Rapid on-site evaluation in percutaneous core needle biopsies of renal masses: Cytologic-histologic concordance and diagnostic challenges.

BACKGROUND: Image-guided fine needle aspirations (FNAs) and core needle biopsies (CNBs) play the critical role in diagnosis of renal lesions. Rapid on-site evaluation (ROSE) can potentially increase the adequacy rate and improve diagnostic yield, while providing additional information for rapid clinical decisions. The aim of this study is to evaluate the diagnostic utility of ROSE in obtaining adequate tissue for diagnosis of renal lesions in our institution. METHODS: We retrospectively reviewed all percutaneous renal CNB cases with available ROSE interpretations for a 11-year period. The ROSE interpretations and CNB diagnoses was compared and the concordance rate was calculated accordingly. The discrepant cases were re-reviewed and the possible causes for discrepancy were analyzed. RESULTS: A total of 189 cases were identified. Definitive diagnoses were rendered in 164 (87%) cases on the final CNBs, including primary renal lesions in 151 cases and metastatic malignancies in 13 cases. At the time of ROSE, samples were deemed to be adequate in the majority of cases (83%). The calculated concordance rate between ROSE interpretations and CNB final diagnoses was 84.6%. Sampling issue and scant tumor cells were the main causes for the discordance between ROSE interpretations and CNB diagnoses. CONCLUSION: Our study showed a relatively high-concordance rate of 84.6% between ROSE interpretations and CNB final diagnoses, suggesting that ROSE is a valuable tool for procurement of adequate renal CNB samples for diagnosis.

Impact of an NCCN-Compliant Multidisciplinary Conference on Treatment Decisions for Localized Prostate Cancer.

BACKGROUND: We sought to investigate the impact of an NCCN-compliant multidisciplinary conference on treatment decisions of patients with localized prostate cancer. METHODS: A retrospective review of our quality assurance localized prostate cancer database was performed. All patients with localized prostate cancer who sought a second opinion at Roswell Park Comprehensive Cancer Center between 2009 and 2019 were presented to the multidisciplinary Localized Prostate Cancer Conference (LPCC) that includes urologists, radiation oncologists, pathologists, and patient advocates. Multivariable regression models were fit to evaluate variables associated with concordance between community recommendations, LPCC recommendations, and treatment received by patients. RESULTS: A total of 1,164 patients were identified, of whom 26% had NCCN very low-/low-risk, 27% had favorable intermediate-risk, 25% had unfavorable intermediate-risk, and 22% had high-/very high-risk prostate cancer. Pathology changed in 11% of patients after genitourinary pathologist review, which caused disease reclassification in 9%. Concordance between community and LPCC recommendations occurred in 78%, with lowest concordance for androgen deprivation therapy (21%) and radiotherapy (53%). Concordance between community recommendations and treatment received occurred in 65%, with lowest concordance for androgen deprivation therapy and radiotherapy; among those who were recommended radiotherapy as the only option by their community urologist, only 26% received it. Concordance between LPCC recommendations and treatment received occurred in 92%. CONCLUSIONS: Community recommendations differed from the multidisciplinary NCCN-compliant recommendations in 22% of patients, primarily for radiotherapy. Multidisciplinary recommendations matched the treatment received in 92% of patients compared with 65% for community recommendations.

FABP5 Inhibition against PTEN-Mutant Therapy Resistant Prostate Cancer.

Resistance to standard of care taxane and androgen deprivation therapy (ADT) causes the vast majority of prostate cancer (PC) deaths worldwide. We have developed RapidCaP, an autochthonous genetically engineered mouse model of PC. It is driven by the loss of PTEN and p53, the most common driver events in PC patients with life-threatening diseases. As in human ADT, surgical castration of RapidCaP animals invariably results in disease relapse and death from the metastatic disease burden. Fatty Acid Binding Proteins (FABPs) are a large family of signaling lipid carriers. They have been suggested as drivers of multiple cancer types. Here we combine analysis of primary cancer cells from RapidCaP (RCaP cells) with large-scale patient datasets to show that among the 10 FABP paralogs, FABP5 is the PC-relevant target. Next, we show that RCaP cells are uniquely insensitive to both ADT and taxane treatment compared to a panel of human PC cell lines. Yet, they share an exquisite sensitivity to the small-molecule FABP5 inhibitor SBFI-103. We show that SBFI-103 is well tolerated and can strongly eliminate RCaP tumor cells in vivo. This provides a pre-clinical platform to fight incurable PC and suggests an important role for FABP5 in PTEN-deficient PC.

Utility of rapid on-site evaluation in procurement of adrenal gland tissue biopsies.

BACKGROUND: While the number of adrenal biopsies has increased due to more "incidentalomas" were detected by widespread use of imaging studies, there have been very limited studies to evaluate the utility of rapid on-site evaluation (ROSE) in obtaining adequate core needle biopsy (CNB) tissue for diagnosis of adrenal lesions. METHODS: We retrospectively reviewed all percutaneous adrenal CNB cases with available ROSE for a 12-year period in our institute in order to assess the usefulness of ROSE in adrenal CNB sampling. RESULTS: A total of 83 cases were identified in our database. The majority of cases (52/83, 63%) were diagnosed as metastatic malignancies with the lung being the most common primary site. Adrenal hyperplasia/adenoma was the most common primary adrenal lesion. The concordance between the ROSE interpretations and CNB final diagnoses is 80%. The interpretation errors accounted for majority (11/17, 65%) of the discordant cases. CONCLUSION: ROSE assessment during adrenal CNB procedures is a helpful tool for obtaining adequate diagnostic tissue. Pathologists should be familiar with adrenal cytology in order to reduce interpretation errors at ROSE.

Case Report: Immune Checkpoint Inhibitor-Induced Exuberant Tumor Inflammation With Accelerated Clinical Deterioration in Metastatic Renal Cell Carcinoma.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy. Nivolumab, an anti-PD-1 monoclonal antibody, markedly improved overall survival in advanced renal cell carcinoma (RCC). However, ICIs can rarely trigger massive inflammation, a phenomenon characterized by rapid acceleration in radiographic tumor growth, the mechanisms underlying which are largely unknown. We report three patients with metastatic RCC who experienced rapid radiographic progression and clinical deterioration following treatment with nivolumab. However, histological analysis revealed no viable cancer despite the evidence of radiological progression. Instead, extensive necrosis and lymphohistiocytic infiltration were noted, as described previously in patients with ICI-induced pseudoprogression. Based on these observations, we postulate that exuberant antitumor inflammatory responses may contribute to adverse clinical outcomes in some patients with ICI-induced radiographic progression. Prospective studies incorporating tumor biopsies may shed more light on this rare phenomenon.

Differential Associations of SLCO Transporters with Prostate Cancer Aggressiveness between African Americans and European Americans.

BACKGROUND: Androgen receptor signaling is crucial to prostate cancer aggressiveness. Members of the solute carrier family of the organic anion transporting peptides (SLCO) are potential regulators of androgen availability in prostate tissue. It remains unknown whether genetic variations in SLCOs contribute to the differences in prostate cancer aggressiveness in African Americans (AA) and European Americans (EA). METHODS: SNPs in 11 SLCO members were selected, with addition of 139 potentially functional SNPs and 128 ancestry informative markers. A total of 1,045 SNPs were genotyped and analyzed in 993 AAs and 1,057 EAs from the North Carolina-Louisiana Prostate Cancer Project. Expression and cellular localization of SLCOs were examined using qRT-PCR, IHC, and in situ RNA hybridization in independent sets of prostate cancer cases. RESULTS: Significant associations with prostate cancer characteristics were found for SNPs in SLCO2A1 and SLCO5A1. The associations differed by race (P interaction < 0.05). SNPs in SLCO2A1 were associated with reduced tumor aggressiveness and low Gleason score in AAs; whereas, SNPs in SLCO5A1 were associated with high clinical stage in EAs. In prostate tissue, SLCO2A1 and SLCO5A1 were the most expressed SLCOs at the mRNA level and were expressed predominantly in prostate endothelial and epithelial cells at the protein level, respectively. CONCLUSIONS: SLCO2A1 and SLCO5A1 play important but different roles in prostate cancer aggressiveness in AAs versus EAs. IMPACT: The finding calls for consideration of racial differences in biomarker studies of prostate cancer and for investigations on functions of SLCO2A1 and SLCO5A1 in prostate cancer.

Patient derived models of bladder cancer enrich the signal of the tumor cell transcriptome facilitating the analysis of the tumor cell compartment.

The evolving paradigm of the molecular classification of bladder cancer requires models that represent the classifications with less heterogeneity. Robust transcriptome based molecular classifications are essential to address tumor heterogeneity. Patient derived models (PDMs) are a powerful preclinical tool to study specific tumor compartments. We tested if the consensus molecular subtype analysis was applicable to PDMs and evaluated the tumor compartment each model represents. PDMs derived from surgical specimens were established as xenografts (PDX), organoids (PDO), and spheroids (PDS). The surgical specimens and PDMs were molecularly characterized by RNA sequencing. PDMs that were established in immune deficient mice or in vitro significantly downregulated transcripts related to the immune and stromal compartments compared to the surgical specimens. However, PDMs upregulate a patient-specific bladder cancer cell signal which allowed for analysis of cancer cell pathways independent of the tumor microenvironment. Based on transcriptomic signatures, PDMs are more similar to their surgical specimen than the model type; indicating that the PDMs retained unique features of the tumor from which the PDM was derived. When comparing models, PDX models were the most similar to the surgical specimen, while PDO and PDS models were most similar to each other. When the consensus molecular subtype classification system was applied to both the surgical samples and the three PDMs, good concordance was found between all samples indicating that this system of classification can be applied to PDO and PDS models. PDMs reduce tumor heterogeneity and allow analysis of tumor cells while maintaining the gene expression profile representative of the original tumor.

Pharmacological polyamine catabolism upregulation with methionine salvage pathway inhibition as an effective prostate cancer therapy.

Prostatic luminal epithelial cells secrete high levels of acetylated polyamines into the prostatic lumen, sensitizing them to perturbations of connected metabolic pathways. Enhanced flux is driven by spermidine/spermine N1-acetyltransferase (SSAT) activity, which acetylates polyamines leading to their secretion and drives biosynthetic demand. The methionine salvage pathway recycles one-carbon units lost to polyamine biosynthesis to the methionine cycle to overcome stress. Prostate cancer (CaP) relies on methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme, to relieve strain. Here, we show that inhibition of MTAP alongside SSAT upregulation is synergistic in androgen sensitive and castration recurrent CaP models in vitro and in vivo. The combination treatment increases apoptosis in radical prostatectomy ex vivo explant samples. This unique high metabolic flux through polyamine biosynthesis and connected one carbon metabolism in CaP creates a metabolic dependency. Enhancing this flux while simultaneously targeting this dependency in prostate cancer results in an effective therapeutic approach potentially translatable to the clinic.

Tumor-associated macrophage expression of interferon regulatory Factor-8 (IRF8) is a predictor of progression and patient survival in renal cell carcinoma.

Tumor-associated macrophages have been well-characterized in solid malignancies, including renal cell carcinoma and generally correlate with poor prognosis. However, the molecular mechanisms which govern intratumoral macrophage behavior and patient outcome are unclear. Here, we investigated whether alterations in macrophage expression of the transcriptional regulator for myeloid commitment and function, interferon regulatory factor-8 (IRF8), could predict survival of clear cell renal cell carcinoma patients. Transcriptional analysis of publicly available data revealed elevated IRF8 expression was associated with prolonged disease-free survival. Evaluation of protein expression within histologic sections of primary clear cell renal cell carcinoma patient samples showed intensity of IRF8 by CD68+ macrophages correlated inversely with stage. Survival outcomes of patients with primary or metastatic disease could be stratified on the basis of IRF8 levels by macrophages. Patients with high levels of IRF8 expression within metastatic sites had prolonged overall survival (log-rank P < 0.01, HR = 0.44, 95% C.I.: 0.23-0.84) compared to patients with low levels of IRF8 expression. When patient cohorts were further separated based on macrophage infiltration within metastatic lesions, patients with a macrophagelo IRF8hi profile had a more than 10 year increase in median overall survival compared to patients with a macrophagelo IRF8lo profile (log-rank, P < 0.001). In summary, we report that macrophage expression of IRF8 is inversely correlated with tumor mass and directly related to survival outcome. These findings support the utilization of IRF8 expression by macrophages to predict patient outcome, which may have important implications for guiding treatment decisions for renal cell carcinoma patients with metastatic disease.

Protein Kinase N1 control of androgen-responsive serum response factor action provides rationale for novel prostate cancer treatment strategy.

Sustained reliance on androgen receptor (AR) after failure of AR-targeting androgen deprivation therapy (ADT) prevents effective treatment of castration-recurrent (CR) prostate cancer (CaP). Interfering with the molecular machinery by which AR drives CaP progression may be an alternative therapeutic strategy but its feasibility remains to be tested. Here, we explore targeting the mechanism by which AR, via RhoA, conveys androgen-responsiveness to serum response factor (SRF), which controls aggressive CaP behavior and is maintained in CR-CaP. Following a siRNA screen and candidate gene approach, RNA-Seq studies confirmed that the RhoA effector Protein Kinase N1 (PKN1) transduces androgen-responsiveness to SRF. Androgen treatment induced SRF-PKN1 interaction, and PKN1 knockdown or overexpression severely impaired or stimulated, respectively, androgen regulation of SRF target genes. PKN1 overexpression occurred during clinical CR-CaP progression, and hastened CaP growth and shortened CR-CaP survival in orthotopic CaP xenografts. PKN1's effects on SRF relied on its kinase domain. The multikinase inhibitor lestaurtinib inhibited PKN1 action and preferentially affected androgen regulation of SRF over direct AR target genes. In a CR-CaP patient-derived xenograft, expression of SRF target genes was maintained while AR target gene expression declined and proliferative gene expression increased. PKN1 inhibition decreased viability of CaP cells before and after ADT. In patient-derived CaP explants, lestaurtinib increased AR target gene expression but did not significantly alter SRF target gene or proliferative gene expression. These results provide proof-of-principle for selective forms of ADT that preferentially target different fractions of AR's transcriptional output to inhibit CaP growth.

Accurate Quantification of Residual Cancer Cells in Pelvic Washing Reveals Association with Cancer Recurrence Following Robot-Assisted Radical Cystectomy.

PURPOSE: Bladder cancer recurrence following cystectomy remains a significant cause of bladder cancer specific mortality. Residual cancer cells contribute to cancer recurrence due to tumor spillage or undetectable preexisting micrometastatic tumor clones. We detected and quantified residual cancer cells in pelvic washing using ultradeep targeted sequencing. We compared the levels of residual cancer cells with clinical variables and cancer recurrence. MATERIALS AND METHODS: The primary tumor specimen was available in 17 patients who underwent robot-assisted radical cystectomy. All tumors had negative surgical margins. Pelvic washes and blood were collected intraoperatively before and after robot-assisted radical cystectomy, after pelvic lymph node dissection and in the suction fluid collected during the procedure. Two-step sequencing, including whole exome sequencing followed by ultradeep targeted sequencing (× greater than 50,000), was done to quantify residual cancer cells in each sample. Eight patients were excluded from study due to sample quality issues. The final analysis cohort comprised 9 patients. The residual cancer cell level was quantified for each sample as the relative cancer cell fraction and compared between time points. The peak relative cancer cell fraction of each patient was correlated with clinical and pathological variables. RESULTS: Residual cancer cells were detected in approximately half of the pelvic washing specimens during or after but not before robot-assisted radical cystectomy. Higher residual cancer cell levels were associated with aggressive variant histology and cancer recurrence. Verifying the feasibility of using residual cancer cells as a novel biomarker for recurrence requires larger cohorts. CONCLUSIONS: Detection of residual cancer cells in intraoperative peritoneal washes of patients with bladder cancer who undergo radical cystectomy may represent a robust biomarker of tumor aggressiveness and metastatic potential.

Adrenal androgens rescue prostatic dihydrotestosterone production and growth of prostate cancer cells after castration.

Adrenal androgens dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) are potential substrates for intracrine production of testosterone (T) and dihydrotestosterone (DHT), or directly to DHT, by prostate cancer (PCa) cells. Production of DHT from DHEAS and DHEA, and the role of steroid sulfatase (STS), were evaluated ex vivo using fresh human prostate tissue and in vitro using human PCa cell lines. STS was expressed in benign prostate tissue and PCa tissue. DHEAS at a physiological concentration was converted to DHT in prostate tissue and PCa cell lines, which was STS-dependent. DHEAS activation of androgen receptor (AR) and stimulation of PCa cell growth were STS-dependent. DHEA at a physiological concentration was not converted to DHT ex vivo and in vitro, but stimulated in vivo tumor growth of the human PCa cell line, VCaP, in castrated mice. The findings suggest that targeting metabolism of DHEAS and DHEA may enhance androgen deprivation therapy.

Inhibition of EZH2 induces NK cell-mediated differentiation and death in muscle-invasive bladder cancer.

Lysine-specific demethylase 6A (KDM6A) and members of the Switch/Sucrose Non-Fermentable (SWI/SNF) family are known to counteract the activity of Enhancer of Zeste Homolog 2 (EZH2), which is often overexpressed and is associated with poor prognosis in muscle-invasive bladder cancer. Here we provide evidence that alterations in chromatin modifying enzymes, including KDM6A and members of the SWI/SNF complex, are frequent in muscle-invasive bladder cancer. We exploit the loss of function mutations in KDM6A and SWI/SNF complex to make bladder cancer cells susceptible to EZH2-based epigenetic therapy that activates an immune response to drive tumor cell differentiation and death. We reveal a novel mechanism of action of EZH2 inhibition, alone and in combination with cisplatin, which induces immune signaling with the largest changes observed in interferon gamma (IFN-γ). This upregulation is a result of activated natural killer (NK) signaling as demonstrated by the increase in NK cell-associated genes MIP-1α, ICAM1, ICAM2, and CD86 in xenografts treated with EZH2 inhibitors. Conversely, EZH2 inhibition results in decreased expression of pluripotency markers, ALDH2 and CK5, and increased cell death. Our results reveal a novel sensitivity of muscle-invasive bladder cancer cells with KMD6A and SWI/SNF mutations to EZH2 inhibition alone and in combination with cisplatin. This sensitivity is mediated through increased NK cell-related signaling resulting in tumor cell differentiation and cell death.

Proteomic Analysis of Charcoal-Stripped Fetal Bovine Serum Reveals Changes in the Insulin-like Growth Factor Signaling Pathway.

Charcoal-stripped fetal bovine serum (CS-FBS) is commonly used to study androgen responsiveness and androgen metabolism in cultured prostate cancer (CaP) cells. Switching CaP cells from FBS to CS-FBS may reduce the activity of androgen receptor (AR), inhibit cell proliferation, or modulate intracellular androgen metabolism. The removal of proteins by charcoal stripping may cause changes in biological functions and has not yet been investigated. Here we profiled proteins in FBS and CS-FBS using an ion-current-based quantitative platform consisting of reproducible surfactant-aided precipitation/on-pellet digestion, long-column nanoliquid chromatography separation, and ion-current-based analysis. A total of 143 proteins were identified in FBS, among which 14 proteins including insulin-like growth factor 2 (IGF-2) and IGF binding protein (IGFBP)-2 and -6 were reduced in CS-FBS. IGF-1 receptor (IGF1R) and insulin receptor were sensitized to IGFs in CS-FBS. IGF-1 and IGF-2 stimulation fully compensated for the loss of AR activity to maintain cell growth in CS-FBS. Endogenous production of IGF and IGFBPs was verified in CaP cells and clinical CaP specimens. This study provided the most comprehensive protein profiles of FBS and CS-FBS and offered an opportunity to identify new protein regulators and signaling pathways that regulate AR activity, androgen metabolism, and proliferation of CaP cells.

Transcriptional changes associated with in vivo growth of muscle-invasive bladder cancer cell lines in nude mice.

Cancer cells set in motion transcriptomic programs allowing for adaptation and growth in immunocompromised mice to form xenografts, a frequently used tool in cancer research. 2D cultures may not be representative of tumors growing in a complex host microenvironment. This can result in different responses to the same agent tested in vitro and in vivo which impedes the process of developing novel therapeutics. Understanding the transition cells undergo from 2D cell culture to a 3D host microenvironment will help in developing and choosing appropriate models for pre-clinical studies. Our study characterized the transcriptome of a three frequently used muscle-invasive bladder cancer cell lines HT1376, T24 and UM-UC-3 grown in culture and xenografts in nude mice. We found that bladder cancer cells undergo few transcriptomic changes when transitioned from 2D cell culture to xenografts in nude mice. UM-UC-3 cells have the least transcriptomic alterations followed by T24 and HT1376 cells. Respective xenografts cluster with their parental cell lines rather than other xenografts or cell lines. We applied established bladder cancer molecular subtypes to our data and found that UM-UC-3, containing the least transcriptomic alterations, most closely resembled the basal-like molecular subtype of bladder cancer. HT1376 and T24 have mixed basal and luminal molecular signatures. Our studies suggest this subset of bladder cancer cell lines and derived xenografts maintain similar transcriptomic profiles in both 2D culture and 3D xenografts and can be used interchangeably in pre-clinical studies.

Inhibition of dihydrotestosterone synthesis in prostate cancer by combined frontdoor and backdoor pathway blockade.

Androgen deprivation therapy (ADT) is palliative and prostate cancer (CaP) recurs as lethal castration-recurrent/resistant CaP (CRPC). One mechanism that provides CaP resistance to ADT is primary backdoor androgen metabolism, which uses up to four 3α-oxidoreductases to convert 5α-androstane-3α,17ß-diol (DIOL) to dihydrotestosterone (DHT). The goal was to determine whether inhibition of 3α-oxidoreductase activity decreased conversion of DIOL to DHT. Protein sequence analysis showed that the four 3α-oxidoreductases have identical catalytic amino acid residues. Mass spectrometry data showed combined treatment using catalytically inactive 3α-oxidoreductase mutants and the 5α-reductase inhibitor, dutasteride, decreased DHT levels in CaP cells better than dutasteride alone. Combined blockade of frontdoor and backdoor pathways of DHT synthesis provides a therapeutic strategy to inhibit CRPC development and growth.

Akt activation by Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in ovarian cancer cells.

Hyperactivation of Akt is associated with oncogenic changes in the growth, survival, and chemoresistance of cancer cells. The PI3K/phosphoinositide-dependent kinase (PDK) 1 pathway represents the canonical mechanism for phosphorylation of Akt at its primary activation site, Thr-308. We observed that Ca2+/calmodulin (CaM)-dependent protein kinase kinase 2 (ß) (CaMKK2) is highly expressed in high-grade serous ovarian cancer, and we investigated its role in Akt activation in ovarian cancer (OVCa) cell lines (OVCAR-3, SKOV-3, and Caov-3). Knockdown or pharmacological inhibition of CaMKK2 produced phenotypes expected of Akt inhibition, including reductions in cell growth and cell viability and in the regulation of Akt downstream targets involved in G1/S transition and apoptosis. CaMKK2 knockdown or inhibition decreased Akt phosphorylation at Thr-308 and Ser-473 to extents similar to those of PDK1 knockdown or PI3K inhibition. Combined CaMKK2 and PDK1 knockdown or CaMKK and PI3K inhibition, respectively, produced additive effects on p-Akt and cell growth, consistent with direct Akt phosphorylation by CaMKK2. This conclusion was supported by the absence of effects of CaMKK2 knockdown/inhibition on alternative means of activating Akt via p-Akt Thr-450, p-PDK1 Ser-241, or p-IRS1 Ser-636/639. Recombinant CaMKK2 directly activated recombinant Akt by phosphorylation at Thr-308 in a Ca2+/CaM-dependent manner. In OVCa cells, p-Akt Thr-308 was significantly inhibited by intracellular Ca2+i chelation or CaM inhibition. Ionomycin-induced Ca2+ influx promoted p-Akt, an effect blocked by PDK1, and/or CaMKK2, siRNAs, and by PI3K and/or CaMKK inhibitors. CaMKK2 knockdown potentiated the effects of the chemotherapeutic drugs carboplatin and PX-866 to reduce proliferation and survival of OVCa cells.

Blinded review of archival radical prostatectomy specimens supports that contemporary Gleason score 6 prostate cancer lacks metastatic potential.

BACKGROUND: Retrospective identification of Gleason pattern 4 in metastatic Gleason score 3 + 3 = 6 (GS6) radical prostatectomy (RP) specimens has suggested true GS6 prostate cancer (CaP) lacks metastatic potential. However, pathologist awareness of study design and metastasis outcomes at the time of RP review might have introduced upgrading bias. We used pathologist-blinded methodology for unbiased characterization of metastasis rates for contemporarily defined pathologic GS6 (pGS6) CaP. METHODS: An institutional RP database was queried to identify pGS6 patients with metastasis or concern for micrometastasis based on: 1) biochemical failure (BF) despite negative surgical margins or 2) incomplete biochemical response to salvage/adjuvant radiation. RP specimens were regraded independently by two genitourinary pathologists blinded to study aims or clinical outcomes. Additional blinding was performed by random inclusion of pGS6 control specimens from BF-free patients. Only upgrading identified independently by both pathologists was considered. RESULTS: Among 451 pGS6 patients identified, none had synchronous lymph node metastases and 43/451 (10%) suffered BF. Two patients (0.4%) developed metachronous metastasis during a 110-month median follow-up for BF patients. Both metastatic cases had Gleason pattern 4 on blinded RP review, as did 88% of cases with concern for micrometastasis versus 38% of control cases (P = 0.02). All BF patients (29/29) undergoing postoperative radiation had a complete biochemical response or Gleason pattern 4 on blinded RP review. CONCLUSIONS: Unbiased pathologist review of archival RP specimens supports absent metastatic potential for contemporarily defined GS6 CaP. Reduced postoperative monitoring is appropriate for pGS6, but may require pathology review to confirm absent Gleason pattern 4.

Exome Sequencing of African-American Prostate Cancer Reveals Loss-of-Function ERF Mutations.

African-American men have the highest incidence of and mortality from prostate cancer. Whether a biological basis exists for this disparity remains unclear. Exome sequencing (n = 102) and targeted validation (n = 90) of localized primary hormone-naïve prostate cancer in African-American men identified several gene mutations not previously observed in this context, including recurrent loss-of-function mutations in ERF, an ETS transcriptional repressor, in 5% of cases. Analysis of existing prostate cancer cohorts revealed ERF deletions in 3% of primary prostate cancers and mutations or deletions in ERF in 3% to 5% of lethal castration-resistant prostate cancers. Knockdown of ERF confers increased anchorage-independent growth and generates a gene expression signature associated with oncogenic ETS activation and androgen signaling. Together, these results suggest that ERF is a prostate cancer tumor-suppressor gene. More generally, our findings support the application of systematic cancer genomic characterization in settings of broader ancestral diversity to enhance discovery and, eventually, therapeutic applications.Significance: Systematic genomic sequencing of prostate cancer in African-American men revealed new insights into prostate cancer, including the identification of ERF as a prostate cancer gene; somatic copy-number alteration differences; and uncommon PIK3CA and PTEN alterations. This study highlights the importance of inclusion of underrepresented minorities in cancer sequencing studies. Cancer Discov; 7(9); 973-83. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 920.

Intratumoral and Intertumoral Genomic Heterogeneity of Multifocal Localized Prostate Cancer Impacts Molecular Classifications and Genomic Prognosticators.

BACKGROUND: Next-generation sequencing is revealing genomic heterogeneity in localized prostate cancer (CaP). Incomplete sampling of CaP multiclonality has limited the implications for molecular subtyping, stratification, and systemic treatment. OBJECTIVE: To determine the impact of genomic and transcriptomic diversity within and among intraprostatic CaP foci on CaP molecular taxonomy, predictors of progression, and actionable therapeutic targets. DESIGN, SETTING, AND PARTICIPANTS: Four consecutive patients with clinically localized National Comprehensive Cancer Network intermediate- or high-risk CaP who did not receive neoadjuvant therapy underwent radical prostatectomy at Roswell Park Cancer Institute in June-July 2014. Presurgical information on CaP content and a customized tissue procurement procedure were used to isolate nonmicroscopic and noncontiguous CaP foci in radical prostatectomy specimens. Three cores were obtained from the index lesion and one core from smaller lesions. RNA and DNA were extracted simultaneously from 26 cores with ≥90% CaP content and analyzed using whole-exome sequencing, single-nucleotide polymorphism arrays, and RNA sequencing. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Somatic mutations, copy number alternations, gene expression, gene fusions, and phylogeny were defined. The impact of genomic alterations on CaP molecular classification, gene sets measured in Oncotype DX, Prolaris, and Decipher assays, and androgen receptor activity among CaP cores was determined. RESULTS AND LIMITATIONS: There was considerable variability in genomic alterations among CaP cores, and between RNA- and DNA-based platforms. Heterogeneity was found in molecular grouping of individual CaP foci and the activity of gene sets underlying the assays for risk stratification and androgen receptor activity, and was validated in independent genomic data sets. Determination of the implications for clinical decision-making requires follow-up studies. CONCLUSIONS: Genomic make-up varies widely among CaP foci, so care should be taken when making treatment decisions based on a single biopsy or index lesions. PATIENT SUMMARY: We examined the molecular composition of individual cancers in a patient's prostate. We found a lot of genetic diversity among these cancers, and concluded that information from a single cancer biopsy is not sufficient to guide treatment decisions.