNA; cDNA DKFZp434M0420 (from clone DKFZp434M0420)
	Hs.326048 AA443976:
868	TP53: tumor protein p53 (Li-Fraumeni syndrome) Hs.1846 R39356 p53
869	FBI1: HIV-1 inducer of short transcripts binding protein Hs.104640 R06252
870	GOT1: glutamic-oxaloacetic transaminase 1, soluble (aspartate aminotransferase 1)
	Hs.597 H22855
871	FLJ21434: hypothetical protein FLJ21434 Hs.298503 AA680129
872	DNMT2: DNA (cytosine-5-)-methyltransferase 2 Hs.97681 R95731
873	ESTs: Hs.55272 W02785
874	H2BFQ: H2B histone family, member Q Hs.2178 AA010223
875	NFIC: nuclear factor I/C (CCAAT-binding transcription factor) Hs.184771 N20996
876	NPTX1: neuronal pentraxin I Hs.84154 H22445
877	TLOC1: translocation protein 1 Hs.8146 AA450205
878	MGC5302: endoplasmic reticulum resident protein 58; hypothetical protein MGC5302
	Hs.44970 N39195
879	ACTR2: ARP2 (actin-related protein 2, yeast) homolog Hs.42915 AA032090
880	AI287555:
881	ABCA7: ATP-binding cassette, sub-family A (ABC1), member 7 Hs.134514
	AI668632
882	COL7A1: collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and
	recessive) Hs.1640 AA598507
883	RFC2: replication factor C (activator 1) 2 (40 kD) Hs.139226 AA663472
884	FLJ22583: hypothetical protein FLJ22583 Hs.287700 AA135836
885	*ESTs,: Weakly similar to ORF2 [M. musculus*] Hs.172208 AI820570
886	ESTs: Hs.21667 R15709
887	RBBP4: retinoblastoma-binding protein 4 Hs.16003 AA705035
888	Homo sapiens mRNA; cDNA DKFZp434J1027 (from clone DKFZp434J1027); partial
	cds Hs.22908 R20166:
889	ESTs: Hs.166539 AI080987
890	NKTR: natural killer-tumor recognition sequence Hs.241493 AA279666 NK-tumor
	recognition protein = cyclophilin-related protein
891	MUC1: mucin 1, transmembrane Hs.89603 AA486365
892	AP4B1: adaptor-related protein complex 4, beta 1 subunit Hs.28298 AA481045
893	ESTs: Hs.94943 AA452165
894	MITF: microphthalmia-associated transcription factor Hs.166017 N66177
895	ESTs: Hs.183299 AA286914 Unknown UG Hs.183299 ESTs sc_id2032
896	BAG3: BCL2-associated athanogene 3 Hs.15259 AI269958
897	INSR: insulin receptor Hs.89695 AA001106
898	TRIP: TRAF interacting protein Hs.21254 AA186426
899	EST: Hs.307975 R22182
900	**Homo sapiens cDNA: FLJ23037 fis, clone LNG02036, highly similar to HSU68019
	Homo sapiens mad protein homolog (hMAD-3) mRNA Hs.288261 W72201:
901	HLA-DNA: major histocompatibility complex, class II, DN alpha Hs.11135
	AA702254 Major histocompatibility complex, class II, DN alpha
902	FLJ10392: **hypothetical protein FLJ10392 Hs.20887 AI261305
903	MPHOSPH1: **M-phase phosphoprotein 1 Hs.240 N63752
904	STAG1: stromal antigen 1 Hs.286148 R36160
905	USP1: ubiquitin specific protease 1 Hs.35086 AA970066
906	ESTs,: Moderately similar to ALU4_HUMAN ALU SUBFAMILY SB2 SEQUENCE
	CONTAMINATION WARNING ENTRY [H. sapiens] Hs.181315 AA448251
907	PA26: p53 regulated PA26 nuclear protein Hs.14125 AA447661
908	ESTs,: Weakly similar to zinc finger protein [H. sapiens ] Hs.71243 N92478
909	SH3PX1: SH3 and PX domain-containing protein SH3PX1 Hs.7905 R69163
910	**Homo sapiens cDNA: FLJ22554 fis, clone HSI01092 Hs.93842 H58317:
911	RPS25: ribosomal protein S25 Hs.113029 AA779404
912	ESTs,: Weakly similar to A49134 Ig kappa chain V-I region [H. sapiens] Hs.5890
	N34799 fra-2 = fos-related antigen 2
913	TXNRD1: thioredoxin reductase 1 Hs.13046 AA453335 Thioredoxin reductase
914	**ESTs: Hs.184378 N77828
915	GCSH: glycine cleavage system protein H (aminomethyl carrier) Hs.77631 R71327
916	Homo sapiens cDNA FLJ11904 fis, clone HEMBB1000048 Hs.285519 AA447098:
917	NCOA3: nuclear receptor coactivator 3 Hs.225977 H51992 AIB1 = Amplified in Breast
	Cancer = TRAM-1 = RAC3 = ACTR = CAGH16 = nucl
918	FLJ20159: hypothetical protein FLJ20159 Hs.288809 R33122
919	IL7R: interleukin 7 receptor Hs.237868 AA487121
920	RAB23: RAB23, member RAS oncogene family Hs.94769 AA134569
921	ESTs: Hs.132493 AA923168
922	ESTs: Hs.87507 AA236015
923	SHC1: SHC (Src homology 2 domain-containing) transforming protein 1 Hs.81972
	R52960
924	KIAA1321: KIAA1321 protein Hs.24336 W37999
925	GLI: glioma-associated oncogene homolog (zinc finger protein) Hs.2693 AI373071
926	ESTs: Hs.183299 AA291137 Unknown UG Hs.183299 ESTs sc_id2032
927	GPRK6: G protein-coupled receptor kinase 6 Hs.76297 AA291284
928	ESTs: Hs.93704 AA702684
929	CAPS: calcyphosine Hs.26685 AA858390
930	Homo sapiens cDNA FLJ10976 fis, clone PLACE1001399 Hs.296323 R27711:
931	C6: complement component 6 Hs.1282 N59396
932	UBE2D3: ubiquitin-conjugating enzyme E2D 3 (homologous to yeast UBC4/5)
	Hs.118797 AA465196
933	DDX8: DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 8 (RNA helicase)
	Hs.171872 AA465387 RNA helicase (HRH1)
934	DKFZP434B168: DKFZP434B168 protein Hs.48604 N62684
935	FLJ10512: hypothetical protein FLJ10512 Hs.93581 T39933
936	Homo sapiens mRNA; cDNA DKFZp564F093 (from clone DKFZp564F093)
	Hs.18724 W87709:
937	F8A: coagulation factor VIII-associated (intronic transcript) Hs.83363 AA463924
938	HSU53209: transformer-2 alpha (htra-2 alpha) Hs.24937 AA465172
939	UBQLN2: ubiquilin 2 Hs.4552 R43580
940	EIF2C2: eukaryotic translation initiation factor 2C, 2 Hs.193053 N93082
941	Homo sapiens mRNA for FLJ00012 protein, partial cds Hs.21051 H17645:
942	KIAA0841: KIAA0841 protein Hs.7426 R20299
943	KCNAB2: potassium voltage-gated channel, shaker-related subfamily, beta member 2
	Hs.298184 H14383
944	KIAA1637: coactivator independent of AF-2 (CIA); KIAA1637 protein Hs.288140
	AA521358
945	ESTs: Hs.27379 H17455
946	FLJ11323: hypothetical protein FLJ11323 Hs.25625 R49707
947	SSP29: acidic protein rich in leucines Hs.84264 AA489201
948	ESTs: Hs.69280 AA486011
949	ADAMTS1: a disintegrin-like and metalloprotease (reprolysin type) with
	thrombospondin type 1 motif, 1 Hs.8230 AA057170
950	ESTs: Hs.43466 N23889
951	MLLT4: myeloid/lymphoid or mixed-lineage leukemia (trithorax (Drosophila)
	homolog); translocated to, 4 Hs.100469 AA010818
952	ESTs: Hs.271034 AA406581
953	LMNB1: lamin B1 Hs.89497 AA983462
954	Homo sapiens cDNA FLJ13547 fis, clone PLACE1007053 Hs.7984 AA629264:
955	PTMS: parathymosin Hs.171814 R10451
956	H2AFL: H2A histone family, member L Hs.28777 AI268551
957	FLJ21603: hypothetical protein FLJ21603 Hs.129691 R72794
958	FLJ13287: hypothetical protein FLJ13287 Hs.53263 AA621725
959	CXCR4: chemokine (C-X-C motif), receptor 4 (fusin) Hs.89414 AA479357
960	INSM1: insulinoma-associated 1 Hs.89584 R38640
961	FREQ: frequenin (Drosophila) homolog Hs.301760 H16821
962	LOC58486: transposon-derived Buster1 transposase-like protein Hs.25726 AA868020
963	SMARCD1: SWI/SNF related, matrix associated, actin dependent regulator of
	chromatin, subfamily d, member 1 Hs.79335 H91691
964	ESTs: Hs.242998 T96522
965	INADL: PDZ domain protein (Drosophila inaD-like) Hs.321197 AA005153
966	ESTs,: Weakly similar to putative p150 [H. sapiens] Hs.37751 AA436174
967	MGC5338: hypothetical protein MGC5338 Hs.99598 H50550
968	W85890:
969	NUCKS: similar to rat nuclear ubiquitous casein kinase 2 Hs.118064 AI053436
970	Homo sapiens clone 25110 mRNA sequence Hs.27262 H18031:
971	AI333214:
972	GAS41: glioma-amplified sequence-41 Hs.4029 T62072
973	LOC51170: retinal short-chain dehydrogenase/reductase retSDR2 Hs.12150 N79745
974	H2BFG: **H2B histone family, member G Hs.182137 R98472
975	ABCC1: **ATP-binding cassette, sub-family C (CFTR/MRP), member 1 Hs.89433
	AA424804
976	EFNA1: ephrin-A1 Hs.1624 AA857015
977	Homo sapiens mRNA; cDNA DKFZp434A1014 (from clone DKFZp434A1014);
	partial cds Hs.278531 H00596:
978	PPP2CA: protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform
	Hs.91773 AA599092
979	ESTs,: Weakly similar to unnamed protein product [H. sapiens] Hs.118338 W85843
980	Homo sapiens cDNA FLJ11643 fis, clone HEMBA1004366 Hs.111496 AA598803:
981	ESTs,: Moderately similar to ALUE_HUMAN !!!! ALU CLASS E WARNING
	ENTRY !!! [H. sapiens] Hs.125407 AA878944
982	ESTs,: Moderately similar to ALU1_HUMAN ALU SUBFAMILY J SEQUENCE
	CONTAMINATION WARNING ENTRY [H. sapiens] Hs.144662 AI191290
983	KIAA0916: KIAA0916 protein Hs.151411 R91388
984	CDC25A: cell division cycle 25A Hs.1634 R09062
985	PRIM2A: primase, polypeptide 2A (58 kD) Hs.74519 R61073
986	DSP: desmoplakin (DPI, DPII) Hs.74316 H90899
987	KIAA0101: KIAA0101 gene product Hs.81892 W68219
988	ESTs,: Weakly similar to putative p150 [H. sapiens] Hs.268026 AA411454
989	ESTs: Hs.18140 T97707
990	H2AFL: H2A histone family, member L Hs.28777 AA457566
991	Homo sapiens mRNA for KIAA1700 protein, partial cds Hs.20281 H00287:
992	STAG3: stromal antigen 3 Hs.20132 AA453028
993	ZNF207: zinc finger protein 207 Hs.62112 N59119
994	BMP6: bone morphogenetic protein 6 Hs.285671 AA424833
995	ESTs,: Moderately similar to sertolin [R. norvegicus] Hs.91192 H60690
996	LOC51064: **glutathione S-transferase subunit 13 homolog Hs.279952 W88497
997	NUCKS: similar to rat nuclear ubiquitous casein kinase 2 Hs.118064 AA927182
998	ESTs,: Weakly similar to T00370 hypothetical protein KIAA0659 [H. sapiens]
	Hs.131899 W93155
999	FLJ13057: hypothetical protein FLJ13057 similar to germ cell-less Hs.243122 R23254
1000	ESTs: Hs.144796 AI219737
1001	FLJ10511: hypothetical protein FLJ10511 Hs.106768 R25877
1002	DKFZP564A122: DKFZP564A122 protein Hs.187991 N31577
1003	ODF2: outer dense fibre of sperm tails 2 Hs.129055 AA400407
1004	AMY2A: amylase, alpha 2A; pancreatic Hs.278399 R64129
1005	*ESTs,: Weakly similar to plakophilin 2b [H. sapiens*] Hs.12705 N91589
1006	CYP1B1: cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1 (glaucoma
	3, primary infantile) Hs.154654 AA029776
1007	CAPN7: calpain 7 Hs.7145 N46420
1008	FLJ20069: hypothetical protein FLJ20069 Hs.273294 AA229966
1009	FLJ10618: hypothetical protein FLJ10618 Hs.42484 AA478847
1010	KIAA1637: **coactivator independent of AF-2 (CIA); KIAA1637 protein Hs.288140
	AA452531
1011	FLJ20004: **hypothetical protein FLJ20004 Hs.17311 AA487895
1012	FLJ12892: hypothetical protein FLJ12892 Hs.17731 AA670363
1013	PLU-1: putative DNA/chromatin binding motif Hs.143323 AA464869
1014	**ESTs: Hs.36828 AA418448
1015	KIAA0586: KIAA0586 gene product Hs.77724 AA905278
1016	MTHFD2: methylene tetrahydrofolate dehydrogenase (NAD+ dependent),
	methenyltetrahydrofolate cyclohydrolase Hs.154672 AA480994
1017	BRF1: **butyrate response factor 1 (EGF-response factor 1) Hs.85155 AA424743
1018	TFAP2A: transcription factor AP-2 alpha (activating enhancer-binding protein 2 alpha)
	Hs.18387 R38044
1019	VIL2: villin 2 (ezrin) Hs.155191 AA411440
1020	SDC1: syndecan 1 Hs.82109 AA074511
1021	RNTRE: related to the N terminus of tre Hs.278526 AA281057
1022	HSPC207: hypothetical protein Hs.75798 H99997
1023	FLJ22376: hypothetical protein FLJ22376 Hs.29341 AI199155
1024	RNF10: ring finger protein 10 Hs.5094 H73586
1025	PNN: pinin, desmosome associated protein Hs.44499 AA707321
1026	FLJ20516: hypothetical protein FLJ20516 Hs.70811 AA122393
1027	RPL13A: ribosomal protein L13a Hs.119122 AI254200
1028	H2BFB: H2B histone family, member B Hs.180779 AA885642
1029	OGT: O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N-
	acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase) Hs.100293 R13317
1030	KIAA0155: KIAA0155 gene product Hs.173288 AA133684
1031	ILF2: interleukin enhancer binding factor 2, 45 kD Hs.75117 H95638
1032	Homo sapiens mRNA; cDNA DKFZp586I1518 (from clone DKFZp586I1518)
	Hs.21739 AA287917:
1033	PKNOX1: PBX/knotted 1 homeobox 1 Hs.158225 AI350546
1034	KMO: **kynurenine 3-monooxygenase (kynurenine 3-hydroxylase) Hs.107318
	AA044326
1035	VCAM1: vascular cell adhesion molecule 1 Hs.109225 H16591 CD106 = VCAM-1
1036	N54811:
1037	KIAA0618: KIAA0618 gene product Hs.295112 H81940
1038	MAFG: v-maf musculoaponeurotic fibrosarcoma (avian) oncogene family, protein G
	Hs.252229 N21609 MafG = basic-leucine zipper transcription factor
1039	MATN2: matrilin 2 Hs.19368 AA071473
1040	HOXB4: homeo box B4 Hs.126666 AA918749
1041	FLJ10466: hypothetical protein FLJ10466 Hs.121073 AA453607
1042	FLJ22557: hypothetical protein FLJ22557 Hs.106101 AA127879
1043	EST: Hs.149260 AI247680
1044	KIAA0677: KIAA0677 gene product Hs.155983 AA026751
1045	EST: Hs.104123 AA197344
1046	UCP4: uncoupling protein 4 Hs.40510 H94680
1047	EST: Hs.144224 N93807
1048	GATA2: GATA-binding protein 2 Hs.760 H00625 GATA-binding protein 2
1049	ESTs: Hs.14743 H61082
1050	EST: Hs.116174 AA626786
1051	ITGB3: integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) Hs.87149
	AA666269
1052	FLJ23399: hypothetical protein FLJ23399 Hs.299883 R19895
1053	ESTs: Hs.21734 N72976
1054	FLJ20425: hypothetical protein FLJ20425 Hs.71040 AA424566
1055	CUL4A: cullin 4A Hs.183874 AA598836
1056	PTP4A1: protein tyrosine phosphatase type IVA, member 1 Hs.227777 R61007
	protein tyrosine phosphatase PTPCAAX1 (hPTPCAAX1)
1057	ESTs: Hs.7913 N35592
1058	GRO1: GRO1 oncogene (melanoma growth stimulating activity, alpha) Hs.789
	W46900
1059	ESTs,: Moderately similar to NRD2 convertase [H. sapiens] Hs.309734 H78796
1060	FLJ10826: hypothetical protein FLJ10826 Hs.24809 AA486738
1061	TOM34: translocase of outer mitochondrial membrane 34 Hs.76927 AA457118
1062	H2AFL: H2A histone family, member L Hs.28777 AA452933
1063	D10S170: **DNA segment, single copy, probe pH4 (transforming sequence, thyroid-
	1, Hs.315591 N35493
1064	SCYA2: small inducible cytokine A2 (monocyte chemotactic protein 1, homologous to
	mouse Sig-je) Hs.303649 T77816 MCP-1 = MCAF = small inducible cytokine
	A2 = JE = chemokine
1065	FLJ10688: hypothetical protein FLJ10688 Hs.118793 AA465358
1066	PTD017: PTD017 protein Hs.274417 AA160498
1067	KIAA0026: MORF-related gene X Hs.173714 AA676604
1068	BMP2: bone morphogenetic protein 2 Hs.73853 AA489383
1069	MNT: MAX binding protein Hs.25497 AA455508
1070	KIAA1170: KIAA1170 protein Hs.268044 H80507
1071	CRYBA1: crystallin, beta A1 Hs.46275 AA487614
1072	KATNA1: katanin p60 (ATPase-containing) subunit A 1 Hs.289099 AA609740
1073	Homo sapiens cDNA FLJ20796 fis, clone COL00301 Hs.113994 N53458:
1074	CEP4: Cdc42 effector protein 4; binder of Rho GTPases 4 Hs.3903 W32509
1075	ESTs: Hs.117261 AA682521
1076	CYP1B1: cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1 (glaucoma
	3, primary infantile) Hs.154654 AA040872
1077	ALTE: Ac-like transposable element Hs.9933 AA630498
1078	RAD51: RAD51 (S. cerevisiae) homolog (E coli RecA homolog) Hs.23044 AA873056
1079	MAN1A2: mannosidase, alpha, class 1A, member 2 Hs.239114 R78501
1080	H53763:
1081	MET: met proto-oncogene (hepatocyte growth factor receptor) Hs.285754 AA410591
1082	DYRK1A: dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A
	Hs.75842 AA676749
1083	ARHGAP8: **Rho GTPase activating protein 8 Hs.102336 AA037410
1084	LMO4: LIM domain only 4 Hs.3844 H27986
1085	ADCY6: adenylate cyclase 6 Hs.12373 AA148044
1086	EST: Hs.135448 AI078552
1087	NCOA3: nuclear receptor coactivator 3 Hs.225977 W46433
1088	DNAJB4: DnaJ (Hsp40) homolog, subfamily B, member 4 Hs.41693 AA081471
1089	NAB1: NGFI-A binding protein 1 (ERG1 binding protein 1) Hs.107474 AA486027
1090	ESTs,: Weakly similar to T08663 hypothetical protein DKFZp547G0910.1 [H. sapiens]
	Hs.172084 N63646
1091	KIAA0735: KIAA0735 gene product; synaptic vesicle protein 2B homolog Hs.8071
	R56082
1092	GNB1: guanine nucleotide binding protein (G protein), beta polypeptide 1 Hs.215595
	AA487912
1093	Homo sapiens mRNA for KIAA1716 protein, partial cds Hs.21446 R49763:
1094	KINESIN: HEAVY CHAIN
1095	CCND1: cyclin D1 (PRAD1: parathyroid adenomatosis 1) Hs.82932 AA487486
	Cyclin D1 = BCL1 = PRAD1 = Translocated in mantle cell leukemia
1096	ESTs: Hs.106129 R56716
1097	AA431931:
1098	PSEN1: presenilin 1 (Alzheimer disease 3) Hs.3260 AA403083
1099	ESTs: Hs.193804 AA010918
1100	DKFZp762P2111: hypothetical protein DKFZp762P2111 Hs.14217 AA429586
1101	KIAA1350: KIAA1350 protein Hs.101799 W37627
1102	FLJ20847: hypothetical protein FLJ20847 Hs.13479 H16996
1103	HDCMA18P: HDCMA18P protein Hs.278635 N64387
1104	FLJ12890: hypothetical protein FLJ12890 Hs.43299 N62475
1105	ESTs: Hs.127453 AA973625
1106	BAIAP2: BAI1-associated protein 2 Hs.7936 R60328
1107	ESTs: Hs.317584 AA191424
1108	DKFZP434J046: DKFZP434J046 protein Hs.116244 AI024401
1109	ESTs: Hs.114055 AA701352
1110	ESTs: Hs.44380 N93122
1111	ESTs: Hs.20142 AA625570
1112	UBL3: ubiquitin-like 3 Hs.173091 T82438
1113	H2AFL: H2A histone family, member L Hs.28777 N50797
1114	SUCLG2: **succinate-CoA ligase, GDP-forming, beta subunit Hs.247309 N68557
1115	ZWINT: ZW10 interactor Hs.42650 AA706968
1116	FLJ10583: hypothetical protein FLJ10583 Hs.105633 R00425
1117	FLJ20552: hypothetical protein FLJ20552 Hs.69554 AA463982
1118	FADD: Fas (TNFRSF6)-associated via death domain Hs.86131 AA430751
	FADD = MORT
1119	SFRS7: splicing factor, arginine/serine-rich 7 (35 kD) Hs.184167 AA418813
1120	RAD54L: RAD54 (S. cerevisiae)-like Hs.66718 AI372035
1121	MYLE: MYLE protein Hs.11902 T68845
1122	LOC51334: mesenchymal stem cell protein DSC54 Hs.157461 R63841
1123	PRIM2A: primase, polypeptide 2A (58 kD) Hs.74519 AA434404
1124	KIAA0056: KIAA0056 protein Hs.13421 AA430545
1125	ESTs,: Moderately similar to ALU7_HUMAN ALU SUBFAMILY SQ SEQUENCE
	CONTAMINATION WARNING ENTRY [H. sapiens] Hs.82590 N53024
1126	ESTs: Hs.117269 AA705050
1127	NSAP1: NS1-associated protein 1 Hs.155489 AA186327
1128	CEACAM5: carcinoembryonic antigen-related cell adhesion molecule 5 Hs.220529
	AA130547
1129	FLJ11021: hypothetical protein FLJ11021 similar to splicing factor, arginine/serine-
	rich 4 Hs.81648 AA291183 Unknown UG Hs.202583 ESTs, Weakly similar to
	arginine-rich
1130	FOSL1: FOS-like antigen-1 Hs.283565 T82817 fra-1 = fos-related antigen 1
1131	U3-55K: U3 snoRNP-associated 55-kDa protein Hs.153768 AA465355
1132	DNAJC6: DnaJ (Hsp40) homolog, subfamily B, member 6 Hs.44896 AA455940
1133	KIAA1382: amino acid transporter 2 Hs.298275 R27255 Similar to transporter protein
1134	PCAF: p300/CBP-associated factor Hs.199061 N74637 P/CAF = p300/CBP-associated
	factor
1135	ESTs: Hs.130460 AA927252
1136	ESTs: Hs.112570 AI014667
1137	FLJ10209: hypothetical protein FLJ10209 Hs.260150 AA454626
1138	ESTs: Hs.99014 AA485679
1139	ESTs: Hs.99621 AA464707
1140	Homo sapiens cDNA FLJ11904 fis, clone HEMBB1000048 Hs.285519 N74617:
1141	AA928536:
1142	SQSTM1: **sequestosome 1 Hs.182248 AA931964
1143	**Homo sapiens cDNA FLJ13700 fis, clone PLACE2000216, highly similar to
	SPECTRIN BETA CHAIN, BRAIN Hs.324648 AA018591:
1144	SLC22A3: solute carrier family 22 (extraneuronal monoamine transporter), member 3
	Hs.81086 AA460012
1145	FLJ22557: hypothetical protein FLJ22557 Hs.106101 H00595
1146	FLJ20539: hypothetical protein FLJ20539 Hs.118552 R36152
1147	AA991624:
1148	TRAP150: thyroid hormone receptor-associated protein, 150 kDa subunit Hs.108319
	W85832
1149	ESTs: Hs.221847 R91557
1150	TCFL1: transcription factor-like 1 Hs.2430 AA443950
1151	ESTs,: Highly similar to oxytocinase splice variant 1 [H. sapiens] Hs.203271
	AA487918
1152	PLAB: prostate differentiation factor Hs.296638 AA450062
1153	RBM14: RNA binding motif protein 14 Hs.11170 AA417283
1154	EGFL5: EGF-like-domain, multiple 5 Hs.5599 W67981
1155	H2AFO: H2A histone family, member O Hs.795 AA047260
1156	ESTs,: Weakly similar to A46661 leukotriene B4 omega-hydroxylase [H. sapiens]
	Hs.169001 N45556
1157	W78784:
1158	TOP3A: topoisomerase (DNA) III alpha Hs.91175 N21546
1159	W73732: Host cell factor-1 = VP16 transactivator interacting protein
1160	CYP1B1: cytochrome P450, subfamily I (dioxin-inducible), polypeptide 1 (glaucoma
	3, primary infantile) Hs.154654 AA448157 Cytochrome P450 IB1 (dioxin-inducible)
1161	ESTs: Hs.135276 AI092102
1162	RHEB2: Ras homolog enriched in brain 2 Hs.279903 AA482117
1163	ESTs,: Highly similar to EF-9 [M. musculus] Hs.8366 H94467
1164	POLA: polymerase (DNA directed), alpha Hs.267289 AA707650
1165	KIAA1008: KIAA1008 protein Hs.323346 AA863115
1166	PIK3CD: phosphoinositide-3-kinase, catalytic, delta polypeptide Hs.162808
	AA281652
1167	T53625:
1168	**Homo sapiens mRNA; cDNA DKFZp434A1114 (from clone DKFZp434A1114)
	Hs.326292 AA417274:
1169	ESTs: Hs.26744 H16988
1170	FLJ13912: hypothetical protein FLJ13912 Hs.47125 W74133
1171	Homo sapiens mRNA; cDNA DKFZp762B195 (from clone DKFZp762B195)
	Hs.284158 AA625574:
1172	SSA2: Sjogren syndrome antigen A2 (60 kD, ribonucleoprotein autoantigen SS-A/Ro)
	Hs.554 AA010351
1173	BK1048E9.5: hypothetical protein bK1048E9.5 Hs.6657 N68512
1174	TOP1: topoisomerase (DNA) I Hs.317 AA232856 Topoisomerase I
1175	ESTs: Hs.15386 H18472
1176	KPNB1: karyopherin (importin) beta 1 Hs.180446 AA121732
1177	MGC861: hypothetical protein MGC861 Hs.208912 N69694
1178	PMS2L8: **postmeiotic segregation increased 2-like 8 Hs.323954 T62577
1179	TSC22: **transforming growth factor beta-stimulated protein TSC-22 Hs.114360
	R16390
1180	C8ORF1: chromosome 8 open reading frame 1 Hs.40539 AA278836
1181	ESTs: Hs.129165 AA989211
1182	DMTF: cyclin D binding Myb-like transcription factor 1 Hs.5671 AA129860
1183	CDC7L1: CDC7 (cell division cycle 7, S. cerevisiae, homolog)-like 1 Hs.28853
	N62245 Cdc7-related kinase
1184	LOC51700: cytochrome b5 reductase b5R.2 Hs.22142 AA425316
1185	FLNA: filamin A, alpha (actin-binding protein-280) Hs.195464 AA598978
1186	FLJ20257: hypothetical protein FLJ20257 Hs.178011 H78675
1187	Homo sapiens cDNA FLJ13604 fis, clone PLACE1010401 Hs.23193 AA406599:
1188	ESTs: Hs.205227 R73480
1189	SCYB14: small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK)
	Hs.24395 AA953842
1190	MAPK8IP2: **mitogen-activated protein kinase 8 interacting protein 2 Hs.80545
	AA418293
1191	ZNF42: zinc finger protein 42 (myeloid-specific retinoic acid- responsive) Hs.169832
	AA932642
1192	ESTs: Hs.127054 AA862450
1193	NUDT4: nudix (nucleoside diphosphate linked moiety X)-type motif 4 Hs.92381
	AA425630
1194	Homo sapiens cDNA FLJ10632 fis, clone NT2RP2005637 Hs.202596 H82421:
1195	LOC51042: zinc finger protein Hs.102419 AA033532
1196	NUMA1: nuclear mitotic apparatus protein 1 Hs.301512 AA679293
1197	ESTs,: Highly similar to A56429 I-kappa-B-related protein [H. sapiens] Hs.144614
	AA293771
1198	ESTs: Hs.127703 AA947258
1199	Homo sapiens cDNA FLJ14214 fis, clone NT2RP3003576 Hs.321236 AA903913:
1200	NFKBIA: nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor,
	alpha Hs.81328 W55872 IkB alpha
1201	ESTs: Hs.120029 AA707598
1202	ESTs,: Moderately similar to A Chain A, Human Glucosamine-6-Phosphate
	Deaminase Isomerase At 1.75 A [H. sapiens] Hs.21398 AA172012
1203	NFIA: nuclear factor I/A Hs.173933 AI912047
1204	RECQL4: RecQ protein-like 4 Hs.31442 AA620446
1205	**ESTs,: Weakly similar to ALU1_HUMAN ALU SUBFAMILY J SEQUENCE
	CONTAMINATION WARNING ENTRY [H. sapiens] Hs.318894 R96212
1206	Homo sapiens cDNA: FLJ21686 fis, clone COL09379 Hs.20787 R11371:
1207	LOC57168: similar to aspartate beta hydroxylase (ASPH) Hs.184390 H17272
1208	ESTs: Hs.26096 R54109
1209	Homo sapiens OSBP-related protein 6 mRNA, complete cds Hs.318775 AA680281:
1210	APACD: ATP binding protein associated with cell differentiation Hs.153884 N80741
1211	VIM: **vimentin Hs.297753 AI668662
1212	Homo sapiens cDNA FLJ13618 fis, clone PLACE1010925 Hs.17448 AA427980:
1213	NR3C1: nuclear receptor subfamily 3, group C, member 1 Hs.75772 N30428
	Glucocorticoid receptor
1214	Homo sapiens cDNA: FLJ21814 fis, clone HEP01068 Hs.289008 R12808:
1215	BRD7: bromodomain-containing 7 Hs.279762 AA488428
1216	MAP3K8: **mitogen-activated protein kinase kinase kinase 8 Hs.248 W42450
1217	ESTs: Hs.23213 H29336
1218	ESTs: Hs.122444 AA939019
1219	TUSP: tubby super-family protein Hs.102237 H78234
1220	KIAA1117: KIAA1117 protein Hs.278398 H01516
1221	Human: clone 137308 mRNA, partial cds Hs.322149 H91303
1222	ESTs: Hs.130214 AA456631
1223	RAB3A: RAB3A, member RAS oncogene family Hs.27744 H14230
1224	AA598795: Protein phosphatase 2 (formerly 2A), regulatory subunit B (P
1225	H2BFC: H2B histone family, member C Hs.137594 AI340654
1226	CFLAR: CASP8 and FADD-like apoptosis regulator Hs.195175 N94588
1227	CD24: CD24 antigen (small cell lung carcinoma cluster 4 antigen) Hs.286124 H59915
1228	EST: Hs.48532 N62402
1229	CCRK: cell cycle related kinase Hs.26322 H17616
1230	HECH: heterochromatin-like protein 1 Hs.278554 AI139106
1231	DKFZp547O146: hypothetical protein DKFZp547O146 Hs.91246 T80848
1232	ESTs: Hs.71574 AA135328
1233	HLXB9: homeo box HB9 Hs.37035 AI459915
1234	AA600222:
1235	SPINK5: serine protease inhibitor, Kazal type, 5 Hs.5476 W92134
1236	RNUT1: RNA, U transporter 1 Hs.21577 AA447799
1237	Homo sapiens cDNA: FLJ23013 fis, clone LNG00740 Hs.13075 AA464543:
1238	KIAA0063: KIAA0063 gene product Hs.3094 T82263
1239	DYRK2: dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 Hs.173135
	R63622
1240	R94947:
1241	Homo sapiens cDNA FLJ14337 fis, clone PLACE4000494 Hs.180187 AA004903:
1242	FLJ20624: hypothetical protein FLJ20624 Hs.52256 AA431909
1243	ESTs: Hs.43838 R38261
1244	FLJ23053: hypothetical protein FLJ23053 Hs.94037 R25654
1245	MGC11266: hypothetical protein MGC11266 Hs.293943 AA400456
1246	ESTs,: Moderately similar to ALU8_HUMAN ALU SUBFAMILY SX SEQUENCE
	CONTAMINATION WARNING ENTRY [H. sapiens] Hs.34174 AA126603
1247	PLAUR: plasminogen activator, urokinase receptor Hs.179657 AA147962
1248	TSG101: tumor susceptibility gene 101 Hs.118910 AA670215
1249	HCNGP: transcriptional regulator protein Hs.27299 AA457232
1250	KIAA0978: KIAA0978 protein Hs.3686 AA857017
1251	ESTs: Hs.61708 AA033867
1252	ESTs: Hs.120734 AA827482
1253	ESTs: Hs.5909 AA972654
1254	CDH24: cadherin-like 24 Hs.155912 AI668564
1255	CCND1: cyclin D1 (PRAD1: parathyroid adenomatosis 1) Hs.82932 T77237
1256	ESTs: Hs.43148 AA284775
1257	ESTs: Hs.222566 T50982
1258	ESTs: Hs.194125 N52822
1259	EST: Hs.154621 AI138644
1260	MAN1A2: mannosidase, alpha, class 1A, member 2 Hs.239114 R22905
1261	MAN2A2: mannosidase, alpha, class 2A, member 2 Hs.295605 AA454175
1262	Human DNA sequence from clone 967N21 on chromosome 20p12.3-13. Contains the
	CHGB gene for chromogranin B (secretogranin 1, SCG1), a pseudogene similar to part
	of KIAA0172, the gene for a novel protein Hs.88959 W94690
1263	ESTs,: Highly similar to CIKG_HUMAN VOLTAGE-GATED POTASSIUM
	CHANNEL PROTEIN KV3.4 [H. sapiens] Hs.106486 H11376
1264	Homo sapiens HT023 mRNA, complete cds Hs.237225 AA169496:
1265	FLJ10339: **hypothetical protein FLJ10339 Hs.203963 H72354
1266	N66278:
1267	ESTs: Hs.6195 AA454745
1268	KIAA1404: KIAA1404 protein Hs.200317 W72798
1269	PMAIP1: phorbol-12-myristate-13-acetate-induced protein 1 Hs.96 AA458838
	APR = immediate-early-response gene = ATL-derived PMA-responsive
1270	G3BP: Ras-GTPase-activating protein SH3-domain-binding protein Hs.220689
	AA598628
1271	Homo sapiens cDNA: FLJ22807 fis, clone KAIA2887 Hs.261734 R26854:
1272	Homo sapiens, clone IMAGE: 3535294, mRNA, partial cds Hs.80449 T57359:
1273	CDC16: CDC16 (cell division cycle 16, S. cerevisiae, homolog) Hs.1592 AA410559
1274	FGA: **fibrinogen, A alpha polypeptide Hs.90765 AA026626
1275	ESTs: Hs.33446 N53560
1276	Homo sapiens cDNA FLJ14175 fis, clone NT2RP2002979 Hs.288613 AA054704:
1277	ESTs: Hs.44243 AA011390
1278	Homo sapiens mRNA full length insert cDNA clone EUROIMAGE 42408 Hs.284123
	R61732:
1279	ESTs: Hs.53455 AA454165
1280	FLJ11264: hypothetical protein FLJ11264 Hs.11260 AI219094
1281	MBD4: methyl-CpG binding domain protein 4 Hs.35947 AA010492
1282	FLJ11305: hypothetical protein FLJ11305 Hs.7049 N94612
1283	Homo sapiens, Similar to CG5057 gene product, clone MGC: 5309, mRNA, complete
	cds Hs.13885 AA460004:
1284	ARHB: ras homolog gene family, member B Hs.204354 H88963
1285	ITPR3: inositol 1,4,5-triphosphate receptor, type 3 Hs.77515 AA865667
1286	HMG20B: high-mobility group 20B Hs.32317 AA775743
1287	ESTs: Hs.146276 AI214204
1288	PTPN9: protein tyrosine phosphatase, non-receptor type 9 Hs.147663 AA434420
1289	Homo sapiens clone FLB9213 PRO2474 mRNA, complete cds Hs.21321 AA486770:
1290	H21107:
1291	HSPC157: HSPC157 protein Hs.279842 N20480
1292	Homo sapiens mRNA; cDNA DKFZp564O2363 (from clone DKFZp564O2363)
	Hs.321403 AA406332:
1293	ESTs: Hs.150623 AA693532
1294	EST: Hs.188697 AA199733
1295	CLECSF2: C-type (calcium dependent, carbohydrate-recognition domain) lectin,
	superfamily member 2 (activation-induced) Hs.85201 H11732 AICL = activation-
	induced C-type lectin
1296	ITPR1: inositol 1,4,5-triphosphate receptor, type 1 Hs.198443 AA035450
1297	CHML: choroideremia-like (Rab escort protein 2) Hs.170129 R91881
1298	CDC42: cell division cycle 42 (GTP-binding protein, 25 kD) Hs.146409 AA668681
1299	FKBP5: **FK506-binding protein 5 Hs.7557 AA872767

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The mere mentioning of the publications and patent applications does not necessarily constitute an admission that they are prior art to the instant application.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

1-26. (canceled)

27. A method of treating prostate cancer in a patient comprising: (1) obtaining a sample comprising prostate cancer tumor tissue from said patient; (2) determining by microarray the expression of a panel of genes comprising ASF1B, ASPM, BIRC5, BUB1B, C18orf24, CDC2, CDC20, CDCA3, CDCA8, CDKN3, CENPF, CENPM, CEP55, DLGAP5, DTL, FOXM1, KIAA0101, KIF11, KIF20A, MCM10, NUSAP1, ORC6L, PBK, PLK1, PRC1, PTTG1, RAD51, RAD54L, RRM2, TK1 and TOP2A in said sample comprising prostate cancer tumor tissue; (3) prognosing said patient as having a decreased likelihood of prostate cancer progression when the average expression of the genes ASF1B, ASPM, BIRC5, BUB1B, C18orf24, CDC2, CDC20, CDCA3, CDCA8, CDKN3, CENPF, CENPM, CEP55, DLGAP5, DTL, FOXM1, KIAA0101, KIF11, KIF20A, MCM10, NUSAP1, ORC6L, PBK, PLK1, PRC1, PTTG1, RAD51, RAD54L, RRM2, TK1 and TOP2A is lower than a threshold index value; and (4) administering to said patient active surveillance based on the prognosis.

28. The method of claim 1, wherein said determining step further comprises determining the expression of one or more housekeeping genes in said sample.

29. The method of claim 2, wherein said determining step further comprises normalizing the expression of the genes ASF1B, ASPM, BIRC5, BUB1B, C18orf24, CDC2, CDC20, CDCA3, CDCA8, CDKN3, CENPF, CENPM, CEP55, DLGAP5, DTL, FOXM1, KIAA0101, KIF11, KIF20A, MCM10, NUSAP1, ORC6L, PBK, PLK1, PRC1, PTTG1, RAD51, RAD54L, RRM2, TK1 and TOP2A using the expression of the housekeeping genes.

30. The method of claim 3, wherein the housekeeping genes comprise the genes RPL38, UBA52, PSMC1, RPL4, RPL37, RPS29, SLC25A3, CLTC, TXNL1, PSMA1, RPL8, MMADHC, RPL13A, LOC728658, PPP2CA and MRFAP1.

31. A method of treating prostate cancer in a patient comprising: (1) obtaining a sample comprising prostate cancer tumor tissue from said patient; (2) determining by microarray the expression of a panel of genes comprising ASF1B, ASPM, BIRC5, BUB1B, C18orf24, CDC2, CDC20, CDCA3, CDCA8, CDKN3, CENPF, CENPM, CEP55, DLGAP5, DTL, FOXM1, KIAA0101, KIF11, KIF20A, MCM10, NUSAP1, ORC6L, PBK, PLK1, PRC1, PTTG1, RAD51, RAD54L, RRM2, TK1 and TOP2A in said sample comprising prostate cancer tumor tissue; (3) prognosing said patient as having an increased likelihood of prostate cancer progression when the average expression of the genes ASF1B, ASPM, BIRC5, BUB1B, C18orf24, CDC2, CDC20, CDCA3, CDCA8, CDKN3, CENPF, CENPM, CEP55, DLGAP5, DTL, FOXM1, KIAA0101, KIF11, KIF20A, MCM10, NUSAP1, ORC6L, PBK, PLK1, PRC1, PTTG1, RAD51, RAD54L, RRM2, TK1 and TOP2A exceeds a threshold index value; and (4) administering to said patient one or more of chemotherapy, radiation therapy or surgery based on the prognosis.

32. The method of claim 5, wherein said determining step further comprises determining the expression of one or more housekeeping genes in said sample.

33. The method of claim 6, wherein said determining step further comprises normalizing the expression of the genes ASF1B, ASPM, BIRC5, BUB1B, C18orf24, CDC2, CDC20, CDCA3, CDCA8, CDKN3, CENPF, CENPM, CEP55, DLGAP5, DTL, FOXM1, KIAA0101, KIF11, KIF20A, MCM10, NUSAP1, ORC6L, PBK, PLK1, PRC1, PTTG1, RAD51, RAD54L, RRM2, TK1 and TOP2A using the expression of the housekeeping genes.

34. The method of claim 7, wherein the housekeeping genes comprise the genes RPL38, UBA52, PSMC1, RPL4, RPL37, RPS29, SLC25A3, CLTC, TXNL1, PSMA1, RPL8, MMADHC, RPL13A, LOC728658, PPP2CA and MRFAP1.