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Title:
MICROARRAY GENE EXPRESSION PROFILING IN CLASSES OF PAPILLARY RENAL CELL CARCINOMA
Document Type and Number:
WIPO Patent Application WO/2006/112867
Kind Code:
A2
Abstract:
A nucleic acid probe or a set of such probes in a microarray is provided. The probe or probe set is used for the prognosis of patients with papillary cell renal cell carcinoma (PRCC), wherein aggressive and non-aggressive PRCC tumor types are characterized by differential expression profiles of genes that hybridize with one or more of these probes. Microarrays and kits for carrying out expression profiling of tumor tissue and methods of using them are disclosed.

Inventors:
TEH BIN TEAN (US)
TAN MINHAN (SG)
Application Number:
PCT/US2005/020840
Publication Date:
October 26, 2006
Filing Date:
June 13, 2005
Export Citation:
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Assignee:
VANANDEL RES INST (US)
TEH BIN TEAN (US)
TAN MINHAN (SG)
International Classes:
C12Q1/68; G16B25/10
Other References:
HIGGINS ET AL.: 'Gene expression patterns in renal cell carcinoma assessed by complementary DNA microarray' AMERICAN JOURNAL OF PATHOLOGY vol. 162, no. 3, March 2003, pages 925 - 932, XP003006396
Attorney, Agent or Firm:
DOLCE, Marcus P. (Heneveld Cooper, DeWitt & Litton, LLP, 695 Kenmoor, S.E., P.O. Box 256, Grand Rapids MI, US)
Download PDF:
Claims:
What is claimed is:

1. A microarray useful as a prognostic composition, comprising a matrix of at least one probe from a set of probes immobilized to a solid surface in predetermined order such that a row of pixels corresponds to replicates of one distinct probe from the set, the probes being any of SEQ ID NO:1- SEQ:ID NO:139, inclusive; and

(a) wherein the probes are complementary to nucleic acid sequences expressed differentially in aggressive as compared to non-aggressive types of papillary cell renal cell carcinoma (PRCC), which nucleic acid sequences specifically hybridize to the probes.

2. The microarray of claim 1 , wherein the set of probes comprises at least 10 probes.

3. The microarray of claim 1, wherein the set of probes comprises at least 100 probes.

4. The microarray of any of claims 1-3, wherein the one or more probes comprise nucleotides having at least one modified phosphate backbone selected from a phosphorothioate, a phosphoridothioate, a phosphoramidothiate, a phosphoamidate, a phosphordiimidate, a methylsphosphonate, an alkyl phosphotriester, 3'aminopropyl, a formacetal, or an analogue thereof.

5. The microarray of claim 1 or 4, wherein each probes comprises at least 15 nucleotides.

6. The microarray of any of claims 1-5, further comprising one or more nucleic acid samples representing expressed genes, each sample from an individual subject's tumor tissue, each sample spotted column- wise on the pixels of the microarray probes.

7. The microarray of claim 6, which has further been subjected to nucleic acid hybridization under stringency conditions such that the nucleic acid samples specifically hybridize to the immobilized probes on which the samples have been spotted.

8. A composition comprising a set of two or more oligonucleotide or polynucleotide probes each of which specifically hybridize to part of or all of a coding sequence that is

differentially expressed in an aggressive type of PRCC compared to a non-aggressive type of PRCC.

9. The composition of claim 8 comprising a set of at least 10 of the probes.

10. The composition of claim 8 comprising a set of at least 100 of the probes.

11. The composition of any of claims 8-10, wherein the coding sequence is up- regulated in the aggressive PRCC compared to the non-aggressive PRCC.

12. The composition of any of claims 8-10, wherein the coding sequence is up- regulated in the non-aggressive PRCC compared to the aggressive PRCC.

13. The composition of any of claims 8-10, wherein the probes are or mammalian origin.

14. The composition of claim 13 wherein the probes are of human origin.

15. A kit comprising:

(a) the microarray of any of claims 1 -7;

(b) reagents that facilitate hybridization of the nucleic acid to the immobilized probes; and

(c) a computer readable storage medium comprising logic which enables a processor to read data representing detection of hybridization.

16. The kit of claim 15 wherein the reagents are ones that facilitate detection of fluorescence.

17. A kit comprising:

(a) the composition of any of claims 8-14; '

(b) means for carrying out hybridization of the nucleic acid to the probes; and

(c) means for reading hybridization data.

18. The kit of claim 16, wherein the hybridization data is in the form of fluorescence data.

19. The kit of claims 17-18 wherein the probes are immobilized to the microarray .

20. A method of assessing aggressive PRCC in a renal tumor tissue sample comprising identifying differential modulation of each cDNA (relative to the expression of the same genes in a population of non-aggressive renal tumor tissue samples) in a combination of genes selected from the group consisting of SEQ ID NO:1-SEQ. ID NO:139.

21. The method of claim 20 wherein there is at least a two-fold difference in the expression of the modulated genes.

22. A method of assessing non-aggressive PRCC in a renal tumor tissue sample comprising identifying differential modulation of each gene (relative to the expression of the same genes in a population of aggressive renal tumor tissue samples) in a combination of genes selected from the group consisting of SEQ ID NO: 1 -SEQ. ID NO: 139.

23. The method of claim 22 wherein there is at least a two-fold difference in the expression of the modulated genes.

24. A prognostic portfolio comprising isolated nucleic acid sequences, their complements, or portions thereof of a combination of genes selected from the group consisting SEQ ID NOS:1-139.

25. The prognostic portfolio of claim 24, in a matrix suitable for identifying the differential expression of the genes contained therein.

26. The prognostic portfolio of claim 25 wherein said matrix is employed in a microarray.

27. The prognostic portfolio of claim 26 wherein said microarray is a cDNA microarray.

28. A method of evaluating the aggressiveness of PRCC in a patient, comprising detecting the level of expression in a renal tumor tissue sample of two or more genes from Table 4; wherein differential expression of the genes in Table 4 indicates whether the PRCC is aggressive or non-aggressive.

29. A method of evaluating the aggressiveness of PRCC in a patient, comprising detecting the level of expression in a renal tumor tissue sample of two or more genes from Table 6; wherein differential expression of the genes in Table 6 indicates whether the PRCC is aggressive or non-aggressive.

30. A method of assessing the aggressiveness of PRCC, the method comprising the steps of:

(a) providing a biological sample from a patient having PRCC;

(b) analyzing the expression of a set of genes selected from the group consisting of

sclerostin domain containing 1 four and a half LIM domains 1 prominin 1 four and a half LIM domains 1 four and a half LIM domains 1 four and a half LIM domains 1 keratin 7 chromosome 6 open reading frame 29

MRNA full length insert cDNA clone EUROIMAGE 2344436

MRNA full length insert cDNA clone EUROIMAGE 2344436 chromosome 9 open reading frame 71

MRNA full length insert cDNA clone EUROIMAGE 2344436 chemokine (C-C motif) ligand 28 chromosome 6 open reading frame 29

Transcribed sequences aryl hydrocarbon receptor claudin 7 asparaginase like 1 hypothetical protein MGCl 1242 asparaginase like 1 contactin 4 solute carrier family 6 (neurotransmitter transporter), member 20 fucosyltransferase 6 (alpha (1,3) fucosyltransferase)

Semaphorin 5A

MRNA full length insert cDNA clone EUROIMAGE 2344436 matrix metalloproteinase 15 (membrane-inserted)

KIAA 1026 protein androgen receptor zinc finger, MYND domain containing 12

Transcribed sequences

EPS8-like 1

Occluding hypothetical protein MGC3222 androgen receptor fucosyltransferase 6 (alpha (1,3) fucosyltransferase) suppression of tumorigenicity 14 (colon carcinoma, matriptase, epithin) hypothetical protein LOC283481

EPS8-like 1

EPS8-like 1

EpIiAl

CDNA FLJ45593 fis, clone BRTHA3014920

Similar to hypothetical protein BC006605 (LOC389389), mRNA

EPS8-like 1 hypothetical protein FLJ34633

RNA binding motif, single stranded interacting protein 2

DKFZp434A0131 protein

Transcribed sequence with moderate similarity to protein ref:NP_308425.1 hypothetical protein LOC146517 hypothetical protein FLJ12476

hypothetical protein MGC29816 molecule interacting with Rabl3 hypothetical protein FLJ12973

and (c) identifying the PRCC as aggressive or non-aggressive based on the expression of the set of genes.

Description:

MICROARRAY GENE EXPRESSION PROFILING IN CLASSES OF PAPILLARY RENAL CELL CARCINOMA

FIELD OF THE INVENTION

[0001] This invention relates to the field of molecular biology and medicine, including gene expression profiling for cancer, specifically, papillary renal cell carcinoma.

BACKGROUND OF THE INVENTION

[0002] Kidney cancer is a heterogenous disease consisting of various subtypes with diverse genetic, biochemical, and morphologic features. Epithelial renal cell carcinoma (RCC) accounts for the vast majority of renal malignancies in adults. Renal cell carcinoma is the tenth most common cancer in the United States. A. Jemal et ah, CA Cancer J CHn 54, 8 (2004). Based on morphological features defined in the WHO International Histological Classification of Kidney Tumors, RCC can be divided into clear cell (conventional), papillary (chromophil), chromophobe, collecting duct and unclassified subtypes. EbIe, J.N., Sauter, G., Epstein, J. L, Sesterhenn, I.A., Pathology and Genetics of Tumours of the Uninary System and Male Genital Organs. World Health Organization Classification of Tumours. 2004, Lyon: IARC Press; Storkel, S., J.N. EbIe, K. Adlakha, M. Amin, M.L. Blute, D.G. Bostwick, M. Darson, B. Delahunt, and K. Iczkowski, Classification of renal cell carcinoma: Workgroup No. 1. Union Internationale Contre Ie Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Cancer, 1997. 80(5): p. 987-9.

[0003] Papillary RCC (PRCC) is the second most common subtype comprising 10-15% of kidney cancers (Kovacs, G., M. Akhtar, BJ. Beckwith, P. Bugert, CS. Cooper, B. Delahunt, J.N. EbIe, S. Fleming, B. Ljungberg, LJ. Medeiros, H. Moch, V.E. Reuter, E. Ritz, G. Roos, D. Schmidt, J.R. Srigley, S. Storkel, E. van den Berg, and B. Zbar, The Heidelberg classification of renal cell tumours. J Pathol, 1997. 183(2): p. 131-3.), with an estimated annual incidence of between 3,500 to 5,000 cases in the United States, based on overall statistics for kidney cancer. Jemal, A., T. Murray, E. Ward, A. Samuels, R.C. Tiwari, A. Ghafoor, EJ. Feuer, and MJ. Thun, Cancer statistics, 2005. CA Cancer J Clin, 2005. 55(1): p. 10-30.

[0004] PRCC is histologically characterized by the presence of fibrovascular cores with tumor cells arranged in a papillary or tubopapillary configuration. The majority of PRCC tumors show indolent behavior, and have a limited risk of progression and mortality, but a distinct subset displays highly aggressive behavior. Amin, M.B., CL.

Corless, A.A. Renshaw, S.K. Tickoo, J. Kubus, and D.S. Schultz, Papillary (chromophil) renal cell carcinoma: histomorphologic characteristics and evaluation of conventional pathologic prognostic parameters in 62 cases. Am J Surg Pathol, 1997. 21(6): p. 621-35. The biological and clinical aspects of this cancer have been recently reviewed. Kuroda, N., M. Toi, M. Hiroi, and H. Enzan, Review of papillary renal cell carcinoma with focus on clinical and pathobiological aspects. Histol Histopathol, 2003. 18(2): p. 487-94.

[0005] The morphologic classification of PRCC into Type 1 and Type 2 tumors has been supported by several histological studies (although there is relatively limited molecular evidence to substantiate this subtyping). In particular, Delahunt and EbIe have proposed that PRCC can be morphologically classified into two subtypes. Delahunt, B. and J.N. EbIe, Papillary renal cell carcinoma: a clinicopathologic and immunohistochemical study of 105 tumors. Mod Pathol, 1997. 10(6): p. 537-44. Type 1 is characterized by the presence of small cuboidal cells covering thin papillae, with a single line of small uniform nuclei and basophilic cytoplasm. Type 2 is characterized by the presence of large tumor cells with eosinophilic cytoplasm and pseudostratification. Generally, Type 2 tumors have a poorer prognosis than Type 1 tumors. Mejean, A., V. Hopirtean, J.P. Bazin, F. Larousserie, H. Benoit, Y. Chretien, N. Thiounn, and B. Dufour, Prognostic factors for the survival of patients with papillary renal cell carcinoma: meaning of histological typing and multifocality. J Urol, 2003. 170(3): p. 764-7.

[0006] There is controversy over the proposed Typel/Type 2 morphological classification system of PRCC, preventing its wide application. As a result, some recent studies of PRCC do not stratify PRCC into Type 1 and Type 2 tumors. Cheville, J.C., CM. Lohse, H. Zincke, A.L. Weaver, and M.L. Blute, Comparisons of outcome and prognostic features among histologic subtypes of renal cell carcinoma. Am J Surg Pathol, 2003. 27(5): p. 612-24; Cheville, J.C., CM. Lohse, H. Zincke, A.L. Weaver, and MX. Blute, Comparisons of outcome and prognostic features among histologic subtypes of renal cell carcinoma. Am J Surg Pathol, 2003. 27(5): p. 612-24. As an example of the controversy of using the Type I/Type 2 system, there is no agreement whether a tumor with eosinophilic cytoplasm but low nuclear grade should be classified as Type 1 or Type 2. In the initial proposal outlining this morphologic subtyping (Delahunt, B. and J.N. EbIe, Papillary renal cell carcinoma: a clinicopathologic and immunohistochemical study of 105 tumors. Mod Pathol, 1997. 10(6): p. 537-44), 63% of Type 2 tumors were assessed as being of low Fuhrman nuclear grade despite pleomorphic nuclei being defined as a characteristic of Type 2 tumors. More recently, Allory et al (Allory, Y., D. Ouazana,

E. Boucher, N. Thiounn, and A. Vieillefond, Papillary renal cell carcinoma. Prognostic value of morphological subtypes in a clinicopathologic study of 43 cases. Virchows Arch, 2003. 442(4): p. 336-42) classified only 1/13 (8%) as low-grade Type 2 tumors using a modified criterion. Furthermore, the high frequency of tumors with coexisting Type 1 and Type 2 components poses difficulties for such a binary classification, the prevalence of such mixed tumors having been reported as high as 28%. Allory, Y., D. Ouazana, E. Boucher, N. Thiounn, and A. Vieillefond, Papillary renal cell carcinoma. Prognostic value of morphological subtypes in a clinicopathologic study of 43 cases. Virchows Arch, 2003. 442(4): p. 336-42. Allory et al chose to classify these tumors with mixed (Type 1 and Type 2) features as Type 1 tumors, an approach in line with the molecular classification of the present invention.

[0007] Despite the moderate incidence of PRCC (comparable to that of chronic myeloid leukemia), there is a disproportionately limited knowledge about the underlying molecular basis for development and progression of PRCC. To date, no effective therapy is available for patients with advanced PRCC (Motzer, R.J., J. Bacik, T. Mariani, P. Russo, M. Mazumdar, and V. Reuter, Treatment outcome and survival associated with metastatic renal cell carcinoma of non-clear-cell histology. J Clin Oncol, 2002. 20(9): p. 2376-81), and patients with PRCC may be excluded from clinical trials which are usually designed for the more common clear cell RCC. It is thus imperative to identify new molecular markers for establishing an accurate diagnosis and prognosis, and for developing effective medical therapies for this cancer.

[0008] Gene expression profiling is a technique that has demonstrated promise in addressing these issues in renal cell carcinoma. Tan, M.H., CG. Rogers, J.T. Cooper, J.A. Ditlev, TJ. Maatman, X. Yang, K.A. Furge, and B.T. Teh, Gene expression profiling of renal cell carcinoma. Clin Cancer Res, 2004. 10(18 Pt 2): p. 6315S-21S. The inventors and other groups of investigators have reported molecular signatures specific for several subtypes of kidney cancer, including PRCC. Higgins, J.P., R. Shinghal, H. Gill, J.H. Reese, M. Terris, RJ. Cohen, M. Fero, J.R. Pollack, M. Van De Rijn, and J.D. Brooks, Gene expression patterns in renal cell carcinoma assessed by complementary DNA microarray. Am J Pathol, 2003. 162(3): p. 925-32; Takahashi, M., D.R. Rhodes, K.A. Furge, H. Kanayama, S. Kagawa, B.B. Haab, and B.T. Teh, Gene expression profiling of clear cell renal cell carcinoma: gene identification and prognostic classification. Proc Natl Acad Sci U S A, 2001. 98(17): p. 9754-91; Takahashi, M., XJ.

Yang, T.T. Lavery, K.A. Furge, B.O. Williams, M. Tretiakova, A. Montag, NJ.

Vogelzang, G.G. Re, AJ. Garvin, S. Soderhall, S. Kagawa, D. Hazel-Martin, A. Nordenskjold, and B.T. Teh, Gene expression profiling of favorable histology Wilms tumors and its correlation with clinical features. Cancer Res, 2002. 62(22): p. 6598-605; Takahashi, M., XJ. Yang, J. Sugimura, J. Backdahl, M. Tretiakova, CN. Qian, S.G. Gray, R. Knapp, J. Anema, R. Kahnoski, D. Nicol, NJ. Vogelzang, K.A. Furge, H. Kanayama, S. Kagawa, and B.T. Teh, Molecular subclassification of kidney tumors and the discovery of new diagnostic markers. Oncogene, 2003. 22(43): p. 6810-8; Young, A.N., M.B. Amin, CS. Moreno, S.D. Lim, C. Cohen, J.A. Petros, F.F. Marshall, and A.S. Neish, Expression profiling of renal epithelial neoplasms: a method for tumor classification and discovery of diagnostic molecular markers. Am J Pathol, 2001. 158(5): p. 1639-51; Boer, J.M., W.K. Huber, H. Sultmann, F. Winner, A. von Heydebreck, S. Haas, B. Korn, B. Gunawan, A. Vente, L. Fuzesi, M. Vingron, and A. Poustka, Identification and classification of differentially expressed genes in renal cell carcinoma by expression profiling on a global human 31,500-element cDNA array. Genome Res, 2001. 11(11): p. 1861-70.

[0009] PRCC can be effectively distinguished from the other major subtypes of RCC using gene classifiers, from which α-methylacyl-CoA racemase (AMACR) has been additionally validated as a useful immunohistochemical marker. Tretiakova, M.S., S. Sahoo, M. Takahashi, M. Turkyilmaz, NJ. Vogelzang, F. Lin, T. Krausz, B.T. Teh, and XJ. Yang, Expression of alpha-methylacyl-CoA racemase in papillary renal cell carcinoma. Am J Surg Pathol, 2004. 28(1): p. 6976. However, no distinct molecular subclasses of PRCC have been identified using this marker.

[00010] The Union Internationale Contre Ie Cancer (UICC) recently developed an improved system for classifying RCC known as the "TNM" classification (referring to tumor, lymph node, and metastasis). T, N, and M categories are determined by physical examination and imaging. Sobin, L.H. et al., eds., TNM classification of malignant tumors. 5 th ed. (John Wiley & Sons, New York 1997). This system is set forth in Table 1 below.

[00011] Table 1

TNM Clinical Classification T — Primary Tnmor

TX Primary tumor cannot be assessed TO No evidence of primary tumor

Tl Tumor is <7.0 cm in greatest dimension, limited to the kidney T2 Tumor is >7.0 cm in greatest dimension, limited to the kidney T3 Tumor extends into major veins or invades adrenal or perinephric tissues but not beyond Gerota fascia

T3a Tumor invades adrenal gland or perinephric tissues but not beyond Gerota fascia

T3b Tumor grossly extends into renal vein(s) or vena cava below diaphragm T3c Tumor grossly extends into vena cava above diaphragm

T4 Tumor invades beyond Gerota fascia

N — Regional Lymph Nodes (hilar, abdominal para-aortic, and varacaval)

NX Regional lymph nodes cannot be assessed

NO No regional lymph node metastasis

Nl Metastasis in a single regional lymph node

N2 Metastasis in more than one regional lymph node

M — Distant Metastasis

MX Distant metastasis cannot be assessed MO No distant metastasis Ml Distant metastasis present pTNM Pathological Classification: corresponds to the T, N, and M categories. G — Histopathological Grading

GX Grade of differentiation cannot be assessed

Gl Well differentiated

G2 Moderately differentiated

G3, 4 Poorly differentiated/undifferentiated

Stage Grouping

M N M

Stage I Tl NO MO

Stage II T2 NO MO

Stage III Tl Nl MO

T2 Nl MO

T3 NO 5 Nl MO

Stage N T4 NO, Nl MO

Any T N2 MO

Any T Any N Ml

Genetic markers of particular interest

00013] Keratins are proteins that compose the 8-nni intermediate filaments in epithelial cells. CK7, or KRT7, is a type II keratin of simple nonkeratinizing epithelia. It is expressed in multiple organs, with substantial expression previously observed in lung, bladder, mesothelium, hair follicle, and ductal structures.

[00014] DNA topoisomerases are enzymes that control and alter the topologic states of

DNA in both prokaryotes and eukaryotes. Topoisomerase II from eukaryotic cells catalyzes the relaxation of supercoiled DNA molecules, catenation, decatenation, knotting, and unknotting of circular DNA. It appears likely that the reaction catalyzed by topoisomerase II involves the crossing-over of 2 DNA segments.

SUMMARY OF THE INVENTION

[00015] Using a clinically well-characterized patient population, the inventors correlated the global gene expression profiling of PRCC with tumor progression and clinical outcome, even in the absence of known cellular or molecular characteristics of these tumors. The inventors identified common features of renal cell tumorigenesis, including, genes that were upregulated when comparing two highly distinct molecular PRCC subclasses with morphological correlation, thus enabling the inventors to identify specific molecular signatures for each subclass of PRCC tumors. The discovery of a set of differentially expressed genes for each subclass provides a basis for explaining differences in tumor aggressiveness and clinical outcome.

[00016] Additionally, the methods and compositions described herein permit identification of proteins whose detection provide an early diagnostic approach to PRCC proteins as well as drug targets for the products of these genes. Thus, by discovering that a particular gene is differentially regulated in one subclass of PRCC, one can focus on developing drugs that suppress up-regulation, act directly on the protein product, or bypass the step in a cellular pathway mediated by the product of this gene.

[00017] The present invention provides a nucleic acid probe or set of probes (preferably between two and 139 in number) and a microarray comprising these markers as probes for the gene expression levels that are characteristic of PRCC tumor tissue. In one embodiment, the presence and levels of mRNA in a tissue being analyzed are screened using methods known in the art (i.e., Southern/Northern/Western blotting, gel electrophoresis, RFLP, SSCP). The invention is further directed to a method of implementing a microarray technology for disease prognosis thereby supplementing currently available prognostic techniques and pathological classification.

[00018] Use of the accurate, objective molecular methods described herein will inform physicians about which patients require heightened observation and additional, e.g., adjuvant therapies — for example, patients presenting with low stage PRCCs that appear on their face to be non-aggressive by conventional criteria, but that have the aggressive type molecular signatures as described herein. Moreover, in the case of patients presenting with higher stage PRCCs that might mistakenly be diagnosed as aggressive, but which have the non-aggressive molecular signature disclosed herein, this invention facilitates withholding of unnecessarily aggressive treatment while maintaining appropriate vigilance.

[00019] The present invention also is directed to a prognostic microarray composition of at least one oligonucleotide or polynucleotide probe from a set of probes immobilized to a solid surface in a predetermined order such that a row of pixels corresponds to replicates of one distinct probe from the set. The probes are complementary to nucleic acid sequences expressed differentially in aggressive as compared to non-aggressive types of PRCC. The probes are preferably any of SEQ ID NOS.:1-139 inclusive. The nucleic acid sequences hybridize to the probes under high stringency conditions.

[00020] The microarray may comprise at least about ten probes, more preferably one hundred probes, which probes are complementary to nucleic acid sequences expressed differentially in aggressive as compared to non-aggressive types of PRCC. These probes are preferably at least about fifteen nucleotides in length.

[00021] The present invention also includes a kit comprising the inventive composition; means for carrying out hybridization of the nucleic acid to the probe(s); and means for reading hybridization data. In one embodiment of the invention, the kit includes the inventive microarray, reagents that facilitate hybridization of the nucleic acid to the immobilized probes, and a computer- readable storage medium comprising logic which enables a processor to read data representing detection of hybridization. These kits are useful for determining the prognosis of a patient having PRCC.

[00022] The present invention also includes a method for assessing the aggressiveness of

PRCC in a renal tumor tissue sample, hi this method, the relative expression of genes in a subject's PRCC tumor tissue is compared to the same genes in a population of renal tumor tissue samples. The genes are selected from the group consisting of SEQ ID NOS.1-139. In one embodiment, there is at least a twofold difference in the relative expression of the genes comparing the subject's renal tumor tissue sample with the same genes in the population of renal tumor tissue samples.

[00023] Another method discovered by the inventors includes evaluating the aggressiveness of PRCC in a patient by detecting the level of expression in a renal tumor tissue sample of two or more genes from Table 4, wherein differential expression of the genes in Table 4 indicates whether the PRCC is aggressive or non-aggressive. In another embodiment of this method invention, the genes in Table 6 are evaluated for aggressiveness of PRCC in a patient.

[00024] The above probes are typically of mammalian, preferably human, origin.

[00025] hi the above methods, the nucleic acids from the tumor and the tissue are detectably labeled, preferably with a fluorescent label prior to the hybridization. With fluorescent labels, hybridization is detected as a fluorescent signal bound to the probe. BRIEF DESCRIPTION OF THE DRAWINGS

[00026] Figures 1 A-ID show images of four categories of PRCC tumors fixed in buffered formalin and hexatoxylin-eosin stain (Type 1, Type 2A, Typel/2A mixed, Type 2B, respectively).

[00027] Figure IE plots expression profiles by first two principal components grouped into

Type 1 (thick ring symbol), Type 2 (solid) and mixed Type I/Type 2 (thin ring symbol) tumors. Figure IF plots expression profiles by first two principal components grouped into Class 1 (solid) and Class 2 (ring) tumors.

[00028] Figure IG shows survival curves of Type 1, Type 2, and mixed Type I/Type 2 tumors, with Type 1 and mixed Type I/Type 2 curves overlapping. Figure IH shows survival curves of Class 1 and Class 2 tumors.

[00029] Figure 2 A is a heatmap showing hierarchical clustering of thirty-four PRCC tumor samples, showing distinct clustering of Class 1 (right) and Class 2 (left) tumors.

[00030] Figure 2B shows a comparative genomic microarray analysis inferred from cytogenic profiles of thirty- four PRCC tumor samples.

[00031] Figures 3A-L show images of formalin-fixed paraffin-embedded PRCC samples by H&E, CK7, and TopIIα immunostaining in Type 1 (3 A-C), Type 2A (3D-F), and Type

2B (3G-L) tumors. [00032] Figure 4A shows a prediction analysis of microarrays, depicting cross-validated misclassification error over a range of shrinking gene thresholds. [00033] Figure 4B shows cross-validated predictions of tumor Class 1 (left) and Class 2

(right).

[00034] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[00035] The preferred embodiments of the present invention may be understood more readily by reference to the following detailed description of specific embodiments and the Examples, Tables, and Sequence Listing included hereinafter.

[00036] The Sequence Listing contained on the compact disc titled "MICROARRAY

GENE EXPRESSION PROFILING IN CLASSES OF PAPILLARY RENAL CELL CARCINOMA SEQUENCE LISTING," with file title "Sequence Listing.ST25.txt," is incorporated by reference. This compact disc (attached) was created on June 10, 2005 and is 498 kilobytes.

[00037] Definitions

[00038] As used in the present application, "a" can mean one or more, depending on the context with which it is used; the acronym "PCR" is used interchangeably with "polymerase chain reaction"; and the term "oligonucleotide" refers to primers, probes, and oligomer fragments.

[00039] As used in the present application, "nucleic acid" and "polynucleotide" are interchangeable and refer to both DNA and RNA (as well as peptide nucleic acids). The term "oligonucleotide" is not intended to be limited to a particular number of nucleotides and therefore overlaps with polynucleotide. Probes for gene expression analysis include those comprising ribonucleotides, deoxyribonucleotides, copies thereof, or their analogues as described below. They may be poly- or oligonucleotides, without limitation of length.

[00040] As used in the present application, the term "specifically hybridize to" refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. The term "stringent conditions" refers to conditions under which a probe will hybridize to its target subsequence, but to no other sequence. Stringent conditions are sequence-dependant and will be different in different circumstances. One skilled in the art knows how to select such conditions. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 degrees Celsius lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm, 50% of the

probes are occupied at equilibrium). ' Typically, stringent conditions will be those in which the salt concentration is at least about 0.01 ato 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3.0 degrees Celsius for short probes (e.g., 10 to 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.

[00041] The present invention uses both oligonucleotide microarrays and cDNA microarrays to probe for, and to determine the relative expression of target genes of interest in a tissue sample of PRCC.

[00042] As set forth in greater detail below, the inventors studied the gene expression profiles of thirty-four cases of PRCC using Affymetrix HGUl 33 Plus 2.0 arrays (54,675 probe sets) using both unsupervised and supervised analysis. Comparative genomic microarray analysis (CGMA) was used to infer cytogenetic aberrations, and pathways were ranked with a curated database. Expression of selected genes was validated by immunohistochemistry in thirty-four samples, with fifteen independent tumors. The inventors thus identified two highly distinct molecular PRCC subclasses with morphologic correlation. Class 1, with excellent survival, corresponded to three histological subtypes: Type 1, low-grade Type 2 and mixed Type 1/low-grade Type 2 tumors. Class 2, with poor survival, corresponded to high-grade Type 2 tumors (n=ll). Dysregulation of Gl/S and G2/M checkpoint genes were found in Class 1 and Class 2 tumors respectively, alongside characteristic chromosomal aberrations. The inventors also identified a seven-transcript predictor that classified samples on cross-validation with 97% accuracy. Immunohistochemistry confirmed high expression of cytokeratin 7 in Class 1 tumors, and of topoisomerase Ilα in Class 2 tumors.

[00043] Also, the inventors have found Class 2 (Type 2B) PRCC may be distinguished from Class 1 (Type 1/ mixed Type 1 and 2A / Type 2A tumors) by the following characteristics: larger gross tumor size, higher nuclear grade (three to four), decreased CK7 staining and increased TopIIα staining, higher rate of metastases at surgery and poorer patient survival. Morphologic findings of less specificity include larger cell size and eosinophilic cytoplasm in Class 2 tumors.

[00044] Microarrays are orderly arrangements of spatially resolved samples or probes (in the present invention oligonucleotides and cDNAs of known sequence) that allow for massively parallel gene expression and gene discovery studies (Lockhart DJ et al, Nature (2000) 405 (6788):827-836).

[00045] The underlying concept of the microarray depends on base-pairing (hybridization) between purine and pyrimidine bases following the rules of Watson-Crick base pairing. DNA microarrays (DNA "chips") are fabricated by high-speed robotics. Microarray technology adds automation to the process of resolving nucleic acids of particular identity and sequence present in an analyte sample by labeling, preferably with fluorescent labels, and subsequent hybridization to their complements immobilized to a solid support in microarray format. An experiment with a single DNA chip can provide simultaneous information on thousands of genes- a dramatic increase in throughput (Reichert et al. (2000) Anal. Chem. 72:6025-6029) when compared to traditional methods.

[00046] Array experiments employ common solid supports such as glass slides, microplates or standard blotting membranes, and can be created by photolithographic synthesis by robotic deposition of samples. Photolithography generally involves attaching synthetic linkers (modified with photochemically removable protecting groups) to a glass substrate and directing light through a photolithographic mask to deprotect specific areas on the surface. The first of a series of hydroxyl-protected deoxynucleotides is incubated with the surface, and chemical binding occurs at the sites previously illuminated. Using a new mask, light then is directed to different regions of the substrate, and the chemical cycle repeated. Probes may be synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. Sample spot sizes in microarrays are typically <200 μm in diameter, and these arrays usually contain thousands of spots.

[00047] Microarrays require specialized robotics and imaging equipment that generally are commercially available and well-known in the art. Microarray analysis generally involves injecting a fluorescently tagged nucleic acid sample into a chamber to hybridize with complementary oligonucleotides on the microarray slide; laser excitation at the interface of the array surface and the tagged sample; collection of fluorescence emission by a lens; optical filtration of the fluorescence emissions; fluorescence detection; and quantification of hybridization intensity. Lipshutz, Robert J., Fodor, Stephen P.A., Gingeras, Thomas R., Lockhart, David J. High density synthetic oligonucleotide arrays. Nature Genetics Supplement. 21:20-23 (1999).

[00048] Oligonucleotide arrays are based on sequence information and are targeted to monitor the expression levels of many genes. Using as little as 200 to 300 bases of a gene, cDNA, or EST sequence, independent 25-mer oligonucleotides are selected (non- overlapping or minimally overlapping) as detectors. Probe selection is based upon

several factors: complementarity of the probe to a selected gene, cDNA, or EST sequence; uniqueness relative to family members and other genes; and an absence of near-complementarity to other common RNAs that may be in the sample. The overall selection of probes is based on "probe redundancy," i.e., using multiple oligonucleotides having different sequences but designed to hybridize to different regions of the same RNA. In the overall probe mix, additional redundancy involves the use of "mismatch control probes" that are identical to their "perfect match" partners except for a single base difference in a central position. Even with low concentrations of RNA, hybridization to the perfect match/mismatch pairs yields identifiable fluorescence patterns. The strength of these patterns indicates the concentration of the RNA in the sample. Lipshutz, Robert J., Fodor, Stephen P.A., Gingeras, Thomas R., Lockhart, David J. High density synthetic oligonucleotide arrays. Nature Genetics Supplement. 21:20-23 (1999).

[00049] Format I: a cDNA probe (500~5,000 bases) is immobilized to a solid surface such as glass using robotic spotting and exposed to a set of targets either separately or in a mixture. This method, traditionally called "DNA microarray," is considered to have been developed at Stanford University (Ekins, R et al, Trends in Biotech (1999) 17:217-218).

[00050] Format II: an array of probes that are "natural" oligo- or polynucleotides (oligomers of 20~80 bases), oligonucleotide analogues e.g., with phosphorothioate, methylphosphonate, phosphoramidate, or 3'-aminopropyl backbones), or peptide-nucleic acids (PNA).

[00051] The array is (1) exposed to an analyte comprising a detectable labeled, preferably fluorescent, sample nucleic acid (typically DNA), (2) allowed to hybridize, and (3) the identity and/or abundance of complementary sequences is determined.

[00052]

[00053] For analysis of the target nucleic acid of primary tumor tissue, the preferred analyte of this invention is isolated from tissue biopsies before they are stored or from

fresh-frozen tumor tissue of the primary tumor which may be stored and/or cultured in standard culture media. For expression studies, total RNA or poly(A)-containing mRNA is isolated using commercially available reagents and kits, e.g., from Invitrogen, Oligotex, or Qiagen. The mRNA is reverse transcribed into cDNA in the presence of labeled nucleotides. cDNA is generally synthesized using reverse transcriptase (e.g., Superscript II reverse-transcription kit from GIBCO-BRL). This may be directly or indirectly labeled by conjugation with a fluorescent dye.

[00054] The materials for a particular application of microarray technology are not necessarily available in convenient in kit form. The present invention provides microarrays and kits useful for analysis and prognosis of PRCC samples. Namely, the present invention includes microarrays comprising one or more nucleic acid probes having hybridizable fragments of any length (from about 15 bases to full coding sequence) for the genes whose expression is to be analyzed. For purposes of the analysis, the full length sequence must not necessarily be known, as those of skill in the art will know how to obtain the full length sequences using the sequence of a given EST and known data mining, bioinformatics, and DNA sequencing methodologies without undue experimentation. Those skilled in the art will appreciate that the probe of choice for a particular gene can be the full length coding sequence or any fragment thereof having at least about 15 nucleotides. Thus, when the full length sequence is known, the practitioner can select any appropriate fragment of that sequence. When the original results are obtained using partial sequence information (e.g., an EST probe), and when the full length sequence of which that EST is a fragment becomes available (e.g., in a genome database), the skilled artisan can select a longer fragment than the initial EST, as long as the length is at least about 15 nucleotides.

[00055] The polynucleotide or oligonucleotide probes of the present invention may be native DNA or RNA molecules or an analogues of DNA or RNA or portions thereof. The present invention is not limited to the use of any particular DNA or RNA analogue or portion thereof; rather any one is useful provided that it is capable of adequate hybridization to the complementary DNA (or mRNA) in a test sample, has adequate resistance to nucleases and stability in the hybridization protocols employed. DNA or RNA may be made more resistant to nuclease degradation in vivo by modifying internucleosite linkages (e.g., methylphosphonates or phosphorothioates) or by incorporating modified nucleosides (e.g., 2'-0-methylribose or 1 '-α-anomers) as described below.

[00056] A poly- or oligonucleotide may comprise at least one modified base moiety, for example, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5- carboxymethylaminomethyl-ω-thiouridine, 5-carboxymethyl-aminomethyl uracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyamino-methyl-2-thiouracil, β-D-mannosylqueosine, 5-methoxy-carboxymethyluracil, S-methoxyuracil^-methylthio-Nό-iso-pentenyladenine, uracil-5-oxyacetic acid, butoxosine, pseudouracil, queuosine, 2-thio-cytosine, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-t-oxyacetic acid, 5-methyl-2-thiouracil, 3(3-amino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine.

[00057] The poly- or oligonucleotide may comprise at least one modified sugar moiety including, but not limited, to arabinose, 2-flourarabinose, xylulose, and hexose.

[00058] In yet another embodiement, the poly- or oligonucleotide probe comprises a modified phosphate backbone synthesized from a nucleotide having, for example, one of the following structures: a phosphorothioate, a phosphoridothioate, a phosphoramidothioate, a phosphoramidate, a phosphordiimidate, a methylsphosphonate, an alkyl phosphotriester, 3'-aminopropyl and a formacetal or analog thereof.

[00059] In yet another embodiment, the poly- or oligonucleotide probe is an α-anomeric oligonucleotide which forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gautier et al,, 1987, Nwc/. Acids Res. 15:6625-6641).

[00060] An oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a hybridization-triggered cleavage agent, etc., all of which are well-known in the art.

[00061] Oligonucleotides of this invention may be synthesized by standard methods known in the art, e.g., by use of an automated DνA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al, (Nucl. Acids Res. (1998) 16:3209, methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymet supports (Sarin et al, Proc. Natl. Acad. ScL USA (1988) 85:7448-7451), etc.

[00062] Detectable Labels for Oligo- or Polynucleotide Probes

[00063] Preferred detectable labels include radionuclides, fluorescers, fluourogens, a chromophore, a chromogen, a phosphoescer, a chemiluminescer or a bioluminescer. Examples of fluorescers or fluorogens are i fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, σ-phthaldehyde, fluorescamine, a fluorescein derivative, Oregon Green, Rhodamine Green, Rhodol Green or Texas Red.

[00064] Common fluorescent labels include fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, ø-phthaldehyde and fluorescamine. Most preferred are the labels described in the Examples, below.

[00065] The fluorophore must be excited by light of a particular wavelength to flouresce.

See, for example, Haugland, Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed., Molecular Probes, Eugene, OR., 1996).

[00066] Fluorescein, fluorescein derivatives and fluorescein-like molecules such as

Oregon Green™ and its derivatives, Rhodamine Green™ and Rhodol Green™, are coupled to amine groups using the isothiocyanate, succinimidyl ester or dichlorotriazinyl- reactive groups. Similarly, fluorophores may also be coupled to thiols using maleimide, iodoacetamide, and aziridine-reactive groups. The long wavelength rhodamines, which are basically Rhodamine Green™ derivatives with substituents on the nitrogens, are among the most photostable fluorescent labeling reagents known. Their spectra are not affected by changes in pH between 4 and 10, an important advantage over the fluouresceins for many biological applications. This group includes the tetramethylrhodamines, X-rhodamines and Texas Red™ derivatives. Other preferred fluorophores are those which are excited by ultraviolet light. Examples include cascade blue, coumarin derivatives, naphthalenes (of which dansyl chloride is a member), pyrenes and pyridyloxazole derivatives.

[00067] The present invention serves as a basis for even broader implementation of microarrays and gene expression in deducing critical pathways implicated in cancer. In the case of PRCC, which is the focus of the present invention, a database of known patient genetic profiles can be used to categorize each new PRCC patient. The gene expression profile of the newly diagnosed PRCC patient is compared to the known PRCC molecular database of patients, such as that described herein based on thirty-four patients in whom complete clim ' cal follow up information is available,. This database will grow with each patient who is subjected to the present analysis as soon as his clinical outcome information becomes available. If the newly diagnosed patient's gene expression profile

most closely resembles the profile of aggressive PRCC, that patient will be so classified and treated accordingly, i.e., with more aggressive measures. Correspondingly, if a newly diagnosed patient's profile is that of the non-aggressive type, he will be treated accordingly, e.g., with less aggressive measures and careful clinical follow-up.

[00068] The composition of the present invention may be used in diagnostic, prognostic, or research procedures in conjunction with any appropriate cell, tissue, organ or biological sample of the desired animal species. By the term "biological sample" is intended any fluid or other material derived from the body of a normal or diseased subject, such as blood, serum, plasma, lymph, urine, saliva, tears, cerebrospinal fluid, milk, amniotic fluid, bile, ascites fluid, pus and the like. Also included within the meaning of this term is an organ or tissue extract and a culture fluid in which any cells or tissue preparation from the subject has been incubated.

[00069] Drug Discovery Based on Gene Expression Profiling

[00070] The molecular profiling information described herein is also harnessed for the purpose of discovering drugs that are selected for their ability to correct or bypass the molecular alterations or derangements that are characteristic of PRCC, particularly those that are associated with its aggressive form. A number of approaches are available.

[00071] In one embodiment, PRCC cell lines are prepared from tumors using standard methods and are profiled using the present methods. Preferred cell lines are those that maintain the expression profile of the primary tumor from which they were derived. One or several PRCC cells lines may be used as a "general" panel; alternatively or additionally, cell lines from individual patients may be prepared and used. These cell lines are used to screen compounds, preferably by high-throughput screening (HTS) methods, for their ability to alter the expression of selected genes. Typically, small molecule libraries available from various commercial sources are tested by HTS protocols.

[00072] The molecular alterations in the cell line cells can be measured at the mRNA level

(gene expression) applying the methods disclosed in detail herein. Alternatively, one may assay the protein product(s) of the selected gene(s). Thus, in the case of secreted or cell- surface proteins, expression can be assessed using immunoassay or other immunological methods including enzyme immunoassays (EIA), radioimmunoassay (RIA), immunofluorescence microscopy or flow cytometry. EIAs are described in greater detail in several references. Butler, J.E., In: Structure of Antigens, Vol. 1 (Van Regenmortel,

M., CRC Press, Boca Raton (1992), pp. 209-259; Butler, J.E., "ELISA," In: van Oss, CJ.

et al. (eds), Immunochemistry, Marcell Dekker, Inc., New York, 1994, pp. 759-803; Butler, J.E. (ed.), Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton, (1991). RIAs are discussed in Kirkjam and Hunter (eds.), Radioimmune Assay Methods, E. & S. Livingstone, Edinburgh, 1970.

[00073] In another approach, antisense RNAs or DNAs that specifically inhibit the transcription and/or translation of the targeted genes can be screened for specificity and efficacy using the present methods. Antisense compositions would be particularly useful for treating tumors in which a particular gene is up-regulated (e.g., the genes in Tables 2 and 3).

[00074] Diagnostic Methods

[00075] The protein products of genes that are upregulated in PRCC (e.g., Table 4) are targets for ealy diagnostic assays of PRCC if the proteins can be detected by some assay means, e.g., immunoassay, in some accessible body fluid or tissue. The most useful diagnostic targets are secreted proteins which reach a measurable level in a body fluid before the tumor presents by other criteria discussed in the Background section. Thus, a sample of a body fluid such as plasma, serum, urine, saliva, cerebrospinal fluid, et cetera, is obtained from the subject being screened. The sample is subject to any known assay for the protein analyte. Alternatively, cells expressing the protein on their surface may be obtained, e.g., blood cells, by simple, conventional means. If the protein is a receptor or other cell surface structure, it can be detected and quantified by well-known methods such as flow cytometry, immunofluorescence, immunocytochemistry or immunohistochemistry, and the like.

[00076] Preferably, an antibody or other protein or peptide ligand for the target protein to be detected is used. In another embodiment where the gene product is a receptor, a peptidic or small molecule ligand for the receptor may be used in known assays as the basis for detection and quantitation.

[00077] In vivo methods with appropriately labeled binding partners for the protein targets, preferably antibodies may also be used for diagnosis and prognosis, for example to image occult metastatic foci or for other types of in situ evaluations. These methods utilize include various radiographic, scintigraphic, and other imaging methods well-known in the art (MRI, PET, et cetera).

[00078] Suitable detectable labels include radioactive, fluorescent, fluorogenic, chromogenic, or other chemical labels. Useful radiolabels, which are detected simply by

gamma counter, scintillation counter, or autoradiography include 3 H, 125 I, 131 I, 35 S, and 14 C.

[00079] Common fluorescent labels include fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. The fluorophore, such as the dansyl group, must be excited by light of a particular wavelength to fluoresce. See, Haugland, Handbook of Fluorescent Probes and Research Chemicals, Sixth Ed., Molecular Probes, Eugene, OR, 1996. Fluorescein, fluorescein derivatives and fluorescein-like molecules such as Oregon Green™ and its derivatives, Rhodamine Green™ and Rhodol Green™, are coupled to amine groups using the isothiocyanate, succinimidyl ester or dichlorotriazinyl-reactive groups. Fluorophores may also be coupled to thiols using maleimide, iodoacetamide, and aziridine-reactive groups. The long wavelength rhodamines include the tetramethylrhodamines, X-rhodamines and Texas Red™ derivatives. Other preferred fluorophores for derivatizing the protein binding partner are those which are excited by ultraviolet light. Examples include cascade blue, coumarin derivatives, naphthalenes (of which dansyl chloride is a member), pyrenes and pyridyloxazole derivatives.

[00080] The protein (antibody or other ligand) can also be labeled for detection using fluorescence-emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the protein using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[00081] For in vivo diagnosis, radionuclides may be bound to protein either directly or indirectly using a chelating agent such as DTPA and EDTA which is chemically conjugated, coupled, or bound (which terms are used interchangeably) to the protein. The chemistry of chelation is well known in the art. The key limiting factor on the chemistry of coupling is that the antibody or ligand must retain its ability to bind the target protein. A number of references disclose methods and compositions for complexing metals to macromolecules including description or useful chelating agents. The metals are preferably detectable metal atoms, including radionuclides, and are complexed to proteins and other molecules. See, for example, U.S. 5,627,286, U.S. 5,618,513, U.S. 5,567,408, U.S. 5,443,816, U.S. 5,561,220, all of which are incorporated by reference herein.

[00082] Any radionuclide having diagnostic (or therapeutic value) can be used. In a preferred embodiment, the radionuclide is a γ-emitting or a β-emitting radionuclides, for example, one selected from the lanthanide or actinide series of the elements. Positron- emitting radionuclides, e.g., 68 Ga or 64 Cu, may also be used. Suitable γ-emitting

radionuclides include those which are useful in diagnostic imaging applications. The gamma-emitting radionuclides preferably have a half-life of from one hour to forty days, preferably from twelve hours to three days. Examples of suitable γ-emitting radionuclides include 67 Ga, 111 In, 99m Tc, 169 Yb and 186 Re. Examples of preferred radionuclides (ordered by atomic number) are 67 Cu, 67 Ga, 68 Ga 5 72 As, 89 Zr 5 90 Y 5 97 Ru, 99 Tc 5 111 In 5 123 I 5 125 I 5 131 I 5 169 Yb 5 186 Re 5 and 201 Tl. Though limited work has been done with positron-emitting radiometals as labels, certain proteins, such as transferrin and human serum albumin, have been labeled with 68 Ga.

[00083] A number of metals (not radioisotopes) useful for MRI include gadolinium, manganese, copper, iron, gold, and europium. Gadolinium is most preferred. Dosage can vary from 0.01 mg/kg to 100 mg/kg.

[00084] In situ detection of the labeled protein may be accomplished by removing a histological specimen from a subject and examining it by microscopy under appropriate conditions to detect the label. Those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

[00085] An alternative diagnostic approach utilizes probes that are complementary to and thereby detect cells in which a gene associated with PRCC is upregulated by in situ hybridization with niRNA in these cells. The present invention provides methods for localizing target mRNA in cells using fluorescent in situ hybridization (FISH) with labeled probes having a sequence that hybridizes with the mRNA of an upregulated gene. The basic principle of FISH is that DNA or RNA in the prepared specimens are hybridized with the probe nucleic acid that is labeled non-isotopically with, for example, a fluorescent die, biotin, or digoxigenin. The hybridized signals are then detected by fiuorimetric or by enzymatic methods, for example, by using a fluorescence or light microscope. The detected signal and image can be recorded on light sensitive film.

[00086] An advantage of using a fluorescent probe is that the hybridized image can be readily analyzed using a powerful confocal microscope or an appropriate image analysis system with a charge-coupled device (CCD) camera. As compared with radioactive methods, FISH offers increased sensitivity, hi addition to offering positional information, FISH allows better observation of cell or tissue morphology. Because of the nonradioactive approach, FISH has become widely used for localization of specific DNA or mRNA in a specific cell or tissue type.

[00087] The in situ hybridization methods and the preparations useful herein are described in Wu, W. et ah, eds., Methods in Gene Biotechnology, CRC Press, 1997, chapter 13, pages 279-289. This book is incorporated by reference in its entirety, as are the references cited therein. A number of patents and papers that describe various in situ hybridization techniques and applications, also incorporated by reference, are: 5,912,165; 5,906,919; 5,885,531; 5,880,473; 5,871,932; 5,856,097; 5,837,443; 5,817,462; 5,784,162; 5,783,387; 5,750,340; 5,759,781; 5,707,797; 5,677,130; 5,665,540; 5,571,673; 5,565,322; 5,545,524; 5,538,869; and 5,501,954; 5,225,326; 4,888,278. Other related references include Jowett, T., Methods Cell. Biol.; 59:63-85 (1999), Pinkel, et al, Cold Spring Harbor Symp. Quant. Biol. LI: 151-157 (1986); Pinkel, D. et ah, Proc. Natl. Acad. ScL (USA) 83:2934-2938 (1986); Gibson, et ah, Nucl. Acids Res. 15:6455-6467 (1987); Urdea et ah, Nucl. Acids Res. 16:4937-4956 (1988); Cook et ah, Nucl. Acids Res. 16:4077-4095 (1988); Telser et ah, J. Am. Chem. Soc. 111:6966-6976 (1989); Allen et ah, Biochemistry 28:4601-4607 (1989); Nederlof, P.M., et ah, Cytometry 10:20-27 (1989); Nederlof, P.M. et ah, Cytometry 11:126-131 (1990); Seibl, R. et ah, Biol. Chem. Hoppe-Seyler 371:939- 951 (Oct. 1990); Wiegant, J. et ah, Nucl. Acids Res. 19:3237-3241 (1991); McNeil J.A., et ah, Genet. Anal. Tech. Apph 8:41-58 (1991); Komminoth et ah, Diagnostic Molecular Biology 1:85-87 (1992); Dauwerse, J.G., et ah, Hum. MoI. Genet. 1:593-598 (1992); Ried, T. et ah, Proc. Natl. Acad. Sd. (USA) 89:1388-1392 (1992); Wiegant, J. et ah, Cytogenet. Cell Genet. 63:73-76 (1993); Glaser, V., Genetic Eng. News, 16:1, 26 (1996); Speicher, M.R., Nature Genet. 12:368-375 (1996).

[00088] Detection of "Unknown" Gene Product

[00089] In an extreme case, in which an upregulated DNA "X" is identified but its protein product "Y" is unknown, one would first examine the expressed DNA X sequence. The full length gene sequence may be obtained by accessing a human genomic database such as that of Celera. m either case, examination of the coding sequence for appropriate motifs will indicate whether the encoded protein Y is secreted protein or a transmembrane protein. If no antibodies specific for protein Y are already available, the peptides of protein Y can be designed and synthesized using known principles of protein chemistry and immunology. The object is to create a set of immunogenic peptides that elicit antibodies specific for epitopes of the protein that reside on its surface. Alternatively, the coding DNA or portions thereof can be expression-cloned to produce a polypeptide or peptide epitope thereof. That protein or peptide can be used as an immunogen to immunize animals for the production of antisera or to prepare monoclonal antibodies

(mAbs). These polyclonal sera or mAbs can then be applied in an immunoassay, preferably an EIA, to detect the presence of protein Y or measure its concentration in a body fluid or cell/tissue sample.

[00090] Therapeutic Methods

[00091] Taking the lead from the drug discovery methods described above, one can exploit the present invention to treat PRCC based on the knowledge of the genes that are up- regulated in a highly predictable manner across PRCC cases. Based on the nature of the deduced protein product, on can devise a menas to inhibit the action of, or remove and upregulated protein. In the case of a receptor, one would treat the upregulated receptor with an antagonist, a soluble receptor or a "decoy" ligand binding site of a receptor. Gershoni J.M., et al, Proc. Natl. Acad. Sd. USA, 1988 85:4087-9; U.S. Patent No. 5,770,572.

[00092] For an under-expressed receptor, an agonist or mimetic would be administered to maximize binding and activation of those receptor molecules which are expressed.

[00093] As for the set of genes that are shown here to be down-regulated in CC-RCC, one can devise a therapy targeted specifically at this form of the cancer, that would be used alone or in combination with known therapeutic approaches as discussed above. A preferred approach would be to stimulate production of the protein by administering an agent that promoted production, enhanced its stability or inhibited its degradation or metabolism. Alternatively, one could design means to bypass the metabolic step or signal pathway step that was affected by this down-regulation. This could be achieved by stimulating downstream steps in such pathways. If a receptor was involved, then, as above, agonists or mimics could be used to heighten responses of cells expressing too little of the receptor.

[00094] Moreover, for the down-regulated genes, gene therapy methods could be used to introduce more copies of the affected gene or more actively expressed genes operatively linked to strong promoters, e.g., inducible promoters, such as an estrogen inducible system. Braselmann, S. et al., Proc. Natl. Acad. ScL USA (1993) 90:1657-1661. Also known are repressible systems driven by the conventional antibiotic, tetracycline. Gossen, M. et al, Proc. Natl. Acad. ScI USA 89:5547-5551 (1992).

[00095] One can exploit the present invention to treat PRCC based on the knowledge of the genes that are upregulated in a highly predicable manner across PRCC cases. Based on the nature of the deduced protein product, one can devise a means to inhibit the action of, or remove an upregulated protein, hi the case of a receptor, one would treat the

upregulated receptor with an antagonist, a soluble receptor or a "decoy" ligand binding site of a receptor (Gershoni J.M. et al, Proc. Natl. Acad. Sd. USA, 1988 85:408709; U.S. Patent No. 5,770,572).

[00096] Antibodies may be administered to a patient to bind and inactivate (or compete with) secreted protein products or expressed cell surface products or upregulated genes.

[00097] In the case of upregulated genes, gene therapy methods could be used to introduce antisense oligonucleotide or polynucleotide constructs that would inhibit gene expression in a highly specific manner. Such constructs could be operatively linked to strong promoters, e.g., inducible promoters, such as an estrogen inducible system (Braselmann, S. et al,. Proc. Natl. Acad. Sd. USA (1993) 90:1657-1661). Also known are repressible systems driven by the conventional antibiotic, tetracycline (Gossen, M. et al, Proc. Natl. Acad. Sd. USA 89:5547-5551 (1992)). Multiple antisense constructs specific for different upregulated genes could be employed together. The sequences of the upregulated genes described herein are used to design the antisense oligonucleotides (Hambor, J.E. et al., J. Exp. Med. 168:1237-1245 (1988); Holt, LT. et al., Proc. Natl. Acad. Sd. 83:4794-4798 (1986); Izant LG. et al., Cell 36:1007-1015 (1984); Izant, LG. et al, Science 229:345- 352 (1985); De Benedetti, A. et al, Proc. Natl. Acad. Sd. USA 84:658-662 (1987)). The antisense oligonucleotides may range from 6 to 50 nucleotides, and may be as large as 100 or 200 nucleotides. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, sing-stranded or double-stranded. The oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone (as discussed above). The oligonucleotide may include other appending groups such as peptides, or agents facilitating transport across the cell membrane (see., e.g., Letsinger et al, 1989, Proc. Natl. Acad. ScL USA 84:684-652; PCT Publication No. WO 88/09810, published December 15, 1988) or blood-brain barrier (e.g., PCT Publication No. WO 89/10134, published April 25, 1988), hybridization-triggered cleavage agents (e.g., Krol et al, 1988, BioTechniques 6:958-976) or intercalating agents (e.g., Zon, 1988, Pharm. Res. 5:539-549).

[00098] The therapeutic methods that require gene transfer and targeting may include virus-mediated gene transfer, for example, with retroviruses (Nabel, E.G. et al, Science 244:1342 (1989), lentiviruses, and recombinant adenovirus vectors (Horowitz, M.S., In: Virology, Fields, B.N. et al, eds. Raven Press, New York, 1990, p. 1679, or current edition; Berkner, K.L., Biotechniques 6:616-919, 1988), Straus, S.E., In: The

Adenoviruses, Ginsberg, H.S., ed., Plenum Press, New York, 1984, or current edition).

Adeno-associated virus (AAV) also is also useful for human gene therapy (Samulski, RJ., et al., EMBO J. 10:3941 (1991); Lebkowskie, J.S. et al, MoI Cell Biol. (1988) 8:3988-3996; Kotin, R.M. et al., Proc. Natl. Acad. Sci. USA (1990) 87:2211-2215; Hermonat, PL et al, J. Virol. (1984) 51: 329-339). Improved efficiency is attained by the used of promoter enhancer elements in the plasmid DNA constructs (Philip R., et al, J. Biol. Chem. (1993) 268:16087-16090).

[00099] In addition to virus-mediated gene transfer in vivo, physical means well-known in the art can be used for direct gene transfer, including administration of plasmid DNA, and particle-bombardment mediated gene transfer, originally described in the transformation of plant tissue (Klein, T.M. et al, Nature 327:70 (1987); Christou, P. et al, Trends Biotechnol. 6:145 (1990)) but also applicable to mammalian tissues in vivo, ex vivo or in vitro (Yang, N.S. et al, Proc. Natl. Acad. Sci. USA 87:9568 (1990); Williams, R.S. et al, Proc. Natl. Acad. Sci. USA 88: 2726 (1991); Zelenin A.v. et al, FEBS Lett. 280:94 (1991); Zwelenin A.v. et al, FEBS Lett. 244:65 (1989); Johnston, S.A., et al, In Vitro Cell. Dev. Biol. 27:11 (1991)). Furthermore, electroporation, a well-known means to transfer genes into cell in vitro, can be used to transfer DNA molecules according to the present invention to tissues in vivo (Titomirov, A.V. et al, Biochim. Biophys. Acta 1088:131 (1991)).

000100] Gene transfer can also be achieved using "carrier mediated gene transfer" (Wu,

CH. et al, J. Biol Chem. 264: 16985 (1989); Wu, G.Y et al, J. Biol Chem. 263:14621 (1988); Soriano, P. et al, Proc. Natl Acad. Sci. USA 80:7128 (1983); Wang, CY. et al, Proc. Natl Acad. Sci. USA 84:7851 (1982); Wilson, J. M et al, J. Biol. Chem. 267:963 (1992)). Preferred carriers are targeted liposomes (Nicolau, C et al, Proc. Natl Acad. Sci. USA 80:1068 (1983); Soriano, et al, supra) such as immunoliposomes, which can incorporate acylated monoclonal antibodies into the lipid bilayer (Wang, et al, supra), or polycations such as asialogycoprotein/polylysine (Wu, et al., 1989, supra). Liposomes have been used to encapsulate and deliver a variety of materials to cells, including nucleic acids and viral particles (Faller, D. V. et al, J. Virol (1984) 49:269-272).

000101] Preformed liposomes that contain synthetic cationic lipids form stable complexes with polyanionic DNA (Feigner, P.L. et al, Proc. Natl Acad. Sci. USA (1987) 84:7413- 7417). Cationic liposomes, liposomes comprising some cationic lipid, that contained a membrane fusion-promoting lipid dioctadecyldimethyl-ammonium-bromide (DDAB) have efficiently transferred heterologous genes into eukaryotic cells (Rose, J.K. et al,

Biotechniques (1991) 10:520-525). Cationic liposomes can mediate high level cellular

expression of transgenes, or mRNA, by delivering them into a variety of cultured cell lines (Malone, R., et al, Proc. Natl. Acad. Sd. USA (1989) 86:6077-6081).

)00102] RT-PCR

) 00103] In addition to microarray analysis of patient tissue samples, other techniques known in the art can be used to assay expression individually of any one of the identified genes. For example, expression level can be detected by quantitative reverse transcription PCR if the the sample DNA is in formalin-fixed paraffin-embedded tissue.

EXAMPLES

300104] The present invention is more particularly described in the following Examples, which are intended as illustrative only, since modifications and variations therein will be apparent to those skilled in the art.

1)00105] Example 1- Patients and tumor samples

DOOl 06] Frozen samples of forty-three primary tumor specimens with a diagnosis of PRCC after routine pathological review were initially collected following nephrectomy. All tumor specimens were collected from participating institutions in the United States except one case from Japan.

000107] Tumor tissue was flash- frozen in liquid nitrogen immediately after nephrectomy and stored at -80 degrees Celsius. Portions of the tumors were fixed in buffered formalin and hematoxylin-eosin stained slide for all cases were centrally reviewed for the diagnosis of PRCC, except for one case (P 30), where slides were not available and histological description from the pathology report was used for subclassification. All samples had at least 60% tumor tissue. Tumor staging and grading were obtained from review of the pathology reports and evaluation of the case notes by individual clinicians. Node status was either assessed intraoperatively by inspection or by pathological evaluation. Tumors with sarcomatoid change were classified as grade four tumors. Functional status at the point of surgery, expressed by Eastern Cooperative Oncology Group (ECOG) performance status was derived by individual clinicians either at the point of initial evaluation, or by retrospective evaluation of the case notes. Tumor size was defined as the maximum tumor dimension upon measurement after resection.

000108] Based on histological features and Fuhrman grading system, the inventors designated four categories of tumors: Type 1 (n=14, Figure IA) characterized by small tumor cells of low Fuhrman grade (<2); Type 2A (n=4, Figure IB) characterized by large eosinophilic tumor cells of low Fuhrman grade (<2); combined Type 1 and Type 2A

(n=5, Figure 1C), and Type 2B (n=ll, Figure ID) characterized by large eosinophilic tumor cells with Fuhrman grade (>3) (Table 2).

)00109] Table 2

Histological Subclassification

300110] Figure IA shows Type 1 PRCC (Class 1) with basophilic cytoplasm (Furhman grade 2). Figure IB shows Type 2A PRCC (Class 1) with eosinophilic cytoplasm (Furhman grade 2). Figure 1C shows mixed Type 1 and 2A PRCC (Class 1) with combined Type 1 (left) and Type 2A (right) components (Furhman grade 2). Figure ID shows Type 2B PRCC (Class 2) with eosinophilic cytoplasm and pseudostratified layers of tumor cells (Furhman grade 4).

DOOlIl] The inventors extracted total RNA from homogenized samples using Trizol reagent (Invitrogen, CA). Total RNA was subsequently purified with an RNEasy kit (Qiagen, CA), and quality was assessed on denaturing gel electrophoresis. Nine specimens were excluded because of degraded RNA quality. Information on metastatic status at surgery was derived by review of pathologic, radiologic, and intra-operative findings. Clinicopathologic features of the final thirty-four cases are in Table 3. Twelve noncancerous kidney cortical specimens were also obtained for comparison of gene expression profiling.

000112] Table 3

Clinico-Pathologic Features with Molecular Classification

*Last known status. DOD: died of disease; DOO: died of other causes; AWC: alive with cancer; NED: no evidence of disease.

to

)00113] For histological evaluation and immunohistochemical analysis, formalin-fixed paraffin-embedded tissue blocks and sections were obtained from a total of thirty-four cases. Nineteen of these cases had undergone expression profiling and the additional fifteen cases were derived from independent patients, whom did not have tumor tissue profiled.

DOOl 14] Example 2- Microarray analysis

DOOl 15] Thirty-four primary tumor samples underwent expression profiling with whole-genome oligonucleotide arrays containing 54,675 probe sets (HGUl 33 Plus 2.0, Affymetrix, CA).

D00116] Oligonucleotide array profiling

J00117] For oligonucleotide expression profiling, 5-20 ug of total RNA was used to prepare antisense biotinylated RNA. A subset of cases was spiked with external poly- A RNA positive controls (Affymetrix, CA). Synthesis of single-stranded and double- stranded complementary DNA was performed with the use of T7-oligo (dT) primer (Affymetrix). In-vitro transcription was performed using Enzo Bioarray Transcript Labeling Kit (Enzo, NY). The biotinylated cRNA was subsequently fragmented, and 120ug was hybridized to each array at forty-five degrees Celsius over sixteen hours. The HGUl 33 Plus 2.0 GeneChips contain 54,675 probe sets, representing approximately 47,000 transcripts and variants. Scanning was performed in a GeneChip 3000 scanner. Quality assessment of performed in GeneChip Operating System (GCOS) 1.1.1 (Affymetrix) using global scaling to a target signal of 500. Quality assessment was performed using denaturing gel electrophoresis. The manufacturer's recommended protocol (GeneChip Expression Analysis Technical Manual, Affymetrix, April 2003) was followed for expression profiling. Median background was seventy-three, median scaling factor was 3.06, and median GADPH 375' ratio was 1.03, indicative of a high overall array and RNA quality. All data will be uploaded to the Gene Expression Omnibus under a pending accession number.

)00118] Data analysis

)00119] Statistical analyses were performed in the statistical environment R 2.0.1, utilizing packages from the Bioconductor project. Gentleman, R.C., VJ. Carey, D.M. Bates, B. Bolstad, M. Dettling, S. Dudoit, B. Ellis, L. Gautier, Y. Ge, J. Gentry, K. Hornik, T. Hothorn, W. Huber, S. Iacus, R. Irizarry, F. Leisch, C. Li, M. Maechler,

AJ. Rossini, G. Sawitzki, C. Smith, G. Smyth, L. Tierney, J. Y. Yang, and J. Zhang,

software development for computational biology and bioinformatics. Genome Biol, 2004. 5(10): p. R80. The robust multichip average (RMA) algorithm was used to perform preprocessing of the CEL files, including background adjustment, quartile normalization and summarization. Principal component analysis was used to visualize the thirty-four expression profiles.

100120] The inventors noted overlap between histological Type 1 and Type 2 tumors, contrary to their expectation of distinct molecular subtypes (Figure IE). Tumors with mixed Type 1 and Type 2 components (n=5) grouped with Type 1 tumors. PAM with ten-fold cross-validation persistently classified three of four low-grade Type 2 tumors with Type 1 tumors over a wide range of shrinking gene thresholds (Figure 4A). The only low-grade Type 2 tumor that persistently classified with the high grade Type 2 tumors was P 30 (the only tumor the inventors were unable to evaluated histologically to confirm a reported grade of 2). These results supported a hypothesis that Type 2 tumors were molecularly heterogeneous. The inventors analyzed the profiles based on this morphologic subtyping into two classes (Class 1 corresponding to Type 1, low-grade Type 2 and mixed Type 1 and low-grade Type 2 tumors, and Class 2 corresponded to high-grade Type 2 tumors) from a molecular viewpoint. Visualization of principal components then demonstrated distinct differential between expression profiles of Class 1 and Class 2 tumors, consistent with distinct tumor subclasses (Figure IF).

)00121] Significance analysis of microarrays (SAM) based on two-class unpaired analysis, assumption of unequal group variances and 10,000 permutations was used to derive a list of genes differentially expressed between tumor subclasses, and ordered by relative fold- change. Tusher, V. G., R. Tibshirani, and G. Chu, Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci U S A, 2001. 98(9): p. 5116-21. 139 transcripts differentially expressed between Class 1 and Class 2 tumors were identified using SAM at a delta of 1.8, with a false discovery rate of 0.01. The top fifty genes relatively upexpressed in each of Class 1 and Class 2 are listed in Table 4.

)00122] Table 4

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00123] *D(i) is a modified t-statistic calculated by SAM.

00124] **Fold-change is shown in terms of a relationship between the tumor with higher expression relative to the tumor with lower expression.

00125] A hierarchical clustering of the tumor samples based on the one hundred up- expressed transcripts are shown in Figure 2A. Hierarchical clustering of tumor samples by the top one hundred differentially expressed genes in each PRCC class is shown in Figure 2A. For the heatmap, rows represent individual oligonucleotide probes and columns represent individual tumor samples. Complete linkage clustering and a Euclidean distance metric was used, and values were scaled by row. The left cluster represents Class 2 tumors, corresponding to all Type 2B papillary tumors. The right cluster represents Class 1 tumors corresponding to all Type 1 and Type 2 A tumors.

100126] For derivation of a small gene classifier, the inventors used prediction analysis of microarrays (PAM), an R implementation of nearest shrunken centroids methodology with ten-fold cross validation over thirty gene thresholds and an offset percentage of 30%. Tibshirani, R., T. Hastie, B. Narasimhan, and G. Chu, Diagnosis of multiple cancer types by shrunken centroids of gene expression. Proc Natl Acad Sci U S A, 2002. 99(10): p. 6567-72. The inventors identified multiple gene classifiers that effectively differentiated Class 1 and Class 2 tumors at 97% accuracy at multiple shrinkage thresholds using PAM (ranging between 7 to 3,881 transcripts) using nearest shrunken centroids methology. Figure 4B shows cross-validated predictions of tumor class. A seven-transcript predictor is shown in Table 5.

)00127] Table 5

Tumor Subclass Predictor

These class scores are linear discriminant scores for each class as described in Tusher, et al. Only the tumor of P 30, initially reported as a Type 2 tumor with grade 2, which the

inventors were unable to confirm histologically, persistently classified as a Class 2 tumor, rather than as a Class 1 tumor, throughout these multiple shrinkage thresholds.

100128] Survival analysis

100129] Survival analysis was performed by fitting to a Cox proportional hazards model, and significance was determined by the likelihood ratio test. Two-tailed Student's t- testing and Fisher's exact testing was used to evaluate correlation between variables and tumor subclassification. For the purpose of this analysis, tumor grade and stage was classified into two categories, corresponding to low grade or stage (1 and 2), versus high grade and stage (3 and 4). Specifically, survival analysis performed (Figure IG, IH) showed that the inventors' refined morphologic and molecular classification system showed a survival prediction that showed a statistically insignificant edge over the previous morphology-based classification approach (Nagelkerke's R = 0.505 and p=0.001 versus R2 = 0.389 and p=0.005). Class 2 tumors were larger in tumor dimension (p= 0.003), of higher grade (p<0.001), higher stage (p<0.001) and were more likely to exhibit distant metastases at initial surgery (p<0.001) than Class 1 tumors. Indeed, all tumors metastatic at initial surgery were Class 2 tumors (n=7). No significant difference in age (p=0.37) or gender (p=0.70) was found between the two classes.

100130] Example 3- Comparative genomic microarray analysis/regional expression biases

100131] Aneuploidy is well established as a key . driver of global gene expression, and regional DNA copy number correlates well with regional expression in cancer (Hughes, T.R., CJ. Roberts, H. Dai, A.R. Jones, M.R. Meyer, D. Slade, J. Burchard, S. Dow, T.R. Ward, MJ. Kidd, S.H. Friend, and MJ. Marton, Widespread aneuploidy revealed by DNA microarray expression profiling. Nat Genet, 2000. 25(3): p. 333-7), which also has been demonstrated in RCC classification. Furge, K.A., K.A. Lucas, M. Takahashi, J. Sugimura, EJ. Kort, H.O. Kanayama, S. Kagawa, P. Hoekstra, J. Curry, XJ. Yang, and B. T. Teh, Robust classification of renal cell carcinoma based on gene expression data and predicted cytogenetic profiles. Cancer Res, 2004. 64(12): p. 4117-21. PRCC typically shows frequent trisomy 7, 12, 16, 17 and 20 (Amin, M.B., CL. Corless, A.A. Renshaw, S.K. Tickoo, J. Kubus, and D. S. Schultz, Papillary (chromophil) renal cell carcinoma: histomorphologic characteristics and evaluation of conventional pathologic prognostic parameters in 62 cases. Am J Surg Pathol, 1997. 21(6): p. 621-35; Kovacs, G., L. Fuzesi, A. Emanual, and H.F. Kung, Cytogenetics of papillary renal cell tumors. Genes Chromosomes Cancer, 1991. 3(4): p. 249-55; Kattar, M.M., DJ. Grignon, T.

Wallis, G.P. Haas, W.A. Sakr, J.E. Pontes, and D.W. Visscher, Clinicopathologic and

interphase cytogenetic analysis of papillary (chromophilic) renal cell carcinoma. Mod Pathol, 1997. 10(11): p. 1143-50). These findings are consistent with the present findings.

00132] In view of the distinct prognostic subtypes of PRCC defined on unsupervised and supervised analysis, the inventors sought to identify the characteristic chromosomal aberrations involved. Regional expression biases are genetic intervals where gene expression is coordinately changed (K. A. Furge et al, Cancer Res 64, 4117 (2004)), and correspond well with cytogenetic aberrations detected by comparative genomic hybridization. K. A. Furge et al, in preparation. Specifically, the inventors inferred cytogenic profiles for the tumors through the use of a refinement of the comparative genomic microarray analysis (CGMA) algorithm (Furge, K.A., K.A. Lucas, M. Takahashi, J. Sugimura, EJ. Kort, H.O. Kanayama, S. Kagawa, P. Hoekstra, J. Curry, X.J. Yang, and B. T. Teh, Robust classification of renal cell carcinoma based on gene expression data and predicted cytogenetic profiles. Cancer Res, 2004. 64(12): p. 4117- 21), which predicts chromosomal alterations based on regional changes in expression. Relative expression profiles R were generated from the single channel tumor expression profiles (T) and the mean expression values of the twelve single channel kidney cortical expression profiles (N) such that R+log 2 (T) — log 2 (N).

100133] Distinct cytogenetic profiles for each tumor were generated using high resolution

CGMA (Figure 2B). CGMA profiles of PRCC were generated from tumor: kidney cortical tissue expression ratios. As shown in Figure 2B, comparative genomic microarray analysis shows inferred cytogenetic profiles of the thirty-four tumor samples. Each block corresponding to a single chromosome represents the chromosomal expression profiles of a group of samples, and each sample is represented by a single vertical line in each block. Group 1 tumors correspond to samples above the white bar, and Group 2 tumors correspond to samples above the black bar.

100134] For PRCC subclassification, the present results are strictly not directly comparable to recent cytogenetic studies which have classified their results by the Type 1 and Type 2 classification. Jiang, F., J. Richter, P. Schraml, L. Bubendorf, T. Gasser, G. Sauter, MJ. Mihatsch, and H. Moch, Chromosomal imbalances in papillary renal cell carcinoma: genetic differences between histological subtypes. Am J Pathol, 1998. 153(5): p. 1467-73; Gunawan, B., A. von Heydebreck, T. Fritsch, W. Huber, R.H. Ringert, G. Jakse, and L. Fuzesi, Cytogenetic and morphologic typing of 58 papillary renal cell carcinomas: evidence for a cytogenetic evolution of type 2 from type 1 tumors. Cancer Res, 2003.

63(19): p. 6200-5. As expected, cytogenetic profiles inferred by the inventors were consistent with previous studies correlating cytogenetic findings with tumor grade (Lager et al), identifying less frequent trisomy of 7 in high grade tumors (Lager, DJ., BJ. Huston, T.G. Timmerman, and S.M. Bonsib, Papillary renal tumors. Morphologic, cytochemical, and genotypic features. Cancer, 1995. 76(4): p. 669-73) and Renshaw reporting that trisomy of 3 was found in a defined subset of low-grade PRCC tumors. Renshaw, A.A. and CL. Corless, Papillary renal cell carcinoma. Histology and immunohistochemistry. Am J Surg Pathol, 1995. 19(7): p. 842-9. In addition to these findings, in demonstrating that loss of 9q occurred more commonly in Class 2 tumors, the present results support a report that loss of heterozygosity at 9q is associated with reduced survival. Renshaw, A.A. and CL. Corless, Papillary renal cell carcinoma. Histology and immunohistochemistry. Am J Surg Pathol, 1995. 19(7): p. 842-9.

100135] Full-length gains in chromosomes 7, 12, 16, 17 and 20 was found both in Class 1 and Class 2 tumors, consistent with the previously reported trisomies observed by using conventional cytogenetic analysis characteristic of PRCC Jiang, Y., W. Zhang, K. Kondo, J.M. Klco, T.B. St Martin, M.R. DufauhyS.L. Madden, W.G. Kaelin, Jr., and M. Nacht, Gene expression profiling in a renal cell carcinoma cell line: dissecting VHL and hypoxia-dependent pathways. MoI Cancer Res, 2003. 1(6): p. 453-62.; Corless, C.L., A.S. Kibel, O. Iliopoulos, and W.G. Kaelin, Jr., Immunostaining of the von Hippel-Lindau gene product in normal and neoplastic human, tissues. Hum Pathol, 1997. 28(4): p. 459- 64. However, in comparison to Class 1 tumors, Class 2 tumors exhibited more frequent gains at Iq, 2, 8q, losses at 3p, 6q, and showed fewer gains of chromosome 3, 7 and 16. More frequent losses of 6q and 14q were also evident.

)00136] Example 4- Patient Case Example

)00137] As an example of the application of the seven-transcript predictor, the inventors use the example of Patient 34, with histologically diagnosed Type 2B PRCC, and who died nine months after surgery. Internal cross-validation within this data set classified Patient 34 as a Class 2 tumor, corresponding with a poor prognosis, consistent with the eventual outcome.

)00138] Example 5- Pathway analysis

)00139] The inventors performed pathway analysis on the differentially expressed genes using Ingenuity Pathway Analysis (Ingenuity Systems, CA), and enrichment of canonical pathways was assessed for significance by a hypergeometric algorithm hat did not correct for multiple testing. Specifically, 203 genes derived from the 139 transcripts were

eligible for generation of networks in pathway analysis. Ranking of canonical pathways yielded 3 pathways that were significantly enriched within these differentially expressed genes: G2/M DNA damage checkpoint regulation (p=0.007), arginine and proline metabolism (p=0.QH) and Gl/S checkpoint regulation (p=0.018). Genes involved in Gl/S checkpoint regulation (cyclin D2, cyclin-dependent kinase 6, retinoblastoma-like 2 and p21Cipl), were relatively upexpressed in Class 1 tumors, whereas genes involved in G2/M checkpoint regulation (cyclin Bl, cyclin B2 and topoisomerase II alpha) were relatively upexpressed in Class 2 tumors. The present invention highlights dysregulation of Gl/S checkpoint genes in Class 1 PRCC and dysregulation of G2/M checkpoint genes in Class 2 PRCC, as the most highly ranked pathways identified in the differentially expressed genes. Details of individual gene expression in the 139 transcripts are shown in Table 6. Table 6

Relative fold-change*

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)00141] Multiple oligonucleotide probe sets corresponding to c-met were identified as being upexpressed in Class 1 tumors, ranging between 2- to 3 -fold upexpression. In familial studies, mutations of the c-met proto-oncogene have been implicated in hereditary Type 1 PRCC (Schmidt, L., K. Junker, G. Weirich, G. Glenn, P. Choyke, I. Lubensky, Z. Zhuang, M. Jeffers, G. Vande Woude, H. Neumann, M. Walther, W.M. Linehan, and B. Zbar, Two North American families with hereditary papillary renal carcinoma and identical novel mutations in the MET proto-oncogene. Cancer Res, 1998. 58(8): p. 1719-22), and a small subset (<10%) of sporadic Type 1 PRCCs (Schmidt, L., K. Junker, N. Nakaigawa, T. Kinjerski, G. Weirich, M. Miller, I. Lubensky, H.P. Neumann, H. Brauch, J. Decker, C. Vocke, J.A. Brown, R. Jenkins, S. Richard, U. Bergerheim, B. Gerrard, M. Dean, W.M. Linehan, and B. Zbar, Novel mutations of the MET proto-oncogene in papillary renal carcinomas. Oncogene, 1999. 18(14): p. 2343- 50). Interestingly, the inventors demonstrated that c-met was differentially expressed, with higher expression in Class 1 tumors (Table 6). From a mechanistic point of view, this associative link between c-met overexpression/mutation and genes associated with Gl /S checkpoint dysregulation is particularly useful as hepatocytes in conditional Met mutant mice exhibit defective exit from quiescence and diminished entry into the S-phase of the cell cycle. Borowiak, M., A.N. Garratt, T. Wustefeld, M. Strehle, C. Trautwein, and C. Birchmeier, Met provides essential signals for liver regeneration. Proc Natl Acad Sci U S A, 2004. 101(29): p. 10608-13.

)00142] The implication of dysregulation of the G2/M checkpoint regulation in Class 2 tumors is particularly useful from a therapeutic point of view. Specifically, based on the present study, DNA topoisomerase II alpha (TopIIα) is a diagnostic marker for Class 2 tumors. As there is no effective medical therapy for advanced PRCC and this enzyme is associated with the more aggressive PRCC subclass, topoisomerase II inhibitors such as doxorubicin and etoposide could be used in a therapeutic trial of PRCC. G2 arrest occurs in response to these agents (Clifford, B., M. Beljin, G.R. Stark, and W.R. Taylor, G2 arrest in response to topoisomerase II inhibitors: the role of p53. Cancer Res, 2003. 63(14): p. 4074-81), and may therefore be particularly appropriate in this setting. While several trials have reported disappointing results for topoisomerase II inhibitors in kidney cancer (Escudier, B., J.P. Droz, F. Rolland, MJ. Terrier-Lacombe, G. Gravis, P. Beuzeboc, B. Chauvet, C. Chevreau, J.C. Eymard, T. Lesimple, Y. Merrouche, S.

Oudard, F. Priou, C. Guillemare, S. Gourgou, and S. Culine, Doxorubicin and ifosfamide in patients with metastatic sarcomatoid renal cell carcinoma: a phase II study of the Genitourinary Group of the French Federation of Cancer Centers. J Urol, 2002. 168(3): p. 959-61; Law, T.M., P. Mencel, and RJ. Motzer, Phase II trial of liposomal encapsulated doxorubicin in patients with advanced renal cell carcinoma. Invest New Drugs, 1994. 12(4): p. 323-5), these trials have predominantly recruited patients with clear cell RCC, a disease that is genetically distinct from PRCC. Further, note that the same enzyme previously has been reported in cDNA microarray study as being the most overexpressed gene in pediatric Wilms' tumor (Takahashi, M., XJ. Yang, T.T. Lavery, K.A. Furge, B.O. Williams, M. Tretiakova, A. Montag, NJ. Vogelzang, G.G. Re, AJ. Garvin, S. Soderhall, S. Kagawa, D. Hazel-Martin, A. Nordenskjold, and B.T. Teh, Gene expression profiling of favorable histology Wilms tumors and its correlation with clinical features. Cancer Res, 2002. 62(22): p. 6598-605), for which current therapeutic regimens consisting primarily of topoisomerase II inhibitors are very effective. Upon establishing an effective therapy for PRCC, a trial using an effective adjuvant therapy post- nephrectomy may then be considered specifically for Class 2 (Type 2B) PRCC, which has a high rate of relapse and metastasis after nephrectomy.

00143] Example 6- Immunohistochemistry

00144] To validate the gene predictor and to derive immunohistochemical markers, the inventors used immunohistochemistry to confirm high protein expression of CK7 in Class 1 tumors and of Topo Ha in Class 2 tumors. CK7 immunoreactivity has been previously reported to the vast majority of PRCC (Renshaw, A. A. and CL. Corless, Papillary renal cell carcinoma. Histology and immunohistochemistry. Am J Surg Pathol, 1995. 19(7): p. 842-9), but more recent studies suggested that CK may differentiate Type 1 and Type 2 tumors.

D0145] Immunostaining was performed on 5-um thick formalin-fixed, paraffin sections using the biotin-avidin system as previously described (Tretiakova, M.S., S. Sahoo, M. Takahashi, M. Turkyilmaz, NJ. Vogelzang, F. Lin, T. Krausz, B.T. Teh, and XJ. Yang, Expression of alpha-methylacyl-CoA racemase in papillary renal cell carcinoma. Am J Surg Pathol, 2004. 28(1): p. 6976) using mouse monoclonal antibodies specific for cytokeratin 7 (CK7, 1 :50 dilution, Dako, Carpinteria, CA) and DNA topoisomerase II alpha (TopIIq, 1/20 dilution, Vector Laboratories, Burlingame, CA).

)0146] To verify the differential value of CK7 and TopIIα, the inventors studied nineteen

PRCC samples which had undergone microarray analysis (ten Class 1 tumors, eight Class

2 tumors), as well as an independent set of fifteen tumors (ten Class 1 tumors, five Class 2 tumors). The twenty-one Class 1 tumors were composed of histological Type 1 (n=15), low-grade Type 2 tumors (n=3) and mixed Type I/low grade Type 2 tumors (n=3). The thirteen Class 2 tumors were all high-grade Type 2 tumors. The CK7 immunoreactivity was graded as negative (<0.1% positive tumor cells), focally positive (0.1-10% positive tumor cells) or positive (>10% positive tumor cells). The TopIIα immunoreactivity was graded as negative (<0.1% positive tumor cells), focally positive (0.1-10% positive tumor cells) or positive (>10% positive tumor cells). The Mann- Whitney test was used to evaluate significance of the differential staining. '

100147] The immunohistochemical findings are reported in Table 7, and are consistent between the sets of profiled and independent tumors. 100148] Table 7

IMMUNOHISTOCHEMICAL FINDINGS

00149] The majority of Class 1 tumors (86%) including Type 1 (Figures 3A-C) and Type

2A (Figures 3D-F) tumors showed strong CK7 immunoreactivity (Figures 3B, 3E), while the majority of Class 2 tumors (Figures 3 G-L) showed absent (77%) or reduced (23%) CK7 immunoreactivity in both in the set of profiled tumors (Figure 3H) and the independent set of tumors (Figure 3K). In contrast, TopIIα immunoreactivity was focally positive (10%) or negative (90%) in Class 1 tumors including both Type 1 tumors (Figure 3C) and Type 2 A tumors (Figure 3F). The majority of Class 2 tumors were positive for

TopIIα (90% positive, 10% focally positive) both in the set of profiled tumors (Figure 31) and the independent set of tumors (Figure 3L). No TopIIα immunoreactivity was detected in normal kidney tissue. There was no apparent difference between Type 1 and low-grade Type 2 (Type 2A) tumors in CK7 and TopIIα immunostaining. Summarizing the results, CK7 immunoreactivity was significantly higher in Class 1 tumors (pO.OOl), and TopIIα immunoreactvity was significantly higher in Class 2 tumors (p<0.001).

)00150] Figures 3A-C show Type 1 tumor stained with H&E (A), CK7 (B) and TopIIα

(C). Figures 3D-F show low-grade Type 2 (Type 2A) tumor stained with H&E (D), CK7 (E) and TopIIα (F). Figures 3G-I show high-grade Type 2 (Type 2B) tumor, which was subjected to microarray analysis, stained with H&E (G), CK7 (H) and TopIIα (I). Note that a renal tubule (arrow, H) stains positive for CK7 as an internal positive control, while all tumor cells are negative. Finally, Figures 3J-L show high-grade Type 2 (Type 2B) tumor, which was not subjected microarray analysis, stained with H&E (J), CK7 (K) and TopIIα (L). Note that a renal tubule (arrow, K) is positive for CK7, while all tumor cells are negative.

300151] The present microarray and immunohistochemical findings were generally consistent with findings using the morphological classification that between 87-100% of Type 1 tumors showed CK7 positivity and approximately 20% of Type 2 tumors showed CK7 positivity. Delahunt, B. and J.N. EbIe, Papillary renal cell carcinoma: a clinicopathologic and immunohistochemical study of 105 tumors. Mod Pathol, 1997. 10(6): p. 537-44; Ono, K., T. Tanaka, T. Tsunoda, O. Kitahara, C. Kihara, A. Okamoto, K. Ochiai, T. Takagi, and Y. Nakamura, Identification by cDNA microarray of genes involved in ovarian carcinogenesis. Cancer Res, 2000. 60(18): p. 5007-11. No immunohistochemical marker has been previously reported as being specifically upexpressed in Type 2 tumors. Thus, the inventors have identified elevated expression of DNA topoisomerase II alpha, an enzyme controlling the topologic state of DNA, in Class 2 tumors, and demonstrated its usefulness as an immunohistochemical marker.