WO2010008895A2 - Per3 as a biomarker for prognosis of er-positive breast cancer - Google Patents

Abstract

The present application demonstrates that PER3 is a prognostic biomarker for hormone receptor positive breast cancer. The invention therefore provides, among other aspects, methods of prognosis for breast cancers, methods of assigning treatment for breast cancer, methods of identifying therapeutic agents for breast cancers, and kits to perform the methods provided herein.

Description

PER3 AS A BIOMARKER FOR PROGNOSIS OF ER-POSITIVE

BREAST CANCER

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] This application claims priority from U.S. Provisional Application No. 61/133,120, filed June 24, 2008, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under grant No. NCI UOl CA084244 awarded by the National Cancer Institute and grant No. BC063443 awarded by the United States Army Medical Research Acquisition Activity. The US Government has certain rights in this invention.

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED ON A COMPACT DISK

[0003] NOT APPLICABLE

BACKGROUND OF THE INVENTION

[0004] Recent evidence suggests that disruption of circadian rhythms in humans and mouse models may play a significant role in tumor development. Epidemiologic studies demonstrate that women with disrupted sleep cycles, such as night shift workers, are at a higher risk of developing breast cancer (Megdal, S. P. et al., Eur J Cancer. 41(13):2023-32 (2005); Schernhammer, E. S. et al., J Natl Cancer Inst, 93(20):1563-8 (2001); 2006). Some cancers possess different circadian cycles from the surrounding normal cells (Fu 2003,Chen, S. T. et al., Carcinogenesis 26(7):1241-6 (2005)), while additional studies have implicated circadian rhythm genes in cell cycle control or DNA damage responses and tumor progression (Hunt, T. et al., Cell, 129(3):461-4 (2007), Fu 2003, Sahar 2007, Lee 2006). However, the mechanisms by which disruption of circadian rhythm pathways can lead to carcinogenesis are largely unknown. Genes of the Period family (Perl , 2, and 3) control circadian rhythm in flies and mammals. When Per 1 or 2 are inactivated in mice, there is evidence for increased tumor susceptibility (Fu, L. et al., Cell, lll(l):41-50 (2002)), but no causal relationship has been reported on a role for PER3 in cancer. Notably, links to breast cancer are supported by reports of association between a polymorphism in the human PER3 gene and breast cancer susceptibility (Zhu, Y. et al., Cancer Epidemiol Biomarkers Prev. 14(l):268-70 (2005)), and by biochemical studies demonstrating the existence of complexes including proteins of the PER family together with the estrogen receptor (Gery, S. et al., Oncogene. 26(57) :7916-20 (2007)).

[0005] Members of the Period family of circadian rhythm genes {Perl and Per 2) have been implicated in cell cycle control, DNA damage responses and tumor progression (Links, X. et al. Cell, 128(l):59-70 (2007 Jan 2); Chen, S. T. et al., Carcinogenesis 26(7):1241-6 (2005); Hunt, T. et al., Cell, 129(3):461-4 (2007); Fu, L. et al., Cell, lll(l):41-50 (2002); Gery, S. et al., MoI Cell. 22(3):375-82 (2006)). Although the third member of this family, PER3, has no obvious circadian phenotype when inactivated in mice (Shearman, L. P. et al., MoI Cell Biol. (17):6269-75 (2000)), it has been proved that mPER3 transcripts showed a clear circadian rhythm both in the suprachiasmatic nucleus (SCN) (Takumi EMBO 1998) and in mouse peripheral tissues (Yamamoto 2004 BMC). Similar data had been shown in human peripheral blood cells where the results for circadian oscillations were more robust for PER3 than for other markers of circadian gene expression including PERl and PER2 (Archer SN 2008 SLEEP, Hida A 2009). The possible functions ofPER3 in tumor development have not been explored, but links to breast cancer are supported by biochemical studies demonstrating the existence of complexes including proteins of the Per family together with the estrogen receptor (Gery, S. et al., Cold Spring Harb Symp Quant Biol. 72:459-64 (2007)) and by reports of association between a polymorphism in the human PER3 gene and breast cancer susceptibility (Zhu, Y. et al., Cancer Epidemiol Biomarkers Prev. 14(l):268-70 (2005)). This polymorphism is a biallelic variable number tandem repeat (VNTR) consisting of 4 or 5 repeats of a 54 base-pair sequence in a region encoding a putative phosphorylation domain (Archer 2000, 2003), subjects with the long variant PER3^5/5 suffered far greater consequences of sleep deprivation in terms of performance, confirming the close association between the processes of circadian rhythms and sleep at the genetic level. (SCHANTZ MV, Journal of Genetics 2008). [0006] The PER3 gene is located within a region of human chromosome Ip36 that is among the most commonly deleted regions in human cancers. Deletion of Ip36 is especially frequent in breast tumors and it has been related with progression and lymph node metastasis (Tsukamoto, K. et al., Cancer. 82(2):317-22 (1998)), poor prognosis (Ragnarsson G 1996; Ragnarsson, G. et al., Br J Cancer. 79(9-10): 1468-74 (1999)) higher rate of recurrence (Han, W. et al., BMC Cancer. 12;6:92 (2006)), larger tumor size and DNA aneuploidy (Borg, A. et al., Genes Chromosomes Cancer. 5(4):311-20 (1992)). However, no direct relationship between breast carcinogenesis or prognosis and any specific tumor suppressor gene on Ip36 has been established. Recent studies have identified CHD5 (Bagchi, A. et al., Cell, 128(3):459-75 (2007)) and more recently KIFlB (Schlisio, S. et al., Genes Dev.22(7): 884-93 (2008 Apr I)) as candidate tumor suppressor genes in this region, but no specific roles for these genes in breast cancer development have been demonstrated.

[0007] Breast cancer is a highly heterogeneous disease, and treatment is often based on the estrogen receptor (ER) status of individual tumors. Estrogen antagonists such as tamoxifen (TMX) that inhibit estrogen receptor action, are standard therapies that lead to improved survival of patients with ER positive tumors. However, an important proportion of patients with ER positive tumors fail to respond to hormonal treatment and succumb to disease.

BRIEF SUMMARY OF THE INVENTION [0008] The present disclosure demonstrates that PER3 is an important tumor suppressor for breast cancer, particularly in patients with tamoxifen-treated ER positive tumors. TaqMan analysis shows a significant correlation (p- value = 0.01) between PER3 gene copy number and prognosis in breast cancer patients. The correlation between the expression values of the PER3 gene was analyzed using the results from two different breast cancer patient datasets (Chin, K. et al., Cancer Cell. 10(6):529-41 (2006); Van de Vijver, M. J. et al., N Engl J Med. 347(25): 1999-2009 (2002)), demonstrating a significant association between low PER3 gene expression and recurrence in ER+ patients (p value= 0.009). Multivariate analysis showed that PER3 expression was associated with disease-free survival and overall survival in these two patient data sets, independently of other variables such as tumor size, lymph node status or age (Table XS). Deletions of PER3 were not associated with prognosis in patients with basal type, ER negative breast cancers, demonstrating a specific association with a subset of ER positive tumors. Importantly, a causal role for PER3 loss in breast cancer development was demonstrated using two independent mouse models involving either carcinogen treatment or over-expression of Her2/Neu to induce breast tumorigenesis. The present findings implicate PER3 as an important tumor suppressor gene for breast cancer on chromosome Ip36. Copy number alterations and/or expression levels of PER3 may serve as a prognostic biomarker of poor survival in breast cancer patients, especially those with ER+, luminal A, non-basal, and/or ERBB2+ tumors. Specifically, this biomarker may predict recurrence of ER positive tumors treated with Tamoxifen, thus identifying a subset of patients who should receive more aggressive therapy. Further mechanistic insight into the role of PER3 in human breast cancer may provide novel therapeutic targets for this subset of breast cancer patients.

[0009] Here PER3 is identified as a human and mouse breast tumor suppressor gene. Deletion and/or low expression of PER3 is directly related to poor prognosis in human breast cancer, particularly in a subset of tumors that are estrogen receptor (ER) positive, and/or luminal A type, ERBB2 positive, or non-basal tumors. Patients with tumors in these categories that carried PER3 deletions were significantly more likely to undergo early recurrence than patients with normal or increased PER3 copy number. Mice deficient in PER3 showed a specific increase in breast cancer, but not in other tumor types, after systemic treatment with the carcinogen 7, 12-dimethyl-benz[a] anthracene (DMBA). In a different mouse model, breast tumors induced by over-expression of Erbb2 (MMTV-Neu mice) developed breast cancers at significantly earlier stages in the absence of functional PER3.

[0010] The results provided herein demonstrate that PER3 is a tumor suppressor gene within the commonly deleted Ip36 region in human breast cancers. PER3 gene copy number or expression level serves as an indicator of survival probability particularly in a subset of patients with ER positive tumors who receive Tamoxifen treatment, and patients with ER+, luminal A, ERBB2+, and/or non-basal tumors.

[0011] In one aspect of the invention, a method of providing a prognosis for a hormone receptor positive breast cancer in a subject is given. The methods provided generally relate to determining the expression level or genotype of a PER3 biomarker as provided herein. In certain embodiments, the methods may comprise contacting a biological sample from the subject with a reagent that specifically binds to a PER3 biomarker.

[0012] In another aspect, the invention relates to a method of assigning treatment to a subject diagnosed with a hormone receptor positive breast cancer. Generally, the methods comprise determining the expression level or genotype of a PER3 biomarker as provided herein. In certain embodiments, the methods may comprise contacting a biological sample from the subject with a reagent that specifically binds to a PER3 biomarker. In some embodiments, the methods involve assigning a treatment regime comprising the administration of Tamoxifen to a subject likely to respond favorably to the treatment.

[0013] In another aspect, the invention provides kits useful for determining the status of a PER3 biomarker in a sample from a subject. In certain embodiments, the kit may find use for providing a prognosis or assigning a treatment for a hormone receptor positive breast cancer in a subject. Generally, the kits provided herein comprise a reagent that specifically binds to a PER3 biomarker.

[0014] In another aspect, the invention relates to methods of identifying an agent useful for treatment of a hormone receptor positive breast cancer. In one embodiment, the methods comprise contacting a cell having a nucleic acid that encodes a PER3 protein with a candidate agent and assaying for a functional effect on the expression or activity of PER3 in said cell. In a second embodiment, the method comprises contacting a cell having reduced or no PER3 protein or PER3 function with a candidate agent and assaying for a functional effect on a pathway known to be associated with PER3. In certain embodiments, the pathway may be, for example, cell cycle regulation, cell cycle and DNA damage response, a DNA damage or ssDNA checkpoint, transcription, transcriptional regulation, signal transduction, circadian rhythm, rhythmic process, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS [0015] Figure 1. (A) TaqMan copy number analysis of PER3 in 180 lymph node negative breast cancer tumors, showing decreased survival of patients with PER3 deletions. (B) 36 patients who received no treatment or (C) 85 who were treated with anthracyclines chemotherapy showed no effect of PER3 deletion. (D) A subset of 59 patients that were ER and/or PGR positive and were treated only with tamoxifen showed strong association between survival and low PER3 copy number.

[0016] Figure 2. PCR genotyping of a 54 base pair polymorphic repeat sequence in the PER3 gene. The polymorphism analysis was done in 289 DNAs from blood samples and 443 tumor DNAs including the 37 breast cancer cell lines. 117 samples were matched tumor and blood DNA from the same breast cancer patients. A statistically significant loss of heterozygosity is seen in tumor PER3 alleles, as compared to blood PER3 alleles. As can be seen, there is significant preferential retention of the "5 repeat" allele, which has been associated with sleep abnormalities and breast cancer in human populations. [0017] Figure 3. Association between Per3 deletion and disease-free survival in breast cancer patients. Frequency plot of all BAC clones representing the whole genome analyses by CGH array. Genomic gains are showed in green and deletions in red. Statistical differences between genome imbalances in tumors from disease free survival patients and recurrent patients are represented in the blue graph. (A) In 95 lymph node negative breast cancer patients who did not receive systemic chemotherapy, BAC clones showing deletion and corresponding to chromosomal region 1 Iq21-q25, were strongly associated with patient relapse (Climent, J. et al., Cancer Res. 67(2):818-26 (2007)). (B) In 59 patients from the previous group (A) who were ER and/or PGR positive and were treated only with tamoxifen, additional clones showing deletion and corresponding to chromosomal region Ip were strongly associated with patient recurrence.

[0018] Figure 4. Breast cancer incidence in a group of mice treated with 7,12- dimethyl-benz[a]anthracene (DMBA) based in the different genotypes (WT +/+, HET +/-, Null -/-). 86 mice carrying normal or inactivated alleles of the Per 3 gene (17 wild-type Per3+/+, 35 heterozygous Per3+/- and 34 null Per3-/-) were treated by oral gavage with 7, 12- dimethylbenz[a]anthracene (DMBA). Those mice that survived throughout the course of the study were used for determining the statistics. Thirty-six percent of Per3-/- treated with DMBA developed breast tumors, while 12% of the Per3+/- mice developed breast tumors. In striking contrast, none of the control Per 3+1+ mice developed a breast tumor. P values were obtained from the log-rank test.

[0019] Figure 5. Kaplan-Meier estimates of probability of Tumor Free Survival in the group of MMTVneu-PER3 mice. 79 MMTV-Neu positive mice were generated, of which 30 (38%) were (Per3+/+), 35 (44%) were Per3+/-, and 14 (18%) were (Per3-/-). All of the Per3-/- mice developed breast tumors, whereas 25 (71%) of the Per3+/-and 14 (47%) of the Per3+/+ mice developed breast tumors (p-value = 0.003, Cox Proportional Hazard test). P values were obtained from the log-rank test.

[0020] Figure 6. Association between PER3 gene expression and survival of breast cancer patients. (A) PER3 low expression (red) was found in 122 (30%) patients from both data sets. Kaplan-Meier analysis for all patients indicates that those patients with tumors with low expression of PER3 (red) have lower disease free survival rates at 10 years than those patients with normal/high expression of PER3 (blue). (B) Comparison OΪPER3 expression with Estrogen Receptor (ER) status. Low expression of PER3 was less common in ER+ tumors, however those patients with ER+ tumors and low PER3 expression show a higher risk of recurrence (lower left panel). No effect was seen in patients with ER- tumors, (right panel)

[0021] Figure 7. Effect of PER3 expression levels on survival according to molecular subtypes. Kaplan-Meier estimates of Disease-Free Survival among the 413 patients, according to the Per3 expression. Patients were stratified using the Sorlie et al. (Sørlie, T. et al., Proc Natl Acad Sci USA. 98(19): 10869-74 (2001); Sorlie, T. et al., Proc Natl Acad Sci U SA. 100(14):8418-23 (2003)) tumor classification. In the Basal Tumors, the low expression of PER3 gene had no effect in patient recurrence however in the Non Basal tumors those patients which tumors had low expression OΪPER3 showed a significant increase of recurrence. P values were obtained from the log-rank test.

[0022] Figure 8. Effect of PER3 expression levels on survival according to molecular subtypes. Kaplan-Meier estimates of Disease-Free Survival among the 413 patients, according to the Per3 expression. Patients were stratified using the Sorlie et al. (Sørlie, T. et al., Proc Natl Acad Sci USA. 98(19): 10869-74 (2001); Sorlie, T. et al., Proc Natl Acad Sci U SA. 100(14):8418-23 (2003))tumor classification. The increase in recurrence was observed mainly in the Luminal A and ERBB2+ subgroup of tumors whereas no significant difference was observed in the Luminal B subgroup. As seen in Table 5, multivariate analysis revealed that PER3 expression was associated with disease-free survival and overall survival independently of other parameters. P values were obtained from the log-rank test. [0023] Figure 9. Network analysis of breast cancer data using an unpublished algorithm for bioinformatic analysis of gene expression identified high expression of Per3 as a marker of good prognosis breast cancer, associated with genomic instability and cell cycle control.

[0024] Figure 10. Four alternative splicing isoforms were identified, (a) exon 3 skipping isoform was found in three breast cell lines, (b) Two differentially expressed isoforms were identified in intron 4. The major isoform contains one more amino acid (Alanine, GCA) than minor form in the beginning of exon 5. All 34 breast cancer cell lines showed higher expression of the major isoform (containing one more Alanine) allele by RT-PCR and sequencing analysis. Interestingly, minor allele is registered as a reference sequence in NCBI data base, (c-d) Additional two alternative splicing eventss were found in fragment 1 and 3, respectively.

[0025] Figure 11. Kaplan-Meier Estimates of Overall Survival. The different expression levels of Per3 were evaluated in all the patients (A) and the different subgroups of patients based on (B) ER positive (C) ER negative, and based on the different molecular subtypes using Sorlie et al ()Sørlie, T. et al., Proc Natl Acad Sci USA. 98(19): 10869-74 (2001); Sorlie, T. et al., Proc Natl Acad Sci USA. 100(14):8418-23 (2003) classification, (D) Basal, (E) Non Basal, (F) ERBB2+, (G) Luminal A and (H) Luminal B tumors. P values were obtained from the log-rank test. [0026] Figure 12. Differences between Kaplan Meier Estimates for Overall Survival according the expression levels of Per3 (left column) and Chd5 (right column) in all patients (top) and three different subgroups of patients based on ER positive, ER negative and basal type tumors. P-values were calculated using log-rank test.

[0027] Figure 13. Differences between Kaplan Meier Estimates for Overall Survival according the expression levels of Per3 (left column) and Chd5 (right column) in 4 different subgroups of patients based on Non basal, ERBB2, Luminal A and Luminal B tumor subtypes. P-values were calculated using log-rank test.

[0028] Figure 14. Summary of PER3 classification data.

DETAILED DESCRIPTION OF THE INVENTION

[0029] Epidemiological evidence suggests that disruption of sleep patterns plays a significant role in increasing susceptibility to breast cancer, but the molecular links between these processes are unknown. Studies of mouse models have shown that inactivation of the circadian rhythm genes Perl and Per2 can lead to increased development of cancers of the colon and lymphoma. The PER3 gene is located within a commonly deleted region of chromosome Ip36 in breast cancers, and therefore may provide a link between the circadian cycle and epidemiological data on breast tumor susceptibility.

[0030] Disruption of the mammalian biological clock elicits a broad range of physiological and molecular responses, some of which have been associated with increased susceptibility to tumor development (Zhu, Y. et al., Cancer Epidemiol Biomarkers Prev. 14(l):268-70 (2005); Chu, L. W. et al., Prostate Cancer Prostatic Dis. 1-7 (2007)). The present disclosure demonstrates that deletion and/or reduced expression of the PER3 circadian clock gene on human chromosome Ip36 is associated with breast cancer recurrence, particularly in ER+ patients treated with Tamoxifen who did not receive chemotherapy. No effect of deletion was seen in patients with basal type ER- breast tumors. Within the ER+ category, the effect was primarily in tumors classified as Luminal A or ERBB2, but not in the Luminal B type which share some expression features with basal tumors (Sørlie, T. et al., Proc Natl Acad Sci USA. 98(19): 10869-74 (2001); Sorlie, T. et al., Proc Natl Acad Sci USA. 100(14):8418-23 (2003)). Direct evidence for a causal role for loss of PER3, rather than an alternative gene in this commonly deleted region of the genome (Bagchi, A. et al, Cell, 128(3):459-75 (2007); Schlisio, S. et al., Genes Dev .22(7):884-93 (2008 Apr I)), comes from analysis of two different mouse models of breast cancer. Both chemically-induced and Neu(ErbB2)-induced breast cancers are increased in frequency and/or reduced in latency in mice carrying inactivated PER3 alleles. These data demonstrate that PER3 is a bonafide tumor suppressor in mouse models, with a key role in breast tissue.

[0031] While disruption of the mouse Period gene family members Perl and Per2 by gene targeting induces biological clock phenotypes (Liu, A. C. et al., Cell, 129(3):605-16 (2007 May 4)), loss of PER3 function induces no obvious circadian alterations (Shearman, L. P. et al., Neuron. 19(6):1261-9 (1997)). Nevertheless, evidence in favor of PER3 involvement both in sleep disruption and in breast cancer comes from studies of a human structural polymorphism in the PER3 coding sequence (5 repeat allele) that has been associated with delayed sleep phase syndrome, diurnal preference and waking performance (Ebisawa, T. et al., EMBO Rep. 2(4):342-6 (2001); Viola, A. U. et al., Curr Biol. 17(7):613-8 (2007)), but also with increased breast cancer risk (Zhu, Y. et al., Cancer Epidemiol Biomarkers Prev. 14(l):268-70 (2005)), particularly in premenopausal women.

[0032] The present disclosure shows that the long isoform (5 repeat allele) of PER3, which has been associated previously with shorter sleep latency, and displayed profound differences in sleep homeostasis (Viola 2003) is enriched in DNA from primary breast cancers and breast cancer cell lines. Patients heterozygous at this locus in normal DNA (PER3^4/5 ) who develop breast cancers showing LOH preferentially lose the common 4 repeat allele and retain the 5 repeat allele . Considering that subjects with PER3^5/5 seems to be more susceptible to performance errors in conditions of sleep deprivation such as jet lag and night shift work (Archer 2000, Groeger 2008, Arendt J 2008), this may be the first evidence linking circadian sleep disruption and breast cancer risk at a genetic level.

[0033] Although the specific molecular mechanisms remain to be elucidated, and without being bound by theory, increasing evidence points to a role for circadian rhythm genes in cell cycle control and DNA damage responses (Hunt, T. et al., Cell, 129(3):461-4 (2007); Collis, S. J. et al. Chromosoma. 116(4):331-9 (2007)) as well as in hormonal control of gene expression (Gery, S. et al., Cold Spring Harb Symp Quant Biol. 72:459-64 (2007); Gery, S. et al., Oncogene. 26(57):7916-20 (2007)). PER2 has been identified as an estrogen-inducible ER co-repressor that forms heterodimers with PER3 to enter the nucleus. Deletion of PER3 prevents nuclear import, and instead promotes accumulation of PER2 in the cytoplasm (Yagita, K. et al., Genes Dev. 14(ll):1353-63 (2000)). Coordinated functional deregulation of all PERIOD family genes may occur in breast cancers.

[0034] There are several clinical implications of the observations made in the present disclosure. First, the presence of PER3 deletions in ER+ tumors may identify patients who should not be treated only with hormone-based therapy, and who may benefit from more aggressive chemo- or other therapeutic regimens. Secondly, previous data from clinical trials of "Chronotherapy" suggest that the timing of cancer treatment during the day may affect individual patient responses (Eriguchi, M. et al., Biomed Pharmacother. 57 Suppl l:92s-95s (2003); Levi, F. Cancer Causes Control. 17(4):611-21 (2006)). Elucidation of the relationship between control of circadian rhythms, PER gene expression and DNA damage responses may provide a mechanistic rationale for the design of additional clinical trials in this area. Finally, small molecule drugs that help to restore the balance of the biological clock in individuals with frequent sleep disruption may have potential as chemopreventive agents for breast and some other cancer types. [0035] It is shown herein that deletion of PER3 is directly related to tumor recurrence in patients with ER-positive breast cancers treated with Tamoxifen. Low expression of PER3 mRNA is associated with poor prognosis, particularly in a subset of tumors that are estrogen receptor (ER) positive. It is also demonstrated for the first time that a polymorphic variant in the human PER3 gene (PER3 5 repeat allele"), which has been linked to sleep homeostasis and cognitive performance, is preferentially retained in primary human breast cancers and in cell lines. Mice deficient in PER3 showed increased susceptibility to breast cancer induced by carcinogen treatment or by over-expression of Erbb2.

[0036] Comparative Genomic Hybridization arrays and Quantitative TaqMan analysis were used to determine the frequency of alterations at Ip36 and PER3 gene copy number status in 180 lymph node negative breast cancers from patients who had received treatment with chemotherapy and/or Tamoxifen. The significance of altered expression levels of PER3 were also analyzed using two independent published datasets of microarray profiles from breast cancer samples. Finally, the effect of loss of PER3 on tumor susceptibility was tested using two different mouse models of breast cancer. [0037] The frequency of genetic alterations at Ip36 and PER3 gene copy number status was analyzed in 180 lymph node negative breast cancers from patients who had received treatment with chemotherapy and/or Tamoxifen. The frequency of a Variation Number Tandem Repeat polymorphism in the coding sequence of PER3 gene was analyzed both in blood and tumoral DNA from about 700 breast cancer samples. The expression levels of PER3 were also analyzed using microarray profiles from more than 400 breast cancer samples. Finally, the effect of loss OΪPER3 on tumor susceptibility was tested using two mouse models of breast cancer. A combination of human breast tumor analysis and mouse models are used herein to show that PER3 is an important tumor suppressor for breast cancer, disruption of which may serve as a prognostic biomarker of tumor recurrence in patients with ER+, Luminal A and/or ERBB2+ tumors.

METHODS OF PROGNOSIS

[0038] In one aspect, the present invention provides methods of providing a prognosis for breast cancer in a subject. Generally, the method comprises determining the status of a PER3 biomarker in a sample taken from the subject, and correlating the status of the PER3 biomarker with a prognosis for the breast cancer in the subject. Generally, a PER3 positive (PER3+) breast cancer will be associated with a good prognosis, while a PER3 negative (PER3-) breast cancer will be associated with a poor prognosis. [0039] In certain embodiments, the methods provided herein may further comprise determining the status of at least a second breast cancer marker. In a particular embodiment, the second breast cancer biomarker is selected from ESRl , ESR2, PGR, ERBB2, and HER2/neu. In some embodiments, the method may further comprise determining whether or not the breast cancer is ER+ or ER-, PR+ or PR-, and/or HER2/neu+ or Her2/neu-. [0040] In one embodiment, a method of providing a prognosis for a hormone receptor positive breast cancer in a subject comprises the steps of contacting a biological sample from the subject with a reagent that specifically binds to a PER3 biomarker, and determining whether or not the biomarker is differentially expressed in the sample by comparing expression in the sample to a control; thereby providing a prognosis for a hormone receptor positive breast cancer. In one embodiment, the hormone receptor positive breast cancer is an estrogen receptor positive breast cancer. In certain embodiments, the PER3 biomarker may be a PER3 mRNA or a PER3 protein.

[0041] In certain embodiments, the methods provide a prognosis for an estrogen receptor positive breast cancer in a subject that has been treated with a estrogen receptor antagonist, including without limitation, tamoxifen, toremifene, and raloxifene. In one specific embodiment, the antagonist is tamoxifen. [0042] In another embodiment, a method of providing a prognosis for breast cancer may comprise the steps of contacting a biological sample from the subject with a reagents that specifically binds to a PER3 biomarker and determining the PER3 genotype of the sample, wherein a PER3 positive (PER3+) genotype indicates a good prognosis. In certain embodiments, the breast cancer may be a hormone receptor positive breast cancer, e.g. an estrogen receptor positive or progesterone receptor positive breast cancer. In some embodiments, the subject may have been treated previously with an estrogen receptor antagonist selected from tamoxifen, toremifene, and raloxifene. In one particular embodiment, the antagonist is tamoxifen. [0043] In certain embodiments, the method may comprise determining the copy number of PER3 genes in the sample. In one embodiment, a PER3 gene copy number of at least two corresponds to a PER3+ genotype, or corresponds with a good prognosis. Similarly, a PER3 gene copy number of one or zero may correspond to a PER3- genotype, or corresponds with a poor prognosis. In a related embodiment, the method may comprise determining the number of functional alleles of PER3 in the sample. For example, a subject who has at least two copies of a functional PER3 gene may be considered PER3+, while a subject with one or zero copies of a functional PER3 gene may be considered PER-.

[0044] In another embodiment, the method may comprise determining the identity of a polymorphic PER3 variant. In one embodiment, the polymorphism is a biallelic variable number tandem repeat (VNTR) consisting of 4 or 5 repeats of a 54 base-pair sequence in a region encoding a putative phosphorylation domain (Archer 2000, 2003). In certain embodiments, a breast cancer may be classified as PER3^4/\ PER3^5/\ PER3^4/5, PER3^4/4, PER3^5/5, PER3⁴⁺, PER3⁵⁺, PER3^4+/5, or PER3^4/5+. In one embodiment, the method may comprise detecting a loss of polymorphic heterozygosity at the PER3 VNTR in a breast tumor or breast tissue sample. As used herein, the nomenclature PER3⁴⁺, PER3⁵⁺, PER3^4+/5, or PER3^{4 5+} refers to a gain of copy genotype, i.e., where 1, 2, 3, 4, or more copies of a PER⁴ or PER⁵ allele have been gained by a gene duplication, an imbalanced chromosomal translocation, a chromosomal or sub-chromosomal duplication, etc.

[0045] In one embodiment, the method may comprise detecting a mutation or polymorphism in a PER3 protein. In certain embodiments, the mutation or polymorphism may be selected from the group consisting of V419M, S445S, I606I, V639G, L697L, T725T, P745P, L827P, P856A, S864S, M1028T, TlOlOT, and Hl 149R. In other embodiments, the method may comprise detecting a mutation or polymorphism in a PER3 gene. In certain embodiments, the mutation or polymorphism may be selected from those found in Table 3. [0046] In yet another embodiment, the method may comprise detecting expression of a particular splicing isoform of PER3. In some embodiments, the method comprises determining the ratio of the expression level of a first PER3 splicing isoform to the expression level of at least a second splicing isoform of PER3. In certain embodiments, the splicing isoform of PER3 may be selected from PER3 isoform 1, PER3 isoform 2, PER3 isoform 3, and PER3 isoform 4. In yet other embodiments, the splice isoform may be any of those known for PER3 (EnsEMBL gene ID: ENSG00000049246).

[0047] In one embodiment, the methods set forth herein provide a prognosis for a luminal A, ERBB2+, or non-basal breast cancer in a subject (Lacroix M., Endocr Relat Cancer. 2006 ec; 13(4): 1033-67; Pinero-Madrona A et al, Cir Esp. 2008 Sep;84(3): 138-45).

[0048] In certain embodiments, wherein the biomarker is a PER3 gene or mRNA, the step of determining the expression level of the marker or PER3 genotype may comprise PCR, quantitative PCR, RT-PCR, a single base extension reaction, mass spectrometry, hybridization, microarray, and the like. In some embodiments, the reagent may comprise a nucleic acid, such as a hybridization probe, a oligonucleotide primer, a set of PCR primers, and the like.

[0049] In other embodiments, wherein the biomarker is a PER3 protein, the step of determining the expression level of the marker or PER3 genotype of the cancer may comprise an ELISA assay, an immunochemistry assay, mass spectrometry, a protein array, and the like. In certain embodiments, the reagent may comprise an antibody or fragment thereof, an aptamer, spiegelmer, and the like.

[0050] In some embodiments, reduced expression of a PER3 biomarker, down-regulation of a PER3 biomarker, or a PER3- genotype, is correlated with a poor prognosis, while increased expression of a PER3 biomarker, up-regulation of a PER3 biomarker, or a PER3+ genotype, is correlated with a good prognosis. In certain embodiments, a poor prognosis is associated with an increased risk of tumor recurrence, a reduced likelihood of long term survival (e.g., 5, 10, 15, or 20 year survival), a reduced likelihood of long term disease-free survival (e.g., 5, 10, 15, or 20 year disease-free survival), a reduced likelihood of favorably responding to treatment comprising a hormone receptor antagonist, and the like. In some embodiments, a good prognosis is associated with a decreased risk of tumor recurrence, an increased likelihood of long term survival (e.g., 5, 10, 15, or 20 year survival), an increased likelihood of long term disease-free survival (e.g., 5, 10, 15, or 20 year disease-free survival), an increased likelihood of favorably responding to treatment comprising a hormone receptor antagonist, and the like.

[0051] Non-limiting examples of samples which may be used in the methods of the present invention include, a sample of a surgically resected breast tumor, a breast tumor biopsy, breast tissue, a biological fluid (e.g. blood, plasma, urine, saliva, etc.), lymph tissue, and the like.

METHODS OF ASSIGNING TREATMENT

[0052] In another aspect of the invention, methods of assigning treatment to a subject diagnosed with breast cancer are provided. Generally, the method comprises determining the status of a PER3 biomarker in a sample taken from the subject, and correlating the status of the PER3 biomarker with a treatment regime comprising administration of a hormone receptor antagonist. Generally, a PER3 positive (PER3+) breast cancer will respond favorably upon administration of a hormone receptor antagonist, while a PER3 negative (PER3-) breast cancer will not respond favorably upon administration of a hormone receptor antagonist. In certain embodiments, the hormone receptor antagonist may be tamoxifen, toremifene, or raloxifene. In one specific embodiment, the antagonist is tamoxifen.

[0053] In some embodiments, a subject with a PER3- breast cancer may be assigned a more aggressive treatment regime than a subject with a PER3+ breast cancer. In one embodiment, a subject with a PER3- breast cancer may be assigned a treatment regime comprising a mastectomy, radiation therapy, and/or chemotherapy. In some embodiments, the treatment may also comprise hormone therapy.

[0054] Examples of Anti-cancer agents acceptable for use in the present invention include, without limitation, alkylating agents, anti-metabolites, plant alkaloids and terpenoids, topoisomerase inhibitors, antineoplastics, hormone therapeutics, photosensitizers, kinase inhibitors, etc.

[0055] Examples of alkylating agents include, without limitation, cisplatin, caroplatin, oxaliplatin, mechlorethamine, cyclophophamide, chlorambucil, busulfan, hexamethylmelamine, thiotepa, cyclophohphamine, uramustine, melphalan, ifosfamide, carmustine, streptozocin, dacarbazine, temozolomide, etc. [0056] Examples of anti-metabolite agents include, without limitation, Aminopterin, Methotrexate, Pemetrexed, Raltitrexed, Cladribine, Clofarabine, Fludarabine, Mercaptopurine, Pentostatin, Thioguanine, Capecitabine, Cytarabine, Decitabine, Fluorouracil, Floxuridine, Gemcitabine, etc.

[0057] Examples of plant alkaloids and terpenoids include, without limitation, Docetaxel, Larotaxel, Paclitaxel, Vinblastine, Vincristine, Vindesine, Vinorelbine, etc. [0058] Examples of topoisomerase inhibitors include, without limitation, Camptothecin, Topotecan, Irinotecan, Rubitecan, Etoposide, Teniposide, etc.

[0059] Examples of antineoplastics include, without limitation, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Pixantrone, Valrubicin, Actinomycin, Bleomycin, Mitomycin, Plicamycin, etc. [0060] E xamples of photosensitizer agents include, without limitation, Aminolevulinic acid, Methyl aminolevulinate, Porfimer sodium, Verteporfin, etc.

[0061] E xamples of kinase inhibitors include, without limitation, Axitinib, Bosutinib, Cediranib, Dasatinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, Lestaurtinib, Nilotinib, Semaxanib, Sorafenib, Sunitinib, Vandetanib, Seliciclib, etc. [0062] Examples of other anti-cancer agents include, without limitation, Alitretinoin,

Tretinoin, Aflibercept, Altretamine, Amsacrine, Anagrelide, Arsenic trioxide, Pegaspargase, Bexarotene, Bortezomib, Celecoxib, Denileukin diftitox, Elesclomol, Estramustine, Irofulven, Ixabepilone, Masoprocol, Mitotane, Oblimersen, Testolactone, Tipifarnib, Trabectedin [0063] In certain embodiments, the methods provided herein may further comprise determining the status of at least a second breast cancer marker. In a particular embodiment, the second breast cancer biomarker is selected from ESRl, ESR2, PGR, ERBB2, and HER2/neu. In some embodiments, the method may further comprise determining whether or not the breast cancer is ER+ or ER-, PR+ or PR-, and/or HER2/neu+ or Her2/neu-. [0064] In one embodiment, a method of assigning treatment to a subject diagnosed with a hormone receptor positive breast cancer comprises the steps of contacting a biological sample from the subject with a reagents that specifically binds to a Per3 biomarker, determining whether or not the expression of the biomarker is down-regulated in the sample by comparing expression in the sample to a control, and assigning a treatment regime comprising a hormone receptor antagonist if the biomarker is not down-regulated. In one embodiment, the hormone receptor positive breast cancer is an estrogen receptor positive breast cancer. In certain embodiments, the PER3 biomarker may be a PER3 mRNA or a PER3 protein. [0065] In another embodiment, a method of assigning treatment to a subject diagnosed with a hormone receptor positive breast cancer comprises the steps of contacting a biological sample from the subject with a reagents that specifically binds to a PER3 biomarker, determining the PER3 genotype of the sample, and assigning a treatment regime comprising a hormone receptor antagonist if the cancer has a PER3+ genotype. In certain embodiments, the breast cancer may be a hormone receptor positive breast cancer, e.g. an estrogen receptor positive or progesterone receptor positive breast cancer. In some embodiments, the subject may have been treated previously with an estrogen receptor antagonist selected from tamoxifen, toremifene, and raloxifene. In one particular embodiment, the antagonist is tamoxifen.

[0066] In certain embodiments, the method is for assigning a treatment to a subject with estrogen receptor positive breast cancer in a subject that has been treated with a estrogen receptor antagonist, including without limitation, tamoxifen, toremifene, and raloxifene. In one specific embodiment, the antagonist is tamoxifen. [0067] In certain embodiments, the method may comprise determining the copy number of PER3 genes in the sample. In one embodiment, a PER3 gene copy number of at least two corresponds to a PER3+ genotype. Similarly, a PER3 gene copy number of one or zero may correspond to a PER3- genotype. In a related embodiment, the method may comprise determining the number of functional alleles of PER3 in the sample. For example, a subject who has at least two copies of a functional PER3 gene may be considered PER3+, while a subject with one or zero copies of a functional PER3 gene may be considered PER-.

[0068] In another embodiment, the method may comprise determining the identity of a polymorphic PER3 variant. In one embodiment, the polymorphism is a biallelic variable number tandem repeat (VNTR) consisting of 4 or 5 repeats of a 54 base-pair sequence in a region encoding a putative phosphorylation domain (Archer 2000, 2003). In certain embodiments, a breast cancer may be classified as PER3^4/-, PER3^5/-, PER3^4/5, PER3^4/4, PER3^5/5, PER3⁴⁺, PER3⁵⁺, PER3^4+/5, or PER3^4/5+.

[0069] In one embodiment, the method may comprise detecting a mutation or polymorphism in a PER3 protein. In certain embodiments, the mutation or polymorphism may be selected from the group consisting of V419M, S445S, I606I, V639G, L697L, T725T, P745P, L827P, P856A, S864S, M1028T, TlOlOT, and Hl 149R. In other embodiments, the method may comprise detecting a mutation or polymorphism in a PER3 gene. In certain embodiments, the mutation or polymorphism may be selected from those found in Table 3. [0070] In yet another embodiment, the method may comprise detecting expression of a particular splicing isoform of PER3. In some embodiments, the method comprises determining the ratio of the expression level of a first PER3 splicing isoform to the expression level of at least a second splicing isoform of PER3. In certain embodiments, the splicing isoform of PER3 may be selected from PER3 isoform 1, PER3 isoform 2, PER3 isoform 3, and PER3 isoform 4.

[0071] In one embodiment, the invention sets forth a method for assigning a treatment to a subject with a luminal A, ERBB2+, or non-basal breast cancer (Lacroix M., Endocr Relat Cancer. 2006 ec; 13(4): 1033-67; Pinero-Madrona A et al, Cir Esp. 2008 Sep;84(3): 138-45). [0072] In certain embodiments, wherein the biomarker is a PER3 gene or mRNA, the step of determining the expression level of the marker or PER3 genotype may comprise PCR, quantitative PCR, RT-PCR, a single base extension reaction, mass spectrometry, hybridization, microarray, and the like. In some embodiments, the reagent may comprise a nucleic acid, such as a hybridization probe, a oligonucleotide primer, a set of PCR primers, and the like.

[0073] In other embodiments, wherein the biomarker is a PER3 protein, the step of determining the expression level of the marker or PER3 genotype of the cancer may comprise an ELISA assay, an immunochemistry assay, mass spectrometry, a protein array, and the like. In certain embodiments, the reagent may comprise an antibody or fragment thereof, an aptamer, spiegelmer, and the like.

[0074] In some embodiments, wherein a sample from a subject with breast cancer has reduced expression of a PER3 biomarker, down-regulation of a PER3 biomarker, or a PER3- genotype, the subject is assigned an aggressive treatment regime. In certain embodiments, the aggressive treatment regime may comprise chemotherapy, radiation therapy, tumor resection, and/or a mastectomy.

[0075] In other embodiments, wherein a sample from a subject with breast cancer has increased expression of a PER3 biomarker, up-regulation of a PER3 biomarker, or a PER3+ genotype, the subject is assigned a treatment regime comprising administration of a hormone receptor antagonist. In certain embodiments, the hormone receptor antagonist may be selected from tamoxifen, toremifene, and raloxifene. In one specific embodiment, the antagonist is tamoxifen [0076] Non-limiting examples of samples which may be used in the methods of the present invention include, a sample of a surgically resected breast tumor, a breast tumor biopsy, breast tissue, a biological fluid (e.g. blood, plasma, urine, saliva, etc.), lymph tissue, and the like. DEFINITIONS

[0077] As used herein, the term "PER3 Biomarker" refers to a mammalian PER3 gene (human PER3; EFOl 5893), a mammalian PER3 gene product, including mRNA (human PER3 mRNA; NM_016831 ) and protein (human PER3 protein; NP_058515; ABM64204), as well as splice variants, mutant sequences, and polymorphic variants thereof. In the context of the present disclosure, determining the status of a PER3 biomarker generally refers to determining the expression level of a PER3 biomarker, determining the gene copy number of a PER3 biomarker, determining the genotype of a PER3 biomarker, detecting a mutation or polymorphism in a PER3 biomarker, determining the relative expression ratio of different PER3 biomarker splice variants, mutants, polymorphisms, or isoforms, or determining the activity of a PER3 biomarker in a sample.

[0078] The terms "PER3" or a "nucleic acid encoding PER3" refer to nucleic acids and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to an amino acid sequence encoded by an PER3 nucleic acid (Accession numbers AF061025 and NM_012318) or amino acid sequence of an PER3 protein (Accession numbers NP_036450 and AAD13138); (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of an PER3 protein (Accession numbers NP_036450 and AAD 13138), and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence encoding an PER3 protein (Accession numbers AF061025 and NM_012318 and Accession numbers NP_036450 and AADl 3138), and conservatively modified variants thereof; (4) have a nucleic acid sequence that has greater than about 95%, preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferably over a region of at least about 25, 50, 100, 200, 500, 1000, or more nucleotides, to an PER3 nucleic acid (Accession numbers AF061025 and NM O 12318). A polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any mammal. The nucleic acids and proteins of the invention include both naturally occurring or recombinant molecules.

[0079] As used herein, a "PER3 positive cancer", a "PER3 positive genotype", or "PER3+" refers to a cancer, tumor, neoplasia, or hyperplasia characterized by a PER3 genotype associated with a good prognosis, a PER3 gene copy number of two or more, or a normal or up-regulated expression level of a PER3 gene product. In certain embodiments, a PER3 positive cancer refers to a cancer, tumor, neoplasia, or hyperplasia in which a PER3 gene product, i.e. mRNA or protein, is expressed at a level equal to or greater than a control sample, or within a half standard deviation from the average expression in a control data set. In another embodiment, a PER3 positive cancer refers to a cancer, tumor, neoplasia, or hyperplasia having a PER3 gene copy number of at least two. In certain embodiments, a PER3+ cancer can classified by determining the PER3 status of a biological fluid from the individual, for example from a blood, plasma, urine, or saliva sample from the subject.

[0080] As used herein, a "PER3 negative cancer", a "PER3 negative genotype", or "PER3- " refers to a cancer, tumor, neoplasia, or hyperplasia characterized by a PER3 genotype associated with a poor prognosis, a PER3 gene copy number of one or less, or a down- regulated expression level of a PER3 gene product. In certain embodiments, a PER3 negative cancer refers to a cancer, tumor, neoplasia, or hyperplasia in which a PER3 gene product, i.e. mRNA or protein, is expressed at a level less than a control sample, or less than a level within a half standard deviation from the average expression in a control data set. In another embodiment, a PER3 negative cancer refers to a cancer, tumor, neoplasia, or hyperplasia having a PER3 gene copy number of one or less. In certain embodiments, a PER3- cancer can classified by determining the PER3 status of a biological fluid from the individual, for example from a blood, plasma, urine, or saliva sample from the subject. [0081] As used herein, a "PER3 genotype" may refer to the copy number of the PER3 gene, the identity of a PER3 allele or set of alleles, the identity of a PER3 mutant gene, and the like.

[0082] As used herein, a "hormone receptor positive breast cancer" refers to a breast cancer, a breast neoplasia, or breast hyperplasia characterized by expression of the estrogen receptor (ER) or progesterone receptor (PR). Generally, a hormone receptor positive breast cancer may be classified as ER+, PR+, or both.

[0083] "Estrogen receptor positive breast cancer" refers to breast cancers that are in the positive or intermediate range for the estrogen receptor protein. For example, when estrogen receptor protein can be measured as femtomoles per milligram of cytosol protein. In this assay, values above 10 are positive, values from 3 to 10 are intermediate, and values less than 3 are negative. Other assays known in the art can be used to determined if the breast cancer is estrogen receptor positive, in particular assays based on antibodies to estrogen receptors alpha and beta and their use in biochemical or histological assays.

[0084] "Cancer" refers to human cancers and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, etc., including solid and lymphoid cancers, kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including B-acute lymphoblastic lymphoma, non-Hodgkin's lymphomas (e.g. , Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's lymphoma, leukemia (including AML, ALL, and CML), and multiple myeloma.

[0085] "Hormonal therapy" refers to drugs or treatments that block the effect of estrogen or progesterone, or alternatively, lower estrogen or progesterone levels, including anti-estrogen or anti-progesterone therapy and estrogen or progesterone ablation therapy.

[0086] "Providing a prognosis" refers to providing a prediction of the likelihood of metastasis, predictions of disease free and overall survival, the probable course and outcome of cancer therapy, or the likelihood of recovery from the cancer, in a subject.

[0087] By "therapeutically effective amount or dose" or "therapeutically sufficient amount or dose" or "effective or sufficient amount or dose" herein is meant a dose that produces therapeutic effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). In sensitized cells, the therapeutically effective dose can often be lower than the conventional therapeutically effective dose for non-sensitized cells.

[0088] The phrase "functional effects" in the context of assays for testing compounds that modulate activity of a PER3 protein includes the determination of a parameter that is indirectly or directly under the influence of an PER3, e.g., a functional, physical, or chemical effect. It includes control of circadian rhythm. "Functional effects" include in vitro, in vivo, and ex vivo activities. [0089] By "determining the functional effect" is meant assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of an PER3 protein, e.g., functional, physical and chemical effects. Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index); hydrodynamic (e.g., shape); chromatographic; or solubility properties for the protein; measuring inducible markers or transcriptional activation of the protein; measuring binding activity or binding assays, e.g. binding to antibodies; measuring changes in ligand binding activity; measuring cellular proliferation; measuring cell surface marker expression; measurement of changes in protein levels for per3 -associated sequences; measurement of RNA stability; phosphorylation or dephosphorylation; signal transduction, e.g., receptor-ligand interactions, second messenger concentrations (e.g., cAMP, IP3, or intracellular Ca²⁺); identification of downstream or reporter gene expression (CAT, luciferase, β-gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and ligand binding assays.

[0090] "Inhibitors", "activators", and "modulators" of per 3 polynucleotide and polypeptide sequences are used to refer to activating, inhibitory, or modulating molecules identified using in vitro and in vivo assays of per 3 polynucleotide and polypeptide sequences. Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of per3 proteins, e.g., antagonists. "Activators" are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate per3 protein activity. Inhibitors, activators, or modulators also include genetically modified versions of per 3 proteins, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, siRNA, antisense molecules, ribozymes, small chemical molecules and the like. Such assays for inhibitors and activators include, e.g., expressing per3 protein in vitro, in cells, or cell membranes, applying putative modulator compounds, and then determining the functional effects on activity, as described above.

[0091] Samples or assays comprising PER3 proteins that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%. Inhibition of PER3 is achieved when the activity value relative to the control is about 80%, preferably 50%, more preferably 25-0%. Activation of PER3 is achieved when the activity value relative to the control (untreated with activators) is 110%, more preferably 150%, more preferably 200- 500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000% higher.

[0092] The term "test compound" or "drug candidate" or "modulator" or "agent" or grammatical equivalents as used herein describes any molecule, either naturally occurring or synthetic, e.g., protein, oligopeptide (e.g., from about 5 to about 25 amino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule, polysaccharide, lipid, fatty acid, polynucleotide, oligonucleotide, etc., to be tested for the capacity to directly or indirectly modulation cellular proliferation. The test compound can be in the form of a library of test compounds, such as a combinatorial or randomized library that provides a sufficient range of diversity. Test compounds are optionally linked to a fusion partner, e.g., targeting compounds, rescue compounds, dimerization compounds, stabilizing compounds, addressable compounds, and other functional moieties. Conventionally, new chemical entities with useful properties are generated by identifying a test compound (called a "lead compound") with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds. Often, high throughput screening (HTS) methods are employed for such an analysis. [0093] A "small organic molecule" refers to an organic molecule, either naturally occurring or synthetic, that has a molecular weight of more than about 50 daltons and less than about 2500 daltons, preferably less than about 2000 daltons, preferably between about 100 to about 1000 daltons, more preferably between about 200 to about 500 daltons.

[0094] As used herein, the term "differentially expressed" or "differentially regulated" refers generally to a gene product, i.e. protein or nucleic acid, that is overexpressed (up- regulated) or underexpressed (down-regulated) in a first biological sample as compared to at least a second sample or a control. In the context of the present invention, the first sample may be, for example, a sample from a subject diagnosed with breast cancer, or with a hormone receptor positive breast cancer, and the second sample may be, for example, a non- cancerous sample from the same subject, a sample from a subject that dose not have breast cancer, a sample from a subject that has a hormone receptor negative breast cancer, and the like. Alternatively, the expression level of the PER3 biomarker may be compared to a control, such as an average level of PER3 expression in a cohort, or a pre-determined threshold level. [0095] The terms "overexpress", "overexpression", "overexpressed", "up-regulate", or "up- regulated" interchangeably refer to a biomarker that is present at a detectably greater level in a biological sample, e.g. a sample from a patient with breast cancer or a hormone receptor positive breast cancer, in comparison to a second biological sample or control. The term includes overexpression in a sample from a patient with cancer due to transcription, post transcriptional processing, translation, post-translational processing, cellular localization (e.g, organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a second sample or control. Overexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, single base extension reaction (SBE), hybridization, etc.) or proteins (i.e., ELISA, immunohistochemical techniques, mass spectrometry,

® Luminex xMAP technology, etc.). Overexpression can be, for example, at least about 10%,

20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a second sample or control. In certain instances, overexpression may be, for example, at least about 1-fold, 2- fold, 3-fold, 4-fold 5, 6, 7, 8, 9, 10, or 15-fold or more higher levels of transcription or translation in comparison to a second sample or control.

[0096] The terms "underexpress," "underexpression", "underexpressed" or "down- regulated" interchangeably refer to a biomarker, usually a protein or nucleic acid, that is present at a detectably lower level in a biological sample, e.g. a sample from a patient with breast cancer or a hormone receptor positive breast cancer, in comparison to a second biological sample or control. The term includes underexpression due to transcription, post transcriptional processing, translation, post-translational processing, cellular localization (e.g., organelle, cytoplasm, nucleus, cell surface), and RNA and protein stability, as compared to a control. Underexpression can be detected using conventional techniques for detecting mRNA (i.e., RT-PCR, PCR, single base extension reaction (SBE), hybridization, etc.) or proteins (i.e., ELISA, immunohistochemical techniques, mass spectrometry,

® Luminex xMAP technology, etc.). Underexpression can be, for example, at least about

10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less in comparison to a second sample or control. In certain instances, underexpression may be, for example, at least about 1-fold, 2-fold, 3 -fold, 4-fold or more lower levels of transcription or translation in comparison to a second sample or control.

[0097] As used herein, a "control sample" may refer to, for example, a non-cancerous sample from a subject diagnosed with a hormone receptor positive breast cancer, a sample from a subject diagnosed with a hormone negative cancer, a subject that does not have breast cancer. Alternatively, the status and expression level of a PER3 biomarker provided by the present invention may be compared to a predetermined threshold level. Threshold levels may be generated as an average status or expression level in a plurality of samples, for example in a cohort of diseased or normal subjects.

[0098] It will be understood by the skilled artisan that biomarkers provided herein may be used singly or in combination with other markers for any of the uses, e.g., prognosis of a breast cancer, assignment of a treatment for breast cancer, and the like.

[0099] As used herein, a "sample" or "biological sample" includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes. Such samples include blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum or saliva, lymph and tongue tissue, cultured cells, e.g., primary cultures, explants, and transformed cells, stool, urine, etc. A biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., human, or a rodent, e.g., guinea pig, rat, mouse, and the like.

[0094] A "biopsy" refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the diagnostic and prognostic methods of the present invention. The biopsy technique applied will depend on the tissue type to be evaluated (e.g., breast, tongue, colon, prostate, kidney, bladder, lymph node, liver, bone marrow, blood cell, etc.), the size and type of the tumor (e.g., solid or suspended, blood or ascites), among other factors. Representative biopsy techniques include, but are not limited to, excisional biopsy, incisional biopsy, needle biopsy, surgical biopsy, and bone marrow biopsy. An "excisional biopsy" refers to the removal of an entire tumor mass with a small margin of normal tissue surrounding it. An "incisional biopsy" refers to the removal of a wedge of tissue that includes a cross-sectional diameter of the tumor. A diagnosis or prognosis made by endoscopy or fluoroscopy can require a "core-needle biopsy" of the tumor mass, or a "fine- needle aspiration biopsy" which generally obtains a suspension of cells from within the tumor mass. Biopsy techniques are discussed, e.g., in Kasper et al., Harrison 's Principles of

Internal Medicine, eds., 16 ed., Chapter 70 and throughout Part V (2005).

[0100] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

[0101] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.

[0102] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide. [0103] The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).

Generally, stringent conditions are selected to be about 5-10 C lower than the thermal melting point (T ) for the specific sequence at a defined ionic strength pH. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_n, 50% of the probes are occupied at equilibrium).

Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C. [0104] For PCR, a temperature of about 36C is typical for low stringency amplification, although annealing temperatures may vary between about 32C and 48C depending on primer length. For high stringency PCR amplification, a temperature of about 62C is typical, although high stringency annealing temperatures can range from about 5OC to about 65C, depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 9OC - 95C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al., PCR Protocols, A Guide to Methods and Applications (Academic Press, Inc., N. Y., 1990).

[0105] "Antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody will be most critical in specificity and affinity of binding. Antibodies can be polyclonal or monoclonal, derived from serum, a hybridoma or recombinantly cloned, and can also be chimeric, primatized, or humanized.

[0106] The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than 10 to 100 times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only those polyclonal antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Luminex® xMAP technology may also be used in conjunction with the present invention.

PROGNOSTIC METHODS

[0107] The present invention provides methods of providing a prognosis for a breast cancer or a hormone receptor positive breast cancer in a subject by detecting, determining the status of, or determining the expression level of a PER3 biomarker. Prognosis involves, in one embodiment, determining the level of a PER3 polynucleotide or polypeptide in a subject and then comparing the level to a baseline or range. Typically, the baseline value is representative of a polynucleotide or polypeptide of the invention in a healthy person not diagnosed with breast cancer, a person diagnosed with a hormone receptor negative breast cancer, or a non-cancerous biological sample, as measured using biological sample such as a tissue sample (e.g., breast tissue, breast tumor biopsy), serum, blood, urine, or saliva sample.

[0108] In one embodiment, real time or quantitative PCR is used to examine expression of a PER3 biomarker in the panel using RNA from a biological sample such as tumor tissue or biological fluid. No microdissection is required. RNA extraction can be performed by any method know to those of skill in the art, e.g., using Trizol and RNeasy. Real time PCR can be performed by any method known to those of skill in the art, e.g., Taqman real time PCR using Applied Biosystem assays. Gene expression is calculated relative to PER3 expression in non-cancerous breast tissue, hormone receptor negative cancerous tissues, or healthy tissue, and expression may be normalized to housekeeping genes. Suitable oligonucleotide primers are selected by those of skill in the art. In one embodiment, the assay is used for stage I, stage II, stage III, or stage IV cancers. In one embodiment, the tissue sample is from a surgically resected tumor.

® [0109] PCR assays such as Taqman allelic discrimination assay, available from Applied Biosystems, can be used to identify RNA. In another embodiment, mass spectroscopy can be used to detect either nucleic acid or protein. Any antibody-based technique for determining a level of expression of a protein of interest can be used. For example, immunoassays such as ELISA, Western blotting, flow cytometry, immunofluorescence, and immunohistochemistry can be used to detect protein in patient samples. Combinations of the above methods, such as

® those employed in the Luminex xMAP technology can also be used in the present invention.

Analysis of a protein or nucleic acid can be achieved, for example, by high pressure liquid chromatography (HPLC), alone or in combination with mass spectrometry (e.g., MALDI/MS, MALDI-TOF/MS, tandem MS, etc.). [0110] Applicable PCR amplification techniques are described in, e.g., Ausubel et al and Innis et al , supra. General nucleic acid hybridization methods are described in Anderson, "Nucleic Acid Hybridization," BIOS Scientific Publishers, 1999. Amplification or hybridization of a plurality of nucleic acid sequences (e.g., genomic DNA, mRNA or cDNA) can also be performed from mRNA or cDNA sequences arranged in a microarray. Microarray methods are generally described in Hardiman, "Microarrays Methods and Applications: Nuts & Bolts," DNA Press, 2003; and Baldi et al, "DNA Microarrays and Gene Expression: From Experiments to Data Analysis and Modeling," Cambridge University Press, 2002. [0111] Analysis of nucleic acid markers can be performed using techniques known in the art including, without limitation, sequence analysis, and electrophoretic analysis. Non- limiting examples of sequence analysis include Maxam-Gilbert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al, Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman et al, Methods MoI. Cell Biol, 3:39-42 (1992)), single base extension sequencing (SBE), pyrosequencing (Ronaghi et al., Science, 281(5375):363-365 (1998)), sequencing with mass spectrometry such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et al, Nat. Biotechnol, 16:381-384 (1998)), and sequencing by hybridization. Chee et al, Science, 274:610-614 (1996); Drmanac et al, Science, 260:1649- 1652 (1993); Drmanac et al, Nat. Biotechnol, 16:54-58 (1998). Non-limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis.

[0112] A detectable moiety can be used in the assays described herein. A wide variety of detectable moieties can be used, with the choice of label depending on the sensitivity required, ease of conjugation with the antibody, stability requirements, and available instrumentation and disposal provisions. Suitable detectable moieties include, but are not limited to, radionuclides, fluorescent dyes (e.g., fluorescein, fluorescein isothiocyanate

(FITC), Oregon Green , rhodamine, Texas red, tetrarhodimine isothiocynate (TRITC), Cy3, Cy5, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, metals, and the like. [0113] In another embodiment, antibody reagents can be used in assays to detect expression levels of protein biomarkers of the invention in patient samples using any of a number of immunoassays known to those skilled in the art. Immunoassay techniques and protocols are generally described in Price and Newman, "Principles and Practice of Immunoassay," 2nd Edition, Grove's Dictionaries, 1997; and Gosling, "Immunoassays: A Practical Approach," Oxford University Press, 2000. A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used (see, e.g., Self et al., Curr. Opin. Biotechnol, 7:60-65 (1996)). The term immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EIA) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), IgM antibody capture ELISA (MAC ELISA), and microparticle enzyme immunoassay (MEIA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (RIA); immunoradiometric assays (IRMA); fluorescence polarization immunoassays (FPIA); and chemiluminescence assays (CL). If desired, such immunoassays can be automated. Immunoassays can also be used in conjunction with laser induced fluorescence (see, e.g., Schmalzing et al.,

Electrophoresis, 18:2184-93 (1997); Bao, J. Chromatogr. B. Biomed. ScI, 699:463-80 (1997)). Liposome immunoassays, such as flow-injection liposome immunoassays and liposome immunosensors, are also suitable for use in the present invention (see, e.g., Rongen et al., J Immunol. Methods, 204:105-133 (1997)). In addition, nephelometry assays, in which the formation of protein/antibody complexes results in increased light scatter that is converted to a peak rate signal as a function of the marker concentration, are suitable for use in the methods of the present invention. Nephelometry assays are commercially available from Beckman Coulter (Brea, CA; Kit #449430) and can be performed using a Behring Nephelometer Analyzer (Fink et al., J. Clin. Chem. Clin. Biochem., 27:261-276 (1989)). [0114] Specific immunological binding of the antibody to a protein can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. An antibody labeled with iodine- 125

12₅

( I) can be used. A chemiluminescence assay using a chemiluminescent antibody specific for the protein marker is suitable for sensitive, non-radioactive detection of protein levels. An antibody labeled with fluorochrome is also suitable. Examples of fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R- phycoerythrin, rhodamine, Texas red, and lissamine. Indirect labels include various enzymes well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), y- galactosidase, urease, and the like. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p- nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm. An urease detection system can be used with a substrate such as urea- bromocresol purple (Sigma Immunochemicals; St. Louis, MO).

[0115] A signal from a direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of I; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked antibodies, a quantitative analysis can be made using a spectrophotometer such as an EMAX Microplate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer's instructions. If desired, the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected simultaneously.

[0116] The antibodies can be immobilized onto a variety of solid supports, such as polystyrene beads, magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), pieces of a solid substrate material or membrane (e.g., plastic, nylon, paper), and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot. [0117] Useful physical formats comprise surfaces having a plurality of discrete, addressable locations for the detection of a plurality of different biomarkers. Such formats include protein microarrays, or "protein chips" (see, e.g., Ng et al., J Cell MoI. Med., 6:329- 340 (2002)) and certain capillary devices (see, e.g., U.S. Pat. No. 6,019,944). In these embodiments, each discrete surface location may comprise antibodies to immobilize one or more protein markers for detection at each location. Surfaces may alternatively comprise one or more discrete particles (e.g., microparticles or nanoparticles) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one or more protein markers for detection. [0118] Analysis of the level of a biomarker can be carried out in a variety of physical formats. For example, the use of microtiter plates or automation could be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate diagnosis or prognosis in a timely fashion. [0119] Alternatively, the antibodies or nucleic acid probes of the invention can be applied to sections of patient biopsies immobilized on microscope slides. The resulting antibody staining or in situ hybridization pattern can be visualized using any one of a variety of light or fluorescent microscopic methods known in the art.

[0120] In another format, the various markers of the invention also provide reagents for in vivo imaging such as, for instance, the imaging of labeled regents that detect the nucleic acids or encoded proteins of the biomarkers of the invention. For in vivo imaging purposes, reagents that detect the presence of proteins encoded by cancer biomarkers, such as antibodies, may be labeled using an appropriate marker, such as a fluorescent marker.

COMPOSITIONS, KITS AND INTEGRATED SYSTEMS [0121] The invention provides compositions, kits and integrated systems for practicing the assays described herein using polynucleotides and polypeptides of the invention, antibodies specific for polypeptides or polynucleotides of the invention, etc.

[0122] The invention provides assay compositions for use in solid phase assays; such compositions can include, for example, one or more polynucleotides or polypeptides of the invention immobilized on a solid support, and a labeling reagent. In each case, the assay compositions can also include additional reagents that are desirable for hybridization. Modulators of expression or activity of polynucleotides or polypeptides of the invention can also be included in the assay compositions.

[0123] The invention also provides kits for carrying out the diagnostic and prognostic assays of the invention. The kits typically include a probe that comprises an antibody or nucleic acid sequence that specifically binds to polypeptides or polynucleotides of the invention, and a label for detecting the presence of the probe. The kits may include several antibodies specific for, or polynucleotide sequences encoding, the polypeptides of the invention. [0124] Optical images viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical images.

[0125] One conventional system carries light from the specimen field to a cooled charge- coupled device (CCD) camera, in common use in the art. A CCD camera includes an array of picture elements (pixels). The light from the specimen is imaged on the CCD. Particular pixels corresponding to regions of the specimen are sampled to obtain light intensity readings for each position. Multiple pixels are processed in parallel to increase speed. The apparatus and methods of the invention are easily used for viewing any sample, e.g., by fluorescent or dark field microscopic techniques. [0126] In one embodiment of the invention, kits are provided for genotyping a PER3 breast cancer, assigning treatment for a PER3 breast cancer, providing a prognosis for a PER3 breast cancer, and the like. In one embodiment, the kits of the invention may comprise a reagent that specifically binds to a PER3 biomarker. In certain embodiments, the reagent may be a nucleic acid, for example a PER3 hybridization probe, an oligonucleotide primer, or a set of oligonucleotide primers. In other embodiments, the reagent may be a PER3 protein binding moiety, for example an antibody or fragment thereof, an aptamer, a spiegelmer, and the like.

METHODS TO IDENTIFY COMPOUNDS

[0127] A variety of methods may be used to identify compounds that prevent or treat hormone receptor positive breast cancer. Typically, an assay that provides a readily measured parameter is adapted to be performed in the wells of multi-well plates in order to facilitate the screening of members of a library of test compounds as described herein. Thus, in one embodiment, an appropriate number of cells can be plated into the cells of a multi-well plate, and the effect of a test compound on the expression of a biomarker can be determined. [0128] The compounds to be tested can be any small chemical compound, or a macromolecule, such as a protein, sugar, nucleic acid or lipid. Typically, test compounds will be small chemical molecules and peptides. Essentially any chemical compound can be used as a test compound in this aspect of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma- Aldrich (St. Louis, MO), Fluka Chemika- Biochemica Analytika (Buchs Switzerland) and the like.

[0129] In one preferred embodiment, high throughput screening methods are used which involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds. Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. In this instance, such compounds are screened for their ability to reduce or increase the expression of the biomarkers of the invention. [0130] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

[0131] Preparation and screening of combinatorial chemical libraries are well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res., 37:487-493 (1991) and Houghton et al, Nature, 354:84-88 (1991)). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g. , PCT Publication No. WO 91/19735), encoded peptides (e.g. , PCT Publication No. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Patent No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al , PNAS USA, 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara et al, J. Amer. Chem. Soc, 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc., 114:9217-9218 (1992)), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc, 116:2661 (1994)), oligocarbamates (Cho et al, Science, 261:1303 (1993)), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem., 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent No. 5,539,083), antibody libraries (see, e.g., Vaughn et al, Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, Science, 274:1520-1522 (1996) and U.S. Patent No. 5,593,853), small organic molecule libraries {see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent No. 5,569,588; thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974; pyrrolidines, U.S. Patent Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent No. 5,506,337; benzodiazepines, 5,288,514, and the like). [0132] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

[0133] In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microliter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 96 modulators. If 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay many plates per day; assay screens for up to about 6,000, 20,000, 50,000, or 100,000 or more different compounds is possible using the integrated systems of the invention.

[0134] In one embodiment, a method of assaying for an agent for treatment of a hormone receptor positive breast cancer is provided. In one embodiment, the method comprises the steps of expressing in a cell a nucleic acid encoding Per3, contacting the cell with a candidate agent, and assaying for the functional effect of the candidate agent on Per3, thereby identifying an agent useful for treatment of a hormone receptor positive breast cancer. In certain embodiments, the hormone receptor positive breast cancer may be an estrogen receptor positive or progesterone receptor positive breast cancer.

EXAMPLES

Example 1 [0135] Copy Number Analysis of PER3: A combination of human breast tumor analysis and mouse models were use to show that PER3 is an important tumor suppressor for breast cancer, particularly in patients with tamoxifen-treated ER positive tumors (see Figure 1). Analysis of CGH array data on 180 Lymph node negative breast cancers from a Spanish cohort showed that deletions of chromosome Ip36 were associated preferentially with recurrence in patients with ER positive tumors (Climent, J. et al., Cancer Res. 67(2):818-26 (2007)). Although the PER3 gene itself was not included in this array, detailed TaqMan analysis of gene copy number showed a significant correlation (p-value =0.01) between PER3 gene copy number and prognosis in the same patients. The correlation between the expression values of the PER3 gene and cancer recurrence was further analyzed using the results from two different breast cancer patient datasets (Chin, K. et al., Cancer Cell. 10(6):529-41 (2006), Van de Vijver, M. J. et al., N Engl J Med 347(25): 1999-2009 (2002)). This analysis demonstrated a significant association between low PER3 gene expression and recurrence in ER+ patients (n= 309/413) (p value= 0.009). Importantly, a causal role for PER3 loss in breast cancer development was demonstrated using two independent mouse models involving either carcinogen treatment or over-expression of Her2/Neu to induce breast tumorigenesis. [0136] All tumor DNA samples were obtained from frozen breast tumors with >50% tumor cells (Climent, J. et al., Cancer Res. 67(2):818-26 (2007)). The genomic sequence OΪPER3 (GenBank accession NM 016831.1) was used to design a set of primers and probe specific to the PER3 gene (Primer Express software version 1.0 (Applied Biosystems)). The primers for PER3 were 5'- GGAGTGAG AAACCGGTGTCTGT-3' (forward; SEQ ID NO:15) and 5'- GCCCGCAGCCTGCTT -3' (reverse; SEQ ID NO: 16). The probe for PER3 was 5'-(6- FAM) - CTGACTGC AA AGTGAG-(TAMRA)-3', (SEQ ID NO: 17) where FAM is 6- carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine. The primers and probe for RNase P used as an endogenous control gene were obtained from Applied Biosystems. The RNase P probe was labeled at 5' end with VIC (Applied Biosystems) instead of FAM. PER3 copy number was determined by relative quantification using the ΔΔCt method normalized to the RNase P copy number of 2 (Mao, J. H. et al., Cancer Cell. ll(2):161-73 (2007)). To analyze the results from the copy number experiment, the TaqMan® Gene Copy Number Assays Macro File (Applied Biosystems) was utilized.

[0137] Genome- wide array CGH profiles of 185 lymph node negative breast cancers from a Spanish cohort, of whom 85 received anthracycline chemotherapy (Chemo group), and 95 received no chemotherapy (non-Chemo group), were previously reported (Climent, J. et al., Cancer Res. 67(2):818-26 (2007)). To search for genetic events related to resistance to hormonal (Tamoxifen) therapy, the non-Chemo group was divided into two subgroups based on whether they had received hormonal treatment or not. Of the 95 patients in the non- Chemo group, 59 patients with ER and/or PgR positive tumors received Tamoxifen, whereas 36 did not receive any treatment. Analysis of CGH profiles for these patients revealed that deletion of chromosome Ip was associated with recurrence in this subgroup of ER+ Tamoxifen treated patients (p < 0.05 adjusted using False Discovery Rate (FDR)) (Figure 14).

[0138] Chromosome Ip36 shows very frequent deletions in many human tumors, but the region of deletion is large, and separate, non-overlapping chromosome fragments have been implicated (Rubio-Moscardo, F. et al, Blood 105(11):4445-54 (2005); Bieche, I. et al, Cancer Res. 53(9):1990-4 (1993); Matsuzaki, M. et al., M J Oncol. 13(6):1229-33 (1998); Benn, D. E. et al., Cancer Res. 60(24):7048-51 (2000); Schleiermacher, G. et al., Genes Chromosomes Cancer. 10(4):275-81 (1994)). These data suggested that multiple tumor suppressor genes are involved, depending on tumor type. PER3 was considered as a good candidate for involvement in breast cancer because of its location within one of the minimal deletion regions on Ip36.2 (Bagchi, A. et al., Cell, 128(3):459-75 (2007)), as well as the epidemiological (Zhu, Y. et al., Cancer Epidemiol Biomarkers Prev. 14(l):268-70 (2005)) and mechanistic (Gery, S. et al., Cold Spring Harb Symp Quant Biol. 72:459-64 (2007)) data linking circadian rhythm genes to hormone status and breast cancer. Therefore the copy number status of PER3 was examined by quantitative TaqMan analysis in DNA samples from 180 breast cancer patients (Climent, J. et al., Cancer Res. 67(2):818-26 (2007)). The relationship between the frequency of deletion or copy number gain, and clinico-pathological characteristics of the patients is shown in Table 1. The number of copies of PER3 showed a significant gene dosage association with recurrence-free survival at 10 years (Figure IA, p= 0.01). The proportion of disease free surviving patients after 10 years was lowest in patients with single copy PER3 deletion (56% ± 8.6; red line) , compared to those with two (75% ± 4.0; blue line) or more (89% ± 5.6; green line) copies of the PER3 gene (Figure IA). Further analysis showed that the effect of PER3 deletion was most pronounced in the Tamoxifen treated group, with no significant association in the non-treated or chemotherapy-treated groups (Figs. IB-D). Among the 59 patients who only received Tamoxifen treatment (Figure ID), patients with single copy PER3 deletions had a significantly lower disease-free survival rate at 10 years (47% ±12) than those with normal PER3 (84%±6) or copy number gains (100% survival) (p=0.007).

Table 1. Frequency of copy number of PER3 related with the clinical data of 180 lymph node negative breast cancer patients from Climent et al 2007.

Example 2

[0139] PER3 Mutational/Polymorphic Status in Breast Cancer Cell Lines: The mutational/polymorphic status ofPER3 in 32 breast cancer cell lines was next examined. No clear pathogenic (nonsense or missense) mutation was identified, however many known (Ebisawa, T. et al., EMBO Rep. 2(4):342-6 (2001)) and some other unknown polymorphism and alternative splicing isoforms were found. Since one of the polymorphisms found in PER3 has been associated in other studies with breast cancer susceptibility (Zhu, Y. et al., Cancer Epidemiol Biomarkers Prev. 14(l):268-70 (2005)), as well as with disruption of sleep homeostasis, the frequency of this polymorphism was examined in both tumor and blood DNA from breast cancer patients and also in breast cancer cell lines (Figure 2). This is the first frequency report of this polymorphism in tumor DNA and also comparing matched samples (blood and tumor) from the same patient. As is seen in Figure 2, a decrease of the heterozygous PER3^4/5 population, mainly in favor of the increase of homozygous PER3^5/5 (p =0.007, Figure IE) is observed. Enrichment for the PER3^5/5 genotype was even higher in breast cancer cell lines, as compared with primary tumor DNA and normal blood DNA (38% vs 23% vs 16%).

[0140] Briefly, mutational/polymorphic screening covering the entire coding region of PER3 was performed by direct Sanger sequencing in 34 breast cancer cell lines. cDNA was synthesized from 34 breast cancer cell lines and RT-PCR was performed with 7 forward and reverse primer sets designed for the PER3 coding region (Table 2). PCR reactions were carried out in a volume of 25 μl containing 100 ng cDNA, 10 pmol of each primer, 250 mM each dNTP, 0.5 U of Taq polymerase and the reaction buffer provided by the supplier (Qiagen, Hilden, Germany). Whole PER3 coding regions were sequenced using the Taq dideoxy terminator cycle sequencing kit and an ABI 3730 DNA sequencer (Applied Biosystems). Several single missense or silent nucleotide polymorphisms (SNPs) were identified, including V419M, S445S, I606I, V639G, L697L, T725T, P745P, L827P, P856A, S864S, TlOlOT, M1028T, and Hl 149R (Table 3). No clear pathogenic mutations, i.e. nonsense and missense changes that could not be attributed to naturally occurring polymorphisms, were identified.

[0141] The analysis of the 54 base-pair repetition allele polymorphism was done by PCR, as previously reported (Zhu, Y. et al., Cancer Epidemiol Biomarkers Prev. 14(l):268-70 (2005)), modifying the annealing temperature of the PCR reaction to 68°C. The polymorphism analysis was done in 289 DNAs from blood samples and 443 tumor DNAs including the 37 breast cancer cell lines. 117 samples were matched tumor and blood DNA from the same breast cancer patients. The tests for deviation from the Hardy- Weinberg equilibrium (Emigh, Ted H. (1980)) were used to analyze the difference in polymorphism frequencies between blood and tumor DNAs. As seen in Figure 2, a total of 59 patients were identified who were heterozygous PER3^{4 5} and for whom tumor DNA was available. 19 of these cases showed loss of one PER3 allele. In 14 of these 19 cases the 4 repeat allele was lost, and in 5 cases the 5 repeat allele was lost. This shows that there is a statistically significant difference in the distribution of PER3 alleles in tumors compared to normal blood DNA. Moreover, tumors that lose one allele preferentially lose the 4 repeat and retain the 5 repeat allele (p=0.02, Fisher test).

Example 3

[0142] PER3 expression was examined in 413 breast tumor expression arrays taken from Van de Vijver (Van de Vijver, M. J. et al, N EnglJ Med. 347(25): 1999-2009 (2002)) 2002 (n=295) and Chin (Chin, K. et al., Cancer Cell. 10(6):529-41 (2006)) 2007 (n=118). In each dataset a sample S₁ in the set S was labeled as "PER3 Low", "PERS normal", or ^UPER3 high" using the rule:

If s, < ( meanfSJ - ^}Λ ^standard deviationfSJ ), assign LOW If s, ≥ (mean [S] + ¹A * standard deviation [S]), assign HIGH

Otherwise, assign NORMAL.

This method allowed for the comparison of relative PER3 expression levels across both data sets fused as a single group of patients.

[0143] PER3 gene expression was analyzed in 413 breast tumor expression arrays taken from two publicly available data sets (Van de Vijver (Van de Vijver, M. J. et al., N Engl J Med. 347(25):1999-2009 (2002)) 2002, n-295 and Chin (Chin, K. et al., Cancer Cell. 10(6):529-41 (2006)) 2007, n=l 18; Table 4). A full description of the stratification of the patients into different subgroups according to PER3 expression together with disease-free survival curves for all patients in each sub-group is shown in Figures 6, 7, and 8. Patients with lower PER3 expression ("PER3 low", n=122) were significantly more likely to recur than those with normal or higher expression ("PER3 normal/high", n=291) (Figure 6 A; P=O-OlS). Disease-free survival analysis showed that PER3 low patients had significantly worse survival rates than PER3 normal/high patients (p<0.001). ER status is an important predictor of recurrence and greatly influences treatment regimes (Khan, S. A. et al., Cancer Res. 54(4):993-7 (19945); Yager, J. D. et al., N Engl J Med. 354(3):270-82 (2006)). If low expression of PER3 segregates with ER status, any effect of low PER3 expression could be confounded with the effect of ER status. Therefore, a subset analysis of PER3 in ER+ and ER- tumors was performed. It was found that low PER3 was significantly associated with recurrence in ER+ (p= 0.01), but not ER- (Figure 7). Disease-free survival analysis indicated that ER+ patients with low PER3 levels had significantly shorter survival times (p<0.001). In contrast, no significant association was found between survival and PER3 status in ER- tumors (Figure 7). Accordingly, the association between low PER3 expression and recurrence in the complete patient sample set was driven by the ER+ tumors, with no effect being detected in the ER- tumors. These data are in agreement with the association between deletion of PER3 and recurrence specifically in the Tamoxifen-treated (ER positive) patients in Fig. ID.

Table 4. Relationship between Per 3 expression levels and clinical-pathological data of the 413 patients from Van de Vijver et al. 2002, and Chin et al. 2006

[0144] Further analysis of the 413 patients in the Van de Vijver et αl. 2002, and Chin et αl. 2006 cohorts showed that PER3 expression was associated with disease-free survival and overall survival in these two patient data sets, independently of other variables such as tumor size, lymph node status or age (Table 5). Table 5 shows the results of the Cox proportional hazard ratio multivariate analysis for the risk of distant recurrence or death among patients with breast cancer.

Table 5. Cox proportional hazard ratio multivariate analysis for distant recurrence or death.

[0145] It was next determined whether stratifying tumors according to their gene expression classification (Sørlie, T. et al, Proc NαtlAcαdSci USA 98(19): 10869-74 (2001); Sorlie, T. et al., Proc Nαtl Acαd Sci USA. 100(14):8418-23 (2003)) could reveal additional information. The tumors were labeled using a nearest centroid classifier and a label was only assigned if correlation with a target class was above 0.1. This resulted in samples labeled Luminal A (n=90), Luminal B (n=68), ERBB2 (n=56), Normal-like (n=17), Basal (n=73), or Unclassified (n=109) (Figure 7). Of these groups, low PER3 expression had significant association with recurrence only in Luminal A-type (p=0.007) or ERBB2-type tumors (p=0.03) (Figure 8). Disease-free survival analysis for Luminal A and ERBB2-type tumors indicated that PER3 low patients had lower disease free survival rates at 10 years than those patients with PER3 normal/high (28%± 10 vs 84%±4) for Luminal A (ρ<0.001) and (30%± 8 vs 68%±8) for ERBB2-type (p= 0.004). There was also a striking effect on overall survival rate at 10 years in all the patients and in the subgroups of ER positive (Figure 7), Luminal A and ERBB2 patients (Figure 8). The ten year overall survival rate for ER+ patients with low PER3 was 55% ± 6 vs. 79% ± 3 for normal/high patients (p < 0.001) (Figure 1 IB). The overall survival rate was 25% ± 8 for ERBB2 patients with low PER3, vs. 70% ± 7 for ERBB2 patients with normal/high PER3 (pO.OOl) (Figure 1 IF). The overall Survival rate at 10 years in Luminal-A patients with low PER3 was 34% ± 11 vs. 83% ± 3 for patients with normal/high PER3 (p<0.001 ) (Figure 11 G).

[0146] Chromosome engineering studies have previously identified CHD5 as a candidate tumor suppressor gene within the minimal deletion region on Ip36.2 (Bagchi, A. et al., Cell, 128(3):459-75 (2007)). Since PER3 is located only 1.2Mb from CHD5, we analyzed CHDJ expression levels in all 413 breast tumors. No association of CHD5 expression with recurrence or survival was found in any of the subgroups of patients analyzed (Figures 12 and 13).

Example 4

[0147] Association between PER3 deletion or PER 3 expression and clinical-pathological parameters was analyzed using Fisher's exact test. All reported P values were two tailed. Significant differences in disease-free and overall survival time were calculated using the Cox proportional hazard (log-rank) test. Statistical analysis was performed using SPSS version 12.0.

Example 5 [0148] In order to investigate a possible causal association between loss of PER3 function and breast tumor development, two studies involving mouse models of breast cancer were performed. A total of 86 mice carrying normal or inactivated alleles of the PER3 gene (17 wild-type PER3^{+ +}, 35 heterozygous PER3^+/- and 34 null PER3^{- -}) were treated by oral gavage with 7, 12-dimethylbenz[a]anthracene (DMBA), a protocol known to induce breast cancer in sensitive strains of mice (Medina, D. et al., Cancer Res. 40(2):368-73 (1980)). Eight mice (two heterozygous and six null) were found dead before the end point and no remaining tissues were collected from them.

[0149] Wild-type (PER3^+/+) and PER3 knockout (PER3^'1') 129/sv mice (provided by Drs. YH Fu and LJ Ptacek, UCSF) were bred and treated according to Laboratory Animal Resource Center (LARC) regulations. 7-week-old female mice from the F₂ intercross population (PER3^+/+, PER3^+/- and PER3^-Λ) were treated with 6 doses of 1 mg of 7, 12- dimethylbenz[a] anthracene (DMBA) diluted in corn oil by weekly oral gavage. A second group of mice was folio wed-up with no treatment as a group control. In a second experiment, male PER3^-7- mice were crossed with female FVB mice containing the neu protooncogene under control of the MMTV 3'-LTR promoter (Muller, W. J. et al., Cell, 54(l):105-15

(1988)) (provided by Dr. Z Werb, UCSF) to generate F₁ transgenic mice heterozygous for PER3 (neu/PER3^+/~). Fj males and females were intercrossed to produce the F₂ generation consisting of neu/ PER3^+/+, neu/ PER3^+/~ and neu/ PER3-A animals.

[0150] In the DMBA gavage experiment, female mice were examined every three days for sickness or symptoms of tumor development for up to 19.7 months. MMTVneu/PEi?3 transgenic female mice were examined weekly for mammary tumor development by palpation for up to 25.8 months. Mice that showed significant weight loss, morbidity or excessive tumor burden were sacrificed by cervical dislocation after being anesthetized according to the UCSF Animal Care and Use (IACUC) protocol. Tumors and tissues were fixed in 4% neutral buffered paraformaldehyde for histological examination. Mice found dead were censored from the study.

[0151] The median follow-up of the remaining 78 mice included in the study was 8.3 months (range 3.8 - 15.0). All of the mice treated with DMBA developed tumors of various kinds including lymphoma and epithelial tumors such as lung, ovarian, and skin (Table 6). However, development of breast tumors was specifically associated with PER3 deficiency. Thirty-six percent of PER3^-/- mice treated with DMBA developed breast tumors, while 12% of the PER3^+/- mice developed breast tumors. In striking contrast, none of the control PER3^+/+ mice developed a breast tumor (p= 0.005) (Figure 4). A group of 65 mice (19 wild- type, 25 heterozygous, and 21 null) were used as controls with no DMBA gavage treatment. Two of the PER3^-/- control mice developed sporadic breast tumors, but none of the remaining mice were found sick or developed any other class of tumor during the time course of this experiment (24 months).

[0152] The second mouse model was based on the observation that low levels of PER3 expression were strongly associated with recurrence in ERBB2-type human breast cancers. MMTV-Neu mice overexpress ErbB2 in the mammary gland, and spontaneously develop breast tumors (Muller, W. J. et al., Cell, 54(l):105-15 (1988)). A total of 79 MMTV-Neu positive mice were generated, of which 30 (38%) were PER3^+/+, 35 (44%) were PER3^+!~, and 14 (18%) were PER3^{' '}. The median follow-up of all mice was 14.9 months (range 6.3 — 25.8). All PERT^1' mice developed breast tumors, whereas 25 (71%) of the PER3^+/-and 14

(47%) of the PER3^+/+ mice developed breast tumors. The proportion of PER3 null mice free of tumors at 15 months (21% ± 8) was significantly lower than the proportion in the heterozygous and the wild-type mice (63% ±6 in both PER3^+I~ and PER3^+/+, p = 0.003). Histological analysis of tumors from both models of breast cancer showed that loss of PER3 did not affect the tumor class or morphology, since both DMBA-induced and MMTV-Neu- induced tumors in PER3-I- mice resembled equivalent tumors from PER3 wild type animals (data not shown)

[0153] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

L A method of providing a prognosis for estrogen receptor positive breast cancer in a subject, the method comprising the steps of: (a) contacting a biological sample from the subject with a reagent that specifically binds to a PER3 biomarker, and (b) determining whether or not the biomarker is differentially expressed in the sample by comparing expression in the sample to a control; thereby providing a prognosis for estrogen receptor positive breast cancer.

2. The method of claim 1, wherein the reagent is a nucleic acid.

3. The method of claim 1 , wherein the step of determining whether or not the biomarker is differentially expressed comprises PCR, a single base extension reaction, or a hybridization assay.

4. The method of claim 1 , wherein the reagent is an antibody.

5. The method of claim 1, wherein the subject has been treated with Tamoxifen.

6. The method of claim 1 , wherein the breast cancer is luminal A, ERBB2+, or non-basal breast cancer.

7. The method of claim 1, wherein normal or increased expression of the PER3 biomarker indicates a good prognosis and reduced expression of the PER3 biomarker indicates a poor prognosis.

8. The method of claim 1, wherein the sample is selected from the group consisting of a sample of a surgically resected breast tumor, a breast tumor biopsy, a sample of breast tissue, and a blood sample.

9. The method of claim 1 , wherein the prognosis provides a high or low risk of tumor recurrence.

10. A method of assigning treatment to a subject diagnosed with estrogen receptor positive breast cancer or progesterone receptor positive breast cancer, the method comprising the steps of: (a) contacting a biological sample from the subject with a reagent that specifically binds to a PER3 biomarker; (b) determining whether or not the expression of the biomarker is down- regulated in the sample by comparing expression in the sample to a control; and (c) assigning a treatment regime comprising administration of a hormone receptor antagonist if the status of the biomarker is if the biomarker is not down-regulated.

11. A method of assigning treatment to a subject diagnosed with estrogen receptor positive breast cancer or progesterone receptor positive breast cancer, the method comprising the steps of: (a) contacting a biological sample from the subject with a reagent that specifically binds to a PER3 biomarker; (b) determining the PER3 genotype; and (c) assigning a treatment regime comprising administration of a hormone receptor antagonist if the genotype is PER3-positive.

12. The method of claim 11 , wherein the step of determining the PER3 genotype comprises determining the PER3 gene copy number.

13. The method of claim 11 , wherein the step of determining the PER3 genotype comprises detecting a PER3 polymorphism.

14. The method of claim 13, wherein the PER3 polymorphism is a 4 or 5 repeat allele.

15. The method of claim 13 , wherein the PER3 polymorphism is a point mutation.

16. The method of claim 11 , wherein the step of determining the PER3 genotype comprises detecting an expressed PER3 splicing isoform.

17. The method of claim 10, wherein the hormone receptor antagonist is Tamoxifen.

18. A method of providing a prognosis for estrogen receptor positive breast cancer in a subject, the method comprising the steps of: (a) contacting a biological sample from the subject with a reagent that specifically binds to a PER3 biomarker; and (b) determining the PER3 genotype of the sample, wherein a PER3-positive genotype indicates a good prognosis.

19. The method of claim 18, wherein determining the PER3 genotype of the sample comprises detecting a polymorphism in the PER3 gene.

20. The method of claim 19, wherein the polymorphism is a point mutation or splice variant.

21. A kit comprising a reagent that specifically binds to a PER3 biomarker.

22. The kit of claim 21 , wherein the reagent comprises a PCR primer, an SBE primer, or an oligonucleotide for a hybridization assay.

23. The kit of claim 21 , wherein the reagent comprises an antibody.

24. A method of assaying for an agent for treatment of ER positive breast cancer that modulates PER3, the method comprising the steps of: (a) expressing in a cell a nucleic acid encoding PER3; (b) contacting the cell with a candidate agent; and (c) assaying for the functional effect of the candidate agent on PER3.

25. A method of assaying for an agent for the treatment of ER positive breast cancer, the method comprising the steps of: (a) isolating a cell having reduced or absent PER3 activity; (b) contacting the cell with a candidate agent; and (c) assaying for an effect on a cellular pathway associated with PER3.