Jeffry Simko

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.

Validation of a Cell-Cycle Progression Gene Panel to Improve Risk Stratification in a Contemporary Prostatectomy Cohort

PURPOSE We aimed to validate a previously described genetic risk score, denoted the cell-cycle progression (CCP) score, in predicting contemporary radical prostatectomy (RP) outcomes. METHODS RNA was quantified from paraffin-embedded RP specimens. The CCP score was calculated as average expression of 31 CCP genes, normalized to 15 housekeeper genes. Recurrence was defined as two prostate-specific antigen levels ≥ 0.2 ng/mL or any salvage treatment. Associations between CCP score and recurrence were examined, with adjustment for clinical and pathologic variables using Cox proportional hazards regression and partial likelihood ratio tests. The CCP score was assessed for independent prognostic utility beyond a standard postoperative risk assessment (Cancer of the Prostate Risk Assessment post-Surgical [CAPRA-S] score), and a score combining CAPRA-S and CCP was validated. RESULTS Eighty-two (19.9%) of 413 men experienced recurrence. The hazard ratio (HR) for each unit increase in CCP score (range, -1.62 to 2.16) was 2.1 (95% CI, 1.6 to 2.9); with adjustment for CAPRA-S, the HR was 1.7 (95% CI, 1.3 to 2.4). The score was able to substratify patients with low clinical risk as defined by CAPRA-S ≤ 2 (HR, 2.3; 95% CI, 1.4 to 3.7). Combining the CCP and CAPRA-S improved the concordance index for both the overall cohort and low-risk subset; the combined CAPRA-S + CCP score consistently predicted outcomes across the range of clinical risk. This combined score outperformed both individual scores on decision curve analysis. CONCLUSION The CCP score was validated to have significant prognostic accuracy after controlling for all available clinical and pathologic data. The score may improve accuracy of risk stratification for men with clinically localized prostate cancer, including those with low-risk disease.

Quantitative analysis of prostate metabolites using 1H HR-MAS spectroscopy

A method was developed to quantify prostate metabolite concentrations using 1H high‐resolution magic angle spinning (HR‐MAS) spectroscopy. T1 and T2 relaxation times (in milliseconds) were determined for the major prostate metabolites and an internal TSP standard, and used to optimize the acquisition and repetition times (TRs) at 11.7 T. At 1°C, polyamines (PAs; T1mean = 100 ± 13, T2mean = 30.8 ± 7.4) and citrate (Cit; T1mean = 237 ± 39, T2mean = 68.1 ± 8.2) demonstrated the shortest relaxation times, while taurine (Tau; T1mean = 636 ± 78, T2mean = 331 ± 71) and choline (Cho; T1mean = 608 ± 60, T2mean = 393 ± 81) demonstrated the longest relaxation times. Millimolal metabolite concentrations were calculated for 60 postsurgical tissues using metabolite and TSP peak areas, and the mass of tissue and TSP. Phosphocholine plus glycerophosphocholine (PC+GPC), total choline (tCho), lactate (Lac), and alanine (Ala) concentrations were higher in prostate cancer ([PC+GPC]mean = 9.34 ± 6.43, [tCho]mean = 13.8 ± 7.4, [Lac]mean = 69.8 ± 27.1, [Ala]mean = 12.6 ± 6.8) than in healthy glandular ([PC+GPC]mean = 3.55 ± 1.53, P < 0.01; [tCho]mean = 7.06 ± 2.36, P < 0.01; [Lac]mean = 46.5 ± 17.4, P < 0.01; [Ala]mean = 8.63 ± 4.91, P = 0.051) and healthy stromal tissues ([PC+GPC]mean = 4.34 ± 2.46, P < 0.01; [tCho]mean = 7.04 ± 3.10, P < 0.01; [Lac]mean = 45.1 ± 18.6, P < 0.01; [Ala]mean = 6.80 ± 2.95, P < 0.01), while Cit and PA concentrations were significantly higher in healthy glandular tissues ([Cit]mean = 43.1 ± 21.2, [PAs]mean = 18.5 ± 15.6) than in healthy stromal ([Cit]mean = 16.1 ± 5.6, P < 0.01; [PAs]mean = 3.15 ± 1.81, P < 0.01) and prostate cancer tissues ([Cit]mean = 19.6 ± 12.7, P < 0.01; [PAs]mean = 5.28 ± 5.44, P < 0.01). Serial spectra acquired over 12 hr indicated that the degradation of Cho‐containing metabolites was minimized by acquiring HR‐MAS data at 1°C compared to 20°C. Magn Reson Med, 2006.

Do adenocarcinomas of the prostate with gleason score (GS)≤6 have the potential to metastasize to lymph nodes?

Although rare, there are cases within reported series of men with Gleason score (GS)⩽6 on radical prostatectomies that show pelvic lymph node (LN) metastases. However, there are no studies on whether pelvic LN metastases occur in tumors with GS⩽6 using the International Society of Urological Pathology (ISUP) updated GS system. We performed a search of the radical prostatectomy databases at 4 large academic centers for cases of GS⩽6. Only prostatectomies submitted and embedded in entirety with pelvic LN dissections were included. A combined total of 14,123 cases were identified, of which 22 cases had a positive LN. Histopathologic review of 19 cases (3 cases unavailable for review) showed higher grade than originally reported by the pathologists in all cases. Of the 17 pre-ISUP reviewed cases, 2 were upgraded to 4+3=7 with both cribriform and poorly formed glands. One case was upgraded to 4+3=7 with tertiary pattern 5 displaying cribriform glands, poorly formed glands, and cords of single cells. Eleven cases were upgraded to 3+4=7 with glomeruloid structures and small to large cribriform glands (1 of these also had features of ductal adenocarcinoma). Two cases had tertiary pattern 4 with small cribriform glands. One case had a prominent colloid component that would currently be graded as 4+5=9 because of large cribriform glands and solid sheets of cells within the mucin. Of the 2 post-ISUP cases, 1 demonstrated tertiary pattern 4, and the other showed GS 3+4=7 with irregular cribriform glands. Undergrading is the primary reason for LN positivity with GS⩽6, which has decreased significantly since the adoption of the ISUP grading system in 2005. Of over 14,000 totally embedded radical prostatectomies from multiple institutions, there was not a single case of a GS⩽6 tumor with LN metastases. In contrast to prevailing assumptions, GS⩽6 tumors do not appear to metastasize to LNs. Rather, Gleason pattern 4 or 5, as better defined by the current ISUP updated grading system, is required for metastatic disease.

Evaluation of lactate and alanine as metabolic biomarkers of prostate cancer using 1H HR-MAS spectroscopy of biopsy tissues

The goal of this study was to investigate the use of lactate and alanine as metabolic biomarkers of prostate cancer using 1H high‐resolution magic angle spinning (HR‐MAS) spectroscopy of snap‐frozen transrectal ultrasound (TRUS)‐guided prostate biopsy tissues. A long‐echo‐time rotor‐synchronized Carr‐Purcell‐Meiboom‐Gill (CPMG) sequence including an electronic reference to access in vivo concentrations (ERETIC) standard was used to determine the concentrations of lactate and alanine in 82 benign and 16 malignant biopsies (mean 26.5% ± 17.2% of core). Low concentrations of lactate (0.61 ± 0.28 mmol/kg) and alanine (0.14 ± 0.06 mmol/kg) were observed in benign prostate biopsies, and there was no significant difference between benign predominantly glandular (N = 54) and stromal (N = 28) biopsies between patients with (N = 38) and without (N = 44) a positive clinical biopsy. In biopsies containing prostate cancer there was a highly significant (P < 0.0001) increase in lactate (1.59 ± 0.61 mmol/kg) and alanine (0.26 ± 0.07 mmol/kg), and minimal overlap with lactate concentrations in benign biopsies. This study demonstrates for the first time very low concentrations of lactate and alanine in benign prostate biopsy tissues. The significant increase in the concentration of both lactate and alanine in biopsy tissue containing as little as 5% cancer could be exploited in hyperpolarized 13C spectroscopic imaging (SI) studies of prostate cancer patients. Magn Reson Med 60:510–516, 2008.

Integration of high-resolution array comparative genomic hybridization analysis of chromosome 16q with expression array data refines common regions of loss at 16q23-qter and identifies underlying candidate tumor suppressor genes in prostate cancer.

We have constructed a high-resolution genomic microarray of human chromosome 16q, and used it for comparative genomic hybridization analysis of 16 prostate tumors. We demarcated 10 regions of genomic loss between 16q23.1 and 16qter that occurred in five or more samples. Mining expression array data from four independent studies allowed us to identify 11 genes that were frequently underexpressed in prostate cancer and that co-localized with a region of genomic loss. Quantitative expression analyses of these genes in matched tumor and benign tissue from 13 patients showed that six of these 11 (WWOX, WFDC1, MAF, FOXF1, MVD and the predicted novel transcript Q9H0B8 (NM_031476)) had significant and consistent downregulation in the tumors relative to normal prostate tissue expression making them candidate tumor suppressor genes.

Quantification of choline- and ethanolamine-containing metabolites in human prostate tissues using 1H HR-MAS total correlation spectroscopy.

A fast and quantitative 2D high‐resolution magic angle spinning (HR‐MAS) total correlation spectroscopy (TOCSY) experiment was developed to resolve and quantify the choline‐ and ethanolamine‐containing metabolites in human prostate tissues in ≈1 hr prior to pathologic analysis. At a 40‐ms mixing time, magnetization transfer efficiency constants were empirically determined in solution and used to calculate metabolite concentrations in tissue. Phosphocholine (PC) was observed in 11/15 (73%) cancer tissues but only 6/32 (19%) benign tissues. PC was significantly higher (0.39 ± 0.40 mmol/kg vs. 0.02 ± 0.07 mmol/kg, z = 3.5), while ethanolamine (Eth) was significantly lower in cancer versus benign prostate tissues (1.0 ± 0.8 mmol/kg vs. 2.3 ± 1.9 mmol/kg, z = 3.3). Glycerophosphocholine (GPC) (0.57 ± 0.87 mmol/kg vs. 0.29 ± 0.26 mmol/kg, z = 1.2), phosphoethanolamine (PE) (4.4 ± 2.2 mmol/kg vs. 3.4 ± 2.6 mmol/kg, z = 1.4), and glycerophosphoethanolamine (GPE) (0.54 ± 0.82 mmol/kg vs. 0.15 ± 0.15 mmol/kg, z = 1.8) were higher in cancer versus benign prostate tissues. The ratios of PC/GPC (3.5 ± 4.5 vs. 0.32 ± 1.4, z = 2.6), PC/PE (0.08 ± 0.08 vs. 0.01 ± 0.03, z = 3.5), PE/Eth (16 ± 22 vs. 2.2 ± 2.0, z = 2.4), and GPE/Eth (0.41 ± 0.51 vs. 0.06 ± 0.06, z = 2.6) were also significantly higher in cancer versus benign tissues. All samples were pathologically interpretable following HR‐MAS analysis; however, degradation experiments showed that PC, GPC, PE, and GPE decreased 7.7 ± 2.2%, while Cho+mI and Eth increased 18% in 1 hr at 1°C and a 2250 Hz spin rate. Magn Reson Med 60:33–40, 2008.

PI(3) kinase is associated with a mechanism of immunoresistance in breast and prostate cancer.

Immune escape describes a critical event whereby tumor cells adopt an immunoresistant phenotype to escape adaptive surveillance. We show that expression of a pivotal negative regulator of T-cell function, B7-H1, correlates with PI(3) kinase activation in breast and prostate cancer patients. B7-H1-mediated immunoresistance can be attenuated by inhibitors of the PI(3) kinase pathway, and is dependent on S6K1-mediated translational regulation of B7-H1 protein. Breast and prostate carcinoma cells with activated PI(3) kinase lose the immunoresistant phenotype after treatment with B7-H1 siRNA. Conversely, breast and prostate carcinoma cells with minimal PI(3) kinase activation adopt an immunoresistant phenotype when engineered to overexpress B7-H1 protein. These observations describe a mechanism for immune escape from tumor dormancy in humans that relates to oncogenesis.

Extended Followup and Risk Factors for Disease Reclassification in a Large Active Surveillance Cohort for Localized Prostate Cancer

PURPOSE Active surveillance to manage prostate cancer provides an alternative to immediate treatment in men with low risk prostate cancer. We report updated outcomes from a long-standing active surveillance cohort and factors associated with reclassification. MATERIALS AND METHODS We retrospectively reviewed data on all men enrolled in the active surveillance cohort at our institution with at least 6 months of followup between 1990 and 2013. Surveillance consisted of quarterly prostate specific antigen testing, repeat imaging with transrectal ultrasound at provider discretion and periodic repeat prostate biopsies. Factors associated with repeat biopsy reclassification and local treatment were determined by multivariate Cox proportional hazards regression. We also analyzed the association of prostate specific antigen density and outcomes stratified by prostate size. RESULTS A total of 810 men who consented to participate in the research cohort were followed on active surveillance for a median of 60 months. Of these men 556 (69%) met strict criteria for active surveillance. Five-year overall survival was 98%, treatment-free survival was 60% and biopsy reclassification-free survival was 40%. There were no prostate cancer related deaths. On multivariate analysis prostate specific antigen density was positively associated with the risk of biopsy reclassification and treatment while the number of biopsies and time between biopsies were inversely associated with the 2 outcomes (each p <0.01). When stratified by prostate volume, prostate specific antigen density remained significantly associated with biopsy reclassification for all strata but prostate specific antigen density was only significantly associated with treatment in men with a smaller prostate. CONCLUSIONS Significant prostate cancer related morbidity and mortality remained rare at intermediate followup. Prostate specific antigen density was independently associated with biopsy reclassification and treatment while on active surveillance.

Incomplete inhibition of phosphorylation of 4E-BP1 as a mechanism of primary resistance to ATP-competitive mTOR inhibitors

The mammalian target of rapamycin (mTOR) regulates cell growth by integrating nutrient and growth factor signaling and is strongly implicated in cancer. But mTOR is not an oncogene, and which tumors will be resistant or sensitive to new adenosine triphosphate (ATP) competitive mTOR inhibitors now in clinical trials remains unknown. We screened a panel of over 600 human cancer cell lines to identify markers of resistance and sensitivity to the mTOR inhibitor PP242. RAS and phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutations were the most significant genetic markers for resistance and sensitivity to PP242, respectively; colon origin was the most significant marker for resistance based on tissue type. Among colon cancer cell lines, those with KRAS mutations were most resistant to PP242, whereas those without KRAS mutations most sensitive. Surprisingly, cell lines with co-mutation of PIK3CA and KRAS had intermediate sensitivity. Immunoblot analysis of the signaling targets downstream of mTOR revealed that the degree of cellular growth inhibition induced by PP242 was correlated with inhibition of phosphorylation of the translational repressor eIF4E-binding protein 1 (4E-BP1), but not ribosomal protein S6 (rpS6). In a tumor growth inhibition trial of PP242 in patient-derived colon cancer xenografts, resistance to PP242-induced inhibition of 4E-BP1 phosphorylation and xenograft growth was again observed in KRAS mutant tumors without PIK3CA co-mutation, compared with KRAS wild-type controls. We show that, in the absence of PIK3CA co-mutation, KRAS mutations are associated with resistance to PP242 and that this is specifically linked to changes in the level of phosphorylation of 4E-BP1.