Multicenter Comparison of 17-Gene Genomic Prostate Score as a Predictor of Outcomes in African American and Caucasian American Men with Clinically Localized Prostate Cancer.

PURPOSE: Adoption of prognostic molecular assays for prostate cancer requires evidence of robust performance in different racial groups. Retrospective analysis was conducted to assess the performance of the Oncotype DX® Genomic Prostate Score® test in African American and Caucasian American men with surgically treated prostate cancer. MATERIALS AND METHODS: We compared the assay results (scale 0-100) and the 4 gene group scores in biopsy specimens from 201 African American and 1,144 Caucasian American men with clinically localized prostate cancer in 6 cohorts. Adverse pathology was defined as high grade (primary Gleason pattern 4 or any pattern 5) and/or nonorgan-confined disease (≥pT3). Binary logistic regression models were used for adverse pathology. Biochemical recurrence was defined as 2 successive prostate specific antigen levels >0.2 ng/ml or initiation of salvage therapy after radical prostatectomy. Cox proportional hazards models evaluated the association of the assay result or racial group with time to biochemical recurrence. RESULTS: Each cohort had different clinical risk distributions and percentages of African Americans, although median and interquartile ranges of the assay results and gene group scores were similar between both racial groups. In a multivariable model with the assay and pathological/clinical features including race, the assay was significantly associated with adverse pathology (p ≤0.004) and biochemical recurrence (p <0.001). Race was not a significant predictor of either end point. CONCLUSIONS: The assay is similarly predictive of outcomes in African American and Caucasian American patients, and improves risk stratification in men with newly diagnosed prostate cancer from both racial groups.

The 17-Gene Genomic Prostate Score Assay Predicts Outcome After Radical Prostatectomy Independent of PTEN Status.

OBJECTIVE: To compare the ability of loss of phosphatase and tensin homolog (PTEN) and Genomic prostate score assay (GPS) in predicting the biochemical-recurrence (BCR) and clinical-recurrence (CR) after radical prostatectomy (RP) for clinically localized prostate cancer (PCa). METHODS: Three hundred seventy seven patients with and without CR were retrospectively selected by stratified cohort sampling design from RP database. PTEN status (by immunohistochemistry [IHC] and fluorescence in situ hybridization [FISH]) and GPS results were determined for RP specimens. BCR was defined as Prostate Specific Antigen (PSA) ≥ 0.2 ng/mL or initiation of salvage therapy for a rising PSA. CR was defined as local recurrence and/or distant metastases. RESULTS: Baseline mean age, PSA, and GPS score for the cohort were 61.1 years, 8 ng/dL, and 32.8. PTEN loss was noted in 38% patients by FISH and 25% by IHC. The concordance between FISH and IHC for PTEN loss was 66% (Kappa coefficient 0.278; P < .001). On univariable analysis, loss of PTEN by FISH or IHC was associated with BCR and CR (P < .05). However, after adjusting for GPS results, PTEN loss was not a significant predictor for CR or BCR (P > .1). The GPS result remained strongly associated with CR and BCR after adjusting for PTEN status (P < .001). PTEN status and GPS results only weakly correlated. GPS was widely distributed regardless of PTEN status indicating the biological heterogeneity of PCa even in PTEN-deficient cases. CONCLUSION: GPS is a significant predictor of aggressive PCa, independent of PTEN status. After adjustment for GPS results, PTEN was not independently associated with recurrence for PCa.

Validation of the 16-Gene Recurrence Score in Patients with Locoregional, High-Risk Renal Cell Carcinoma from a Phase III Trial of Adjuvant Sunitinib.

Purpose: Adjuvant sunitinib prolonged disease-free survival (DFS; HR, 0.76) in patients with locoregional high-risk renal cell carcinoma (RCC) in the S-TRAC trial (ClinicalTrials.gov NCT00375674). The 16-gene Recurrence Score (RS) assay was previously developed and validated to estimate risk for disease recurrence in patients with RCC after nephrectomy. This analysis further validated the prognostic value of RS assay in patients from S-TRAC and explored the association of RS results with prediction of sunitinib benefit.Patients and Methods: The analysis was prospectively designed with prespecified genes, algorithm, endpoints, and analytical methods. Primary RCC was available from 212 patients with informed consent; primary analysis focused on patients with T3 RCC. Gene expression was quantitated by RT-PCR. Time to recurrence (TTR), DFS, and renal cancer-specific survival (RCSS) were analyzed using Cox proportional hazards regression.Results: Baseline characteristics were similar between patients with and those without RS results, and between the sunitinib and placebo arms among patients with RS results. RS results predicted TTR, DFS, and RCSS in both arms, with the strongest results observed in the placebo arm. When high versus low RS groups were compared, HR for recurrence was 9.18 [95% confidence interval (CI), 2.15-39.24; P < 0.001) in the placebo arm; interaction of RS results with treatment was not significant.Conclusions: The strong prognostic performance of the 16-gene RS assay was confirmed in S-TRAC, and the RS assay is now supported by level IB evidence. RS results may help identify patients at high risk for recurrence who may derive higher absolute benefit from adjuvant therapy. Clin Cancer Res; 24(18); 4407-15. ©2018 AACR.

Gene expression in normal-appearing tissue adjacent to prostate cancers are predictive of clinical outcome: evidence for a biologically meaningful field effect.

PURPOSE: We evaluated gene expression in histologically normal-appearing tissue (NT) adjacent to prostate tumor in radical prostatectomy specimens, assessing for biological significance based on prediction of clinical recurrence (cR - metastatic disease or local recurrence). RESULTS: A total of 410 evaluable patients had paired tumor and NT. Forty-six genes, representing diverse biological pathways (androgen signaling, stromal response, stress response, cellular organization, proliferation, cell adhesion, and chromatin remodeling) were associated with cR in NT (FDR < 20%), of which 39 concordantly predicted cR in tumor (FDR < 20%). Overall GPS and its stromal response and androgen-signaling gene group components also significantly predicted time to cR in NT (RM-corrected HR/20 units = 1.25; 95% CI: 1.01-1.56; P = 0.024). EXPERIMENTAL DESIGN: Expression of 732 genes was measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) separately in tumor and adjacent NT specimens from 127 patients with and 374 without cR following radical prostatectomy for T1/T2 prostate cancer. A 17-gene expression signature (Genomic Prostate Score [GPS]), previously validated to predict aggressive prostate cancer when measured in tumor tissue, was also assessed using pre-specified genes and algorithms. Analysis used Cox proportional hazards models, Storey's false discovery rate (FDR) control, and regression to the mean (RM) correction. CONCLUSIONS: Gene expression profiles, including GPS, from NT adjacent to tumor can predict prostate cancer outcome. These findings suggest that there is a biologically significant field effect in primary prostate cancer that is a marker for aggressive disease.

A 16-gene assay to predict recurrence after surgery in localised renal cell carcinoma: development and validation studies.

BACKGROUND: The likelihood of tumour recurrence after nephrectomy in localised clear cell renal cell carcinoma is well characterised by clinical and pathological parameters. However, these assessments can be improved and personalised by the addition of molecular characteristics of each patient's tumour. We aimed to develop and validate a prognostic multigene signature to improve prediction of recurrence risk in clear cell renal cell carcinoma. METHODS: In the development stage, we investigated the association between expression of 732 genes, measured by reverse-transcription PCR, and clinical outcome in 942 patients with stage I-III clear cell renal cell carcinoma who had undergone a nephrectomy at the Cleveland Clinic (OH, USA). 516 genes were associated with recurrence-free interval. 11 of these genes were selected by further statistical analyses, and were combined with five reference genes (ie, 16 genes in total), from which a recurrence score algorithm was developed. The recurrence score was then validated in an independent cohort of 626 patients from France with stage I-III clear cell renal cell carcinoma who had also undergone nephrectomy. The association between the recurrence score and the risk of recurrence and cancer-specific survival in the first 5 years after surgery was assessed using Cox proportional hazard regression, stratified by tumour stage (stage I vs stage II vs III). FINDINGS: In our primary univariate analysis, the continuous recurrence score (median 37, IQR 31-45) was significantly associated with recurrence-free interval (hazard ratio 3·91 [95% CI 2·63-5·79] for a 25-unit increase in score, p<0·0001). In multivariable analyses, the recurrence score was significantly associated with the risk of tumour recurrence (hazard ratio per 25-unit increase in the score 3·37 [95% CI 2·23-5·08], p<0·0001) after stratification by stage and adjustment for tumour size, grade, or Leibovich score. The recurrence score was able to identify a clinically significant number of both high-risk stage I and low-risk stage II-III patients. A heterogeneity study on separate samples showed little to no intratumoural variability among the 16 genes. INTERPRETATION: Our findings validate the recurrence score as a predictor of clinical outcome in patients with stage I-III clear cell renal cell carcinoma, providing a more accurate and individualised risk assessment beyond existing clinical and pathological parameters. FUNDING: Genomic Health Inc and Pfizer Inc.

A Biopsy-based 17-gene Genomic Prostate Score Predicts Recurrence After Radical Prostatectomy and Adverse Surgical Pathology in a Racially Diverse Population of Men with Clinically Low- and Intermediate-risk Prostate Cancer.

BACKGROUND: Biomarkers that are validated in independent cohorts are needed to improve risk assessment for prostate cancer (PCa). OBJECTIVE: A racially diverse cohort of men (20% African American [AA]) was used to evaluate the association of the clinically validated 17-gene Genomic Prostate Score (GPS) with recurrence after radical prostatectomy and adverse pathology (AP) at surgery. DESIGN, SETTING, AND PARTICIPANTS: Biopsies from 431 men treated for National Comprehensive Cancer Network (NCCN) very low-, low-, or intermediate-risk PCa between 1990 and 2011 at two US military medical centers were tested to validate the association between GPS and biochemical recurrence (BCR) and to confirm the association with AP. Metastatic recurrence (MR) was also evaluated. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Cox proportional hazards models were used for BCR and MR, and logistic regression was used for AP. Central pathology review was performed by one uropathologist. AP was defined as primary Gleason pattern 4 or any pattern 5 and/or pT3 disease. RESULTS AND LIMITATIONS: GPS results (scale: 0-100) were obtained in 402 cases (93%); 62 men (15%) experienced BCR, 5 developed metastases, and 163 had AP. Median follow-up was 5.2 yr. GPS predicted time to BCR in univariable analysis (hazard ratio per 20 GPS units [HR/20 units]: 2.9; p<0.001) and after adjusting for NCCN risk group (HR/20 units: 2.7; p<0.001). GPS also predicted time to metastases (HR/20 units: 3.8; p=0.032), although the event rate was low (n=5). GPS was strongly associated with AP (odds ratio per 20 GPS units: 3.3; p<0.001), adjusted for NCCN risk group. In AA and Caucasian men, the median GPS was 30.3 for both, the distributions of GPS results were similar, and GPS was similarly predictive of outcome. CONCLUSIONS: The association of GPS with near- and long-term clinical end points establishes the assay as a strong independent measure of PCa aggressiveness. Tumor aggressiveness, as measured by GPS, and outcomes were similar in AA and Caucasian men in this equal-access health care system. PATIENT SUMMARY: Predicting outcomes in men with newly diagnosed prostate cancer is challenging. This study demonstrates that a new molecular test, the Genomic Prostate Score, which can be performed on a patient's original prostate needle biopsy, can predict the aggressiveness of the cancer and help men make decisions regarding the need for immediate treatment of their cancer.

A 17-gene assay to predict prostate cancer aggressiveness in the context of Gleason grade heterogeneity, tumor multifocality, and biopsy undersampling.

BACKGROUND: Prostate tumor heterogeneity and biopsy undersampling pose challenges to accurate, individualized risk assessment for men with localized disease. OBJECTIVE: To identify and validate a biopsy-based gene expression signature that predicts clinical recurrence, prostate cancer (PCa) death, and adverse pathology. DESIGN, SETTING, AND PARTICIPANTS: Gene expression was quantified by reverse transcription-polymerase chain reaction for three studies-a discovery prostatectomy study (n=441), a biopsy study (n=167), and a prospectively designed, independent clinical validation study (n=395)-testing retrospectively collected needle biopsies from contemporary (1997-2011) patients with low to intermediate clinical risk who were candidates for active surveillance (AS). OUTCOME MEASURES AND STATISTICAL ANALYSIS: The main outcome measures defining aggressive PCa were clinical recurrence, PCa death, and adverse pathology at prostatectomy. Cox proportional hazards regression models were used to evaluate the association between gene expression and time to event end points. Results from the prostatectomy and biopsy studies were used to develop and lock a multigene-expression-based signature, called the Genomic Prostate Score (GPS); in the validation study, logistic regression was used to test the association between the GPS and pathologic stage and grade at prostatectomy. Decision-curve analysis and risk profiles were used together with clinical and pathologic characteristics to evaluate clinical utility. RESULTS AND LIMITATIONS: Of the 732 candidate genes analyzed, 288 (39%) were found to predict clinical recurrence despite heterogeneity and multifocality, and 198 (27%) were predictive of aggressive disease after adjustment for prostate-specific antigen, Gleason score, and clinical stage. Further analysis identified 17 genes representing multiple biological pathways that were combined into the GPS algorithm. In the validation study, GPS predicted high-grade (odds ratio [OR] per 20 GPS units: 2.3; 95% confidence interval [CI], 1.5-3.7; p<0.001) and high-stage (OR per 20 GPS units: 1.9; 95% CI, 1.3-3.0; p=0.003) at surgical pathology. GPS predicted high-grade and/or high-stage disease after controlling for established clinical factors (p<0.005) such as an OR of 2.1 (95% CI, 1.4-3.2) when adjusting for Cancer of the Prostate Risk Assessment score. A limitation of the validation study was the inclusion of men with low-volume intermediate-risk PCa (Gleason score 3+4), for whom some providers would not consider AS. CONCLUSIONS: Genes representing multiple biological pathways discriminate PCa aggressiveness in biopsy tissue despite tumor heterogeneity, multifocality, and limited sampling at time of biopsy. The biopsy-based 17-gene GPS improves prediction of the presence or absence of adverse pathology and may help men with PCa make more informed decisions between AS and immediate treatment. PATIENT SUMMARY: Prostate cancer (PCa) is often present in multiple locations within the prostate and has variable characteristics. We identified genes with expression associated with aggressive PCa to develop a biopsy-based, multigene signature, the Genomic Prostate Score (GPS). GPS was validated for its ability to predict men who have high-grade or high-stage PCa at diagnosis and may help men diagnosed with PCa decide between active surveillance and immediate definitive treatment.

Analytical validation of the Oncotype DX prostate cancer assay - a clinical RT-PCR assay optimized for prostate needle biopsies.

BACKGROUND: The Oncotype DX Prostate Cancer Assay is a multi-gene RT-PCR expression assay that was developed for use with fixed paraffin-embedded (FPE) diagnostic prostate needle biopsies containing as little as 1 mm of prostate tumor in the greatest dimension. The assay measures expression of 12 cancer-related genes representing four biological pathways and 5 reference genes which are algorithmically combined to calculate the Genomic Prostate Score (GPS). This biopsy-based assay has been analytically and subsequently clinically validated as a predictor of aggressive prostate cancer. The aim of this study was to validate the analytical performance of the Oncotype DX Prostate Cancer Assay using predefined acceptance criteria. RESULTS: The lowest quartile of RNA yields from prostate needle biopsies (six 5 µm sections) was between 19 and 34 ng. Analytical validation of the process requiring as little as 5 ng of RNA met all pre-defined acceptance criteria. Amplification efficiencies, analytical sensitivity, and accuracy of gene assays were measured by serially diluting an RNA sample and analyzing features of the linear regression between RNA expression measured by the crossing point (Cp) versus the log2 of the RNA input per PCR assay well. Gene assays were shown to accurately measure expression over a wide range of inputs (from as low as 0.005 ng to 320 ng). Analytical accuracy was excellent with average biases at qPCR inputs representative of patient samples <9.7% across all assays while amplification efficiencies were within ±6% of the median. Assessments of reproducibility and precision were performed by testing 10 prostate cancer RNA samples over multiple instruments, reagent lots, operators, days (precision), and RNA input levels (reproducibility) using appropriately parameterized linear mixed models. The standard deviations for analytical precision and reproducibility were 1.86 and 2.11 GPS units (100-unit scale) respectively. CONCLUSIONS: The Oncotype DX Prostate Cancer Assay, a clinical RT-PCR assay specifically designed for use with prostate needle biopsies, has been analytically validated using very limited RNA inputs. The assay requirements and analytical performance will provide physicians with test results from a robust and reliable assay which will enable improved treatment decisions for men diagnosed with early-stage prostate cancer.

Elevated MDM2 boosts the apoptotic activity of p53-MDM2 binding inhibitors by facilitating MDMX degradation.

The p53 tumor suppressor is a powerful growth suppressive and pro-apoptotic molecule frequently inactivated in human cancer. Many tumors overproduce its negative regulator MDM2, a specific p53 ubiquitin ligase and transcriptional inhibitor, to disable p53 function. Therefore, p53 activation by inhibiting MDM2 has been proposed as a novel strategy for cancer therapy in tumors expressing wild-type p53. Recently developed small-molecule p53-MDM2 binding inhibitors, the nutlins, selectively activate p53 function and induce cell cycle arrest and apoptosis in cancer cells. By stabilizing p53, nutlins also elevate the cellular level of its transcriptional target MDM2. Here, we present evidence that nutlin-induced MDM2 retains its ubiquitin ligase activity and contributes to the anti-tumor activity of p53-MDM2 binding inhibitors by facilitating the degradation of another p53 inhibitor, MDMX. MDM2 and MDMX levels were analyzed in a panel of 12 randomly selected solid tumor cell lines. In the presence of nutlin-3, MDM2 increased in all and MDMX decreased in most of the cell lines. MDMX was resistant to nutlin-induced degradation in 2/12 cell lines. In these cells, MDMX appears to be a major suppressor of the apoptotic response to p53 activation although this effect was only partially p53-dependent. Doxorubicin facilitated MDMX degradation through DNA damage response pathways and restored their sensitivity to nutlin, suggesting that combination therapy may be an effective way to overcome nutlin resistance in cancers with MDMX aberrations.

Bcl-2 is the target of a UV-inducible apoptosis switch and a node for UV signaling.

Sunlight's UVB radiation triggers cell signaling at multiple sites to induce apoptosis. The integration of these signal entry sites is not understood. Here we show that P53 and E2f1 constitute a UV-inducible apoptosis switch. At low-UV doses, wild-type cells resemble the OFF state of an siP53-treated cell, whereas at high-UV doses, the apoptosis frequency transitions to the fully ON behavior of an siE2f1-treated cell. The switch's target is Bcl-2: Rapid Bcl-2 down-regulation in response to UVB-induced DNA photoproducts is lost in P53-deficient cells, but, as for apoptosis, is restored when both P53 and its inhibited target E2f1 are absent. P53's down-regulation of Bcl-2 is mediated entirely through E2f1. Bcl-2 is also down-regulated by a separate pathway triggered by DNA photoproducts in the absence of P53 and E2f1. Four UV pathways terminating on Bcl-2 contribute to apoptosis after UVB irradiation. The apoptosis lost in p53(-/-) is completely restored by siBcl-2, implying that Bcl-2 is a rate-limiting member of this network. These results identify Bcl-2 as an integrator of several UV-induced proapoptotic signals and show that it, in turn, suppresses a direct UV-apoptosis pathway. UV-induced apoptosis requires both UV activation of the direct pathway and a separate UV disinhibition of this pathway through P53-E2f1-Bcl-2.

Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1.

CDK1 is a nonredundant cyclin-dependent kinase (CDK) with an essential role in mitosis, but its multiple functions still are poorly understood at a molecular level. Here we identify a selective small-molecule inhibitor of CDK1 that reversibly arrests human cells at the G(2)/M border of the cell cycle and allows for effective cell synchronization in early mitosis. Inhibition of CDK1 during cell division revealed that its activity is necessary and sufficient for maintaining the mitotic state of the cells, preventing replication origin licensing and premature cytokinesis. Although CDK1 inhibition for up to 24 h is well tolerated, longer exposure to the inhibitor induces apoptosis in tumor cells, suggesting that selective CDK1 inhibitors may have utility in cancer therapy.

Role of E2F1 in apoptosis: a case study in feedback loops.

E2F1 is involved in both cell cycle and apoptosis, but we still fail to understand whether these are distinct E2F1 states and what controls the transition between states. Studies in cell cultures are often contradictory showing opposite effects of E2F1; even further, some phenotypes in animals lacking or overexpressing E2F1 would not be predicted from in vitro findings. In this review, we analyze current literature on the role of E2F1 in apoptosis and use engineering concepts and a systems biology approach to align the overwhelming amount of contradictory data on this gene.

Inactivating E2f1 reverts apoptosis resistance and cancer sensitivity in Trp53-deficient mice.

The E2f1 transcription factor, which regulates genes required for S-phase entry, also induces apoptosis by transcriptional and post-translational mechanisms. As E2f1 is inducible by DNA damage we investigated its importance in vivo in ultraviolet (UV)-induced apoptosis, a protective mechanism that prevents the epidermis from accumulating UV-induced mutations. Contrary to expectation, E2f1-/- mice demonstrated enhanced keratinocyte apoptosis after UVB exposure, whereas apoptosis was suppressed by epidermis-specific overexpression of human E2F1. Apoptosis induced by -radiation was also repressed by E2f1. E2f1-/-;Trp53-/- double knockout mice exhibited the elevated UVB-induced apoptosis of E2f1-/- alone, rather than the profound apoptosis defect seen in Trp53-/- mice, indicating that Trp53 (p53) lies functionally upstream of E2f1. Transfecting E2F1 into E2f1-/-;Trp53-/- primary fibroblasts suppressed UVB-induced apoptosis and this suppression was relieved by Trp53. The double knockout also reverted the abnormal sex ratio and early-onset tumours of Trp53-/- mice. These results imply that E2f1 functions as a suppressor of an apoptosis pathway that is initiated by DNA photoproducts and perhaps genetic abnormalities; p53 relieves this suppression.