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Genomic studies have demonstrated a high level of intra-tumoral heterogeneity in prostate cancer. There is strong evidence suggesting that individual tumor foci can arise as genetically distinct, clonally independent lesions. However, recent studies have also demonstrated that adjacent Gleason pattern (GP) 3 and GP4 lesions can originate from the same clone but follow divergent genetic and morphologic evolution. The clonal relationship of adjacent GP3 and GP5 lesions has thus far not been investigated. Here we analyzed a cohort of 14 cases-11 biopsy and 3 radical prostatectomy specimens-with a Gleason score of 3 + 5 = 8 or 5 + 3 = 8 present in the same biopsy or in a single dominant tumor nodule at radical prostatectomy. Clonal and subclonal relationships between GP3 and GP5 lesions were assessed using genetically validated immunohistochemical assays for ERG, PTEN, and P53. 9/14 (64%) cases showed ERG reactivity in both GP3 and GP5 lesions. Only 1/14 (7%) cases showed a discordant pattern with ERG staining present only in GP3. PTEN expression was lost in 2/14 (14%) cases with perfect concordance between GP5 and GP3. P53 nuclear reactivity was present in 1/14 (7%) case in both GP5 and GP3. This study provides first evidence that the majority of adjacent GP3 and GP5 lesions share driver alterations and are clonally related. In addition, we observed a lower-than-expected rate of PTEN loss in GP5 in the context of Gleason score 3 + 5 = 8 or 5 + 3 = 8 tumors.

GPNMB (glycoprotein nonmetastatic B) and other TFE3/TFEB transcriptional targets have been proposed as markers for microphthalmia (MiT) translocation renal cell carcinomas (tRCCs). We recently demonstrated that constitutive mTORC1 activation via TSC1/2 loss leads to increased activity of TFE3/TFEB, suggesting that the pathogenesis and molecular markers for tRCCs and TSC1/2-associated tumors may be overlapping. We examined GPNMB expression in human kidney and angiomyolipoma (AML) cell lines with TSC2 and/or TFE3/TFEB loss produced using CRISPR-Cas9 genome editing as well as in a mouse model of Tsc2 inactivation-driven renal tumorigenesis. Using an automated immunohistochemistry (IHC) assay for GPNMB, digital image analysis was employed to quantitatively score expression in clear cell RCC (ccRCC, n = 87), papillary RCC (papRCC, n = 53), chromophobe RCC (chRCC, n = 34), oncocytoma (n = 4), TFE3- or TFEB-driven tRCC (n = 56), eosinophilic solid and cystic RCC (ESC, n = 6), eosinophilic vacuolated tumor (EVT, n = 4), and low-grade oncocytic tumor (LOT, n = 3), as well as AML (n = 29) and perivascular epithelioid cell tumors (PEComas, n = 8). In cell lines, GPNMB was upregulated following TSC2 loss in a MiT/TFE- and mTORC1-dependent fashion. Renal tumors in Tsc2

Supplementary Figure S1: Metastasis-free survival of surgically-treated patients stratified by ATM status on immunohistochemistry Supplementary Figure S2: ATM immunostaining of primary and metastatic tumor from case ATM44.

Purpose:The ATM (ataxia telangiectasia mutated) gene is mutated in a subset of prostate cancers, and ATM mutation may confer specific therapeutic vulnerabilities, although ATM-deficient prostate cancers have not been well-characterized.Experimental Design:We genetically validated a clinical grade IHC assay to detect ATM protein loss and examined the frequency of ATM loss among tumors with pathogenic germline ATM mutations and genetically unselected primary prostate carcinomas using tissue microarrays (TMAs). Immunostaining results were correlated with targeted somatic genomic sequencing and clinical outcomes.Results:ATM protein loss was found in 13% (7/52) of primary Gleason pattern 5 cancers with available sequencing data and was 100% sensitive for biallelic ATM inactivation. In a separate cohort with pathogenic germline ATM mutations, 74% (14/19) had ATM protein loss of which 70% (7/10) of evaluable cases had genomic evidence of biallelic inactivation, compared with zero of four of cases with intact ATM expression. By TMA screening, ATM loss was identified in 3% (25/831) of evaluable primary tumors, more commonly in grade group 5 (17/181; 9%) compared with all other grades (8/650; 1%; P < 0.0001). Of those with available sequencing, 80% (4/5) with homogeneous ATM protein loss and 50% (6/12) with heterogeneous ATM protein loss had detectable pathogenic ATM alterations. In surgically treated patients, ATM loss was not significantly associated with clinical outcomes in random-effects Cox models after adjusting for clinicopathologic variables.Conclusions:ATM loss is enriched among high-grade prostate cancers. Optimal evaluation of ATM status requires both genomic and IHC studies and will guide development of molecularly targeted therapies.

Neoadjuvant immune checkpoint blockade represents a novel approach for potentially decreasing the risk of recurrence in patients with nonmetastatic renal cell carcinoma (RCC). In this early phase clincal tiral, we evaluated the safety and tolerability of neoadjuvant treatment with the programmed cell death protein 1 (PD-1) inhibitor nivolumab in patients with nonmetastatic high-risk RCC. Nonprimary endpoints included objective radiographic tumor response rate, immune-related pathologic response rate, quality of life alterations, and metastasis-free and overall survival. In total, 17 patients were enrolled in this study and underwent surgery without a delay after receiving three every-2-wk doses of neoadjuvant nivolumab. Adverse events (AEs) of any grade occurred in 14 (82.4%) patients, with two (11.8%) experiencing grade 3 events. Ten (58.8%) patients experienced an AE of any grade potentially attributable to nivolumab (all grade 1–2), and no grade 4–5 AEs occurred regardless of treatment attribution. The most common AEs were grade 1 fatigue (41.2%), grade 1 pruritis (29.4%), and grade 1 rash (29.4%). All evaluable patients had stable disease as per established radiographic criteria, with one (6.7%) demonstrating features of an immune-related pathologic response. Quality of life remained stable during treatment, with improvements relative to baseline noted at ≥6 mo postoperatively. Metastasis-free survival and overall survival were 85.1% and 100% at 2 yr, respectively. Patient summary In this study, we evaluated the safety and tolerability of preoperative administration of three doses of the immune checkpoint inhibitor nivolumab in patients with clinically localized high-risk renal cell carcinoma. We demonstrated the safety of this approach and found that, although most patients will not experience a radiographic response to treatment, a subset may have features of an immune-related pathologic response.

Prostate cancer (PCa) is associated with several genetic alterations which play an important role in the disease heterogeneity and clinical outcome including gene fusion between TMPRSS2 and members of the ETS family of transcription factors specially ERG. The expanding wealth of pathology whole slide images (WSIs) and the increasing adoption of deep learning (DL) approaches offer a unique opportunity for pathologists to streamline the detection of ERG:TMPRSS2 fusion status. Here, we used two large cohorts of digitized H&E-stained slides from radical prostatectomy specimens to train and evaluate a DL system capable of detecting the ERG fusion status and also detecting tissue regions of high diagnostic and prognostic relevance. Slides from the PCa TCGA dataset were split into training (n=318), validation (n=59), and testing sets (n=59) with the training and validation sets being used for training the model and optimizing its hyperparameters, respectively while the testing set was used for evaluating the performance. Additionally, we used an internal testing cohort consisting of 314 WSIs for independent assessment of the model’s performance. The ERG prediction model achieved an Area Under the Receiver Operating Characteristic curve (AUC) of 0.72 and 0.73 in the TCGA testing set and the internal testing cohort, respectively. In addition to slide-level classification, we also identified highly attended patches for the cases predicted as either ERG-positive or negative which had distinct morphological features associated with ERG status. We subsequently characterized the cellular composition of these patches using HoVer-Net model trained on the PanNuke dataset to segment and classify the nuclei into five main categories. Notably, a high ratio of neoplastic cells in the highly-attended regions was significantly associated with shorter overall and progression-free survival while high ratios of immune, stromal and stromal to neoplastic cells were all associated with longer overall and metastases-free survival. Our work highlights the utility of deploying deep learning systems on digitized histopathology slides to predict key molecular alteration in cancer together with their associated morphological features which would streamline the diagnostic process.

The homologous recombination deficiency (HRD) score integrates three DNA-based measures of genomic instability, and has been understudied in prostate cancer. Given the recent FDA approval of two PARP inhibitors for prostate cancer, HRD score analysis could help to refine treatment selection. We assessed HRD score (defined as the sum of loss-of-heterozygosity, telomeric allelic imbalance, and large-scale state transitions) in three cohorts of primary prostate cancer, including a Johns Hopkins University (JHU) cohort with germline mutations in BRCA2, ATM, or CHEK2 (n = 64), the TCGA cohort (n = 391), and the PROGENE cohort (n = 102). In the JHU cohort, tumors with germline BRCA2 mutations had higher HRD scores (median = 27) than those with germline ATM or CHEK2 mutations (median = 16.5 [p = 0.029] and 9 [p

Summary The spectrum of neuroendocrine (NE) tumors in the genitourinary tract ranges from the aggressive large and small cell carcinomas to the often benign paraganglioma and well-differentiated neuroendocrine tumor (WD-NET). At least 15 pure lower urinary tract (LUT) WD-NETs have been described. Owing to the rarity of WD-NET in the LUT and the limited number of reported cases, a better definition of their biologic long-term behavior is warranted. Herein, we aim to describe 10 new cases of WD-NET arising in the LUT and expand on follow-up findings. Ten consultation cases were identified and included 6 men and 4 women who ranged from 45 to 73 years of age. Seven cases arose in the bladder with one located in the bladder neck, 1 arose in the prostatic urethra, 1 arose in the female urethra, and 1 arose in the left ureteral orifice. All lesions were confined to the lamina propria, and tumor architecture was pseudoglandular in all cases. Associated cystitis cystica et glandularis was identified in 5 cases; urothelial papilloma and florid von Brunn's nests were found in 2 additional cases. Immunohistochemical staining for synaptophysin and chromogranin was diffusely positive in 9 cases and focal in 1 case, and the Ki-67 proliferation index was 5% or less in all tumors. Follow-up ranged from 37 to 137 months (mean = 82; median = 77), and there was no evidence of residual disease or recurrence in any of the 10 patients during the follow-up period.

This study leveraged a gene-protein assay to assess MYC and PTEN status at prostate cancer biopsy and examined the association with adverse outcomes after surgery. MYC gain and PTEN loss were simultaneously assessed by chromogenic in situ hybridization and immunohistochemistry, respectively, using 277 Grade Group 2 needle biopsies that were followed by prostatectomy. The maximal size of cribriform Gleason pattern 4 carcinoma (CRIB), the presence of intraductal carcinoma (IDC), and percentage of Gleason pattern 4 carcinoma at biopsy were also annotated. MYC gain or PTEN loss was present in 19% and 18% of biopsies, respectively, whereas both alterations were present in 9% of biopsies. Tumors with one or both alterations were significantly more likely to have non–organ-confined disease (NOCD) at radical prostatectomy. In logistic regression models, including clinical stage, tumor volume on biopsy, and presence of CRIB/IDC, cases with MYC gain and PTEN loss remained at higher risk for NOCD (odds ratio, 6.23; 95% CI, 1.74–24.55; P = 0.005). The area under the curve for a baseline model using CAPRA variables (age, prostate-specific antigen, percentage of core involvement, clinical stage) was increased from 0.68 to 0.69 with inclusion of CRIB/IDC status and to 0.75 with MYC/PTEN status. Dual MYC/PTEN status can be assessed in a single slide and is independently associated with increased risk of NOCD for Grade Group 2 biopsies.

AIMS Prostatic intraepithelial neoplasia-like (PIN-like) ductal carcinoma is a rare tumour characterised by often cystically dilated glands architecturally resembling high-grade PIN, but lacking basal cells. These tumours are frequently accompanied by grade group 1 acinar cancer and behave relatively indolently. In contrast, conventional ductal adenocarcinoma of the prostate is an aggressive variant comparable to grade group 4 acinar cancer. Here, we used targeted next-generation sequencing to molecularly profile PIN-like ductal carcinoma cases at radical prostatectomy. METHODS AND RESULTS Five PIN-like ductal carcinoma samples at radical prostatectomy with sufficient tumour tissue available were analysed for genomic alterations by targeted next-generation sequencing using the Johns Hopkins University (JHU) solid tumour panel. DNA was captured using SureSelect for 640 genes and sequenced on the Illumina HiSeq platform. Three of five (60%) of the PIN-like ductal carcinomas showed activating mutations in the RAS/RAF pathways, which are extraordinarily rare in conventional primary prostate carcinoma (

Purpose:The ATM (ataxia telangiectasia mutated) gene is mutated in a subset of prostate cancers, and ATM mutation may confer specific therapeutic vulnerabilities, although ATM-deficient prostate cancers have not been well-characterized.Experimental Design:We genetically validated a clinical grade IHC assay to detect ATM protein loss and examined the frequency of ATM loss among tumors with pathogenic germline ATM mutations and genetically unselected primary prostate carcinomas using tissue microarrays (TMAs). Immunostaining results were correlated with targeted somatic genomic sequencing and clinical outcomes.Results:ATM protein loss was found in 13% (7/52) of primary Gleason pattern 5 cancers with available sequencing data and was 100% sensitive for biallelic ATM inactivation. In a separate cohort with pathogenic germline ATM mutations, 74% (14/19) had ATM protein loss of which 70% (7/10) of evaluable cases had genomic evidence of biallelic inactivation, compared with zero of four of cases with intact ATM expression. By TMA screening, ATM loss was identified in 3% (25/831) of evaluable primary tumors, more commonly in grade group 5 (17/181; 9%) compared with all other grades (8/650; 1%; P < 0.0001). Of those with available sequencing, 80% (4/5) with homogeneous ATM protein loss and 50% (6/12) with heterogeneous ATM protein loss had detectable pathogenic ATM alterations. In surgically treated patients, ATM loss was not significantly associated with clinical outcomes in random-effects Cox models after adjusting for clinicopathologic variables.Conclusions:ATM loss is enriched among high-grade prostate cancers. Optimal evaluation of ATM status requires both genomic and IHC studies and will guide development of molecularly targeted therapies.

Summary The grading and prognosis of prostatic adenocarcinoma with Paneth cell–like differentiation (PanEC) is controversial with limited available data. We identified 80 cases, not previously published, of PanEC first identified on biopsy (n = 69), transurethral resection of the prostate (n = 1), and radical prostatectomy (RP) (n = 10). Of 69 biopsies, 22 did not have a grade assigned. In the remaining 47 biopsies, the Grade Groups (GGs) of the associated usual prostatic adenocarcinoma were GG1–2 (n = 34) and GG3–5 (n = 13). Of 10 RPs, the GGs were as follows: GG1–2 (n = 8), GG4 (n = 1), and no grade due to treatment effect (n = 1); pathological stages were pT2 (n = 8) and pT3a (n = 2), all with negative lymph nodes. We analyzed 19 cases with available follow-up only associated with GG1–2 conventional cancer; 9 underwent RP, and GGs at RP were as follows: GG1–2 (n = 7), no grade due to treatment effect (n = 1), and missing data (n = 1); pathologic stages were pT2 (n = 6) and pT3a (n = 3); there were no positive regional lymph nodes; 3 were managed with active surveillance, without follow-up progression; 5 patients underwent radiation therapy with or without hormone therapy; none showed follow-up progression; 2 (10.5%) patients were recommended to undergo radiotherapy, with no further follow-up. Of the cases with available follow-up, 9 were not associated with conventional adenocarcinoma; the majority of these cases were treated with radiation therapy or active surveillance without evidence of progression. In summary, although a minority of PanECs are associated with conventional higher grade adenocarcinoma and have progression after treatment, the majority have favorable findings, justifying the consideration of them as more indolent tumors despite cases in which PanEC resembles Gleason pattern 5 adenocarcinoma.

Summary The prognostic significance of the Gleason grading system has been well established. However, individual Gleason patterns comprise heterogeneous morphologies which might add additional prognostic information. Recent evidence suggests that Gleason pattern 4 with cribriform growth pattern is associated with an adverse prognosis. To determine the association between cribriform pattern on biopsies and pathological findings on subsequent prostatectomies, we evaluated the presence of cribriform architecture in a prospective cohort of 367 men from 2014 to 2018 treated at a single institution. Cribriform architecture was present in 63.5% of all biopsies and was correlated with the overall extent of Gleason pattern 4. In addition, cribriform morphology on biopsy showed a statistically significant association with higher Gleason grade and increased pathological stage and nodal metastasis. In a subset analysis of cases with Grade Group 2 (Gleason score 3 + 4, n = 208), these associations did not reach statistical significance, but the presence of cribriform growth in this subgroup showed a trend toward increased upgrading to Grade Group 5 (Gleason score 9/10) (1 [0.5%] vs. 5 [2.4%], P = 0.06). This large prospective study comparing biopsy and prostatectomy finding of cribriform architecture demonstrates that cribriform pattern 4 is associated with adverse prognostic features and highlights the relevance for recognizing specific morphologies with distinct biological and clinical features.

Pathogenic mutations in homologous recombination (HR) DNA repair genes may be associated with increased tumor mutational burden and numbers of tumor-infiltrating lymphocytes (TIL). Though HR-deficient prostate tumors have been anecdotally associated with improved responses to immunotherapy, it is unclear whether HR mutations or HR deficiency (HRD) scores predict for increased T-cell densities in this cancer. We evaluated 17 primary prostate tumors from patients with pathogenic germline BRCA2 mutations (gBRCA2) and 21 primary prostate tumors from patients with pathogenic germline ATM (gATM) mutations, which were compared to 19 control tumors lacking HR gene mutations, as well as the TCGA prostate cancer cohort. HRD score was estimated by targeted sequencing (gBRCA2 and gATM) or by SNP microarray (TCGA). Tumor-associated T-cell densities were assessed using validated automated digital image analysis of CD8 and FOXP3 immunostaining (gBRCA2 or gATM) or by methylCIBERSORT (TCGA). CD8 + and FOXP3 + T-cell densities were significantly correlated with each other in gBRCA2 and gATM cases. There was no significant difference between CD8 + or FOXP3 + TIL densities in gBRCA2 or gATM cases compared to controls. In the TCGA cohort, HRD score was associated with predicted CD8 + and FOXP3 + TILs. Associations were also seen for HRD score and TIL density among the germline-mutated cases. In contrast to mismatch repair-deficient primary prostate tumors, cancers from germline BRCA2 or ATM mutation carriers do not appear to be associated with elevated TIL density. However, measures of genomic scarring, such as HRD score, may be associated with increased tumor-infiltrating T-cells.

Noninvasive approaches for early detection of bladder cancer are actively being investigated. We recently developed a urine- based molecular assay for the detection and surveillance of bladder neoplasms (UroSEEK). UroSEEK is designed to detect alterations in 11 genes that include most common genetic alterations in bladder cancer. In this study, we analyzed 527 cases, including 373 noninvasive and 154 invasive urothelial carcinomas of bladder from transurethral resections or cystectomies performed at four institutions (1991–2016). Two different mutational analysis assays of a representative tumor area were performed: first, a singleplex PCR assay for evaluation of the TERT promoter region (TERTSeqS) and second, a multiplex PCR assay using primers designed to amplify regions of interest of 10 (FGFR3, PIK3CA, TP53, HRAS, KRAS, ERBB2, CDKN2A, MET, MLL, and VHL) genes (UroSeqS). Overall, 92% of all bladder tumors were positive for at least one genetic alteration in the UroSEEK panel. We found TERT promoter mutations in 77% of low-grade noninvasive papillary carcinomas, with a relatively lower incidence of 65% in high-grade noninvasive papillary carcinomas and carcinomas in situ; p = 0.017. Seventy-two percent of pT1 and 63% of muscle-invasive bladder tumors harbored TERT promoter mutations with g.1295228C>T alteration being the most common in all groups. FGFR3 and PIK3CA mutations were more frequent in low-grade noninvasive papillary carcinomas compared with high-grade noninvasive papillary carcinomas and carcinomas in situ (p

Prostate cancer (PCa) is associated with several genetic alterations which play an important role in the disease heterogeneity and clinical outcome. These alterations involve gene fusion between TMPRSS2 and members of the ETS family of transcription factors like ERG, ETV1, and ETV4 together with mutations or deletions in tumor suppressors like TP53 and PTEN. The expanding wealth of digital whole slide images (WSIs) and the increasing adoption of deep learning approaches offer a unique opportunity for pathologists to streamline the detection of these alterations. Here, we used 736 haematoxylin and eosin-stained WSIs from 494 primary PCa patients to identify several key genetic alterations including ERG, ETV1, and ETV4 fusion, PTEN loss, and TP53 and SPOP mutations. Using a custom segmentation pipeline, we identified tissue regions and tiled them into high-resolution (10X magnification) patches (256X256 pixels) which were passed to our deep multiple instance learning framework. Using a pre-trained ResNet50 model, we extracted informative features which were subsequently used for training to predict slide-level labels and to detect slide regions with high diagnostic relevance. Using a 10-folds cross validation approach, we divided the data into training (80%), validation (10%) and testing (10%) sets. The training and validation data were used for training the model and hyperparameters tuning, respectively while the testing data was used to provide an unbiased evaluation of the models’ performance using the mean Area Under the Receiver Operating Characteristic (AUROC) across the ten testing folds as evaluation metric. We managed to accurately detect key molecular alterations including ERG fusion, ETV1 fusion, ETV4 fusion, and PTEN loss. Additionally, we were able to detect mutations in TP53 and SPOP together with the presence of androgen-receptor splice variant 7 (ARv7). In addition to slide-level classification, we also identified subregions with high attention score which can help pathologists identify the distinct morphological features associated with each genetic alteration. Finally, in order to examine the cellular structure associated with each genetic alteration, we used Hover-Net model to segment and classify the nuclei in the high-attention tiles. Our work highlights the utility of using WSIs to accurately identify key molecular alteration in cancer and their associated morphological and cellular features on the slide which would streamline the diagnostic process. To the best of our knowledge, this is the first study that uses routine WSIs to predict and characterize key genetic alterations in PCa. Citation Format: Mohamed Omar, Zhuoran Xu, Ryan Carelli, Jacob Rosenthal, David Brundage, Daniela C. Salles, Eddie L. Imada, Renato Umeton, Edward M. Schaeffer, Brian D. Robinson, Tamara L. Lotan, Massimo Loda, Luigi Marchionni. Using attention-based deep multiple instance learning to identify key genetic alterations in prostate cancer from whole slide images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 462.

Background: Intraductal (IDC) and cribriform (CRIB) histologies in prostate cancer have been associated with germline BRCA2 (gBRCA2) mutations in small retrospective series, leading to the recommendation of genetic testing for patients with IDC in the primary tumour. Patients and methods: To examine the association of gBRCA2 mutations and other tumour molecular features with IDC and/or cribriform (CRIB) histologies, we conducted a case -control study in which primary prostate tumours from 58 gBRCA2 carriers were matched (1:2) by Gleason Grade Group and specimen type to 116 non-carriers. Presence/absence of IDC and CRIB morphologies was established by two expert uropathologists blinded to gBRCA2 status. Fluorescent in-situ hybridization (FISH) and next-generation sequencing (NGS) were used to detect BRCA2 alterations, PTEN deletions and TMPRSS2-ERG fusions. Chi-squared tests were used to compare the frequency of IDC and CRIB in gBRCA2 carriers and controls and to assess associations with other variables. Logistic regression models were constructed to identify independent factors associated with both histology patterns. Results: No significant differences between gBRCA2 carriers and non-carriers were observed in the prevalence of IDC (36% gBRCA2 versus 50% non-carriers, p = 0.085) or CRIB (53% gBRCA2 versus 43% non-carriers p = 0.197) patterns. However, IDC histology was independently associated with bi-allelic BRCA2 alterations (OR 4.3, 95%CI 1.1-16.2) and PTEN homozygous loss ( OR 5.2, 95% CI 2.1-13.1). CRIB morphology was also independently associated with bi-allelic BRCA2 alterations (OR 5.6, 95%CI 1.7-19.3). Conclusions: While we found no association between gBRCA2 mutations and IDC or CRIB histologies, bi- allelic BRCA2 loss in primary prostate tumours was significantly associated with both variant morphologies, independently of other clinical-pathologic factors. (C) 2021 Elsevier Ltd. All rights reserved.

Background Patients diagnosed with high risk localized prostate cancer have variable outcomes following surgery. Trials of intense neoadjuvant androgen deprivation therapy (NADT) have shown lower rates of recurrence among patients with minimal residual disease after treatment. The molecular features that distinguish exceptional responders from poor responders are not known. Objective To identify genomic and histologic features associated with treatment resistance at baseline. Design, setting, and participants Targeted biopsies were obtained from 37 men with intermediate- to high-risk prostate cancer before receiving 6 mo of ADT plus enzalutamide. Biopsy tissues were used for whole-exome sequencing and immunohistochemistry (IHC). Outcome measurements and statistical analysis We assessed the relationship of molecular features with final pathologic response using a cutpoint of 0.05 cm3 for residual cancer burden to compare exceptional responders to incomplete and nonresponders. We assessed intratumoral heterogeneity at the tissue and genomic level, and compared the volume of residual disease to the Shannon diversity index for each tumor. We generated multivariate models of resistance based on three molecular features and one histologic feature, with and without multiparametric magnetic resonance imaging estimates of baseline tumor volume. Results and limitations Loss of chromosome 10q (containing PTEN) and alterations to TP53 were predictive of poor response, as were the expression of nuclear ERG on IHC and the presence of intraductal carcinoma of the prostate. Patients with incompletely and nonresponding tumors harbored greater tumor diversity as estimated via phylogenetic tree reconstruction from DNA sequencing and analysis of IHC staining. Our four-factor binary model (area under the receiver operating characteristic curve [AUC] 0.89) to predict poor response correlated with greater diversity in our cohort and a validation cohort of 57 Gleason score 8–10 prostate cancers from The Cancer Genome Atlas. When baseline tumor volume was added to the model, it distinguished poor response to NADT with an AUC of 0.98. Prospective use of this model requires further retrospective validation with biopsies from additional trials. Conclusions A subset of prostate cancers exhibit greater histologic and genomic diversity at the time of diagnosis, and these localized tumors have greater fitness to resist therapy. Patient summary Some prostate cancer tumors do not respond well to a hormonal treatment called androgen deprivation therapy (ADT). We used tumor volume and four other parameters to develop a model to identify tumors that will not respond well to ADT. Treatments other than ADT should be considered for these patients.

PURPOSE: The ATM gene is mutated in a subset of prostate cancers, and ATM mutation may confer specific therapeutic vulnerabilities, though ATM-deficient prostate cancers have not been well-characterized. EXPERIMENTAL DESIGN: We genetically validated a clinical-grade immunohistochemistry (IHC) assay to detect ATM protein loss and examined the frequency of ATM loss among tumors with pathogenic germline ATM mutations and genetically unselected primary prostate carcinomas using tissue microarrays (TMA). Immunostaining results were correlated with targeted somatic genomic sequencing and clinical outcomes. RESULTS: ATM protein loss was found in 13% (7/52) of primary Gleason pattern 5 cancers with available sequencing data and was 100% sensitive for bi-allelic ATM inactivation. In a separate cohort with pathogenic germline ATM mutations, 74% (14/19) had ATM protein loss of which 70% (7/10) of evaluable cases had genomic evidence of bi-allelic inactivation, compared to 0/4 of cases with intact ATM expression. By TMA screening, ATM loss was identified in 3% (25/831) of evaluable primary tumors, more commonly in Grade Group 5 (17/181; 9%) compared to all other grades (8/650; 1%) (P

Purpose: The potential biological determinants of aggressive prostate cancer in African American (AA) men are unknown. Here we characterize prostate cancer genomic alterations in the largest cohort to date of AA men with clinical follow-up for metastasis, with the aim to elucidate the key molecular drivers associated with poor prognosis in this population. Experimental Design: Targeted sequencing was retrospectively performed on 205 prostate tumors from AA men treated with radical prostatectomy (RP) to examine somatic genomic alterations and percent of the genome with copy-number alterations (PGA). Cox proportional hazards analyses assessed the association of genomic alterations with risk of metastasis. Results: At RP, 71% (145/205) of patients had grade group ≥3 disease, and 49% (99/202) were non–organ confined. The median PGA was 3.7% (IQR = 0.9%–9.4%) and differed by pathologic grade (P < 0.001) and stage (P = 0.02). Median follow-up was 5 years. AA men with the highest quartile of PGA had increased risks of metastasis (multivariable: HR = 13.45; 95% CI, 2.55–70.86; P = 0.002). The most common somatic mutations were SPOP (11.2%), FOXA1 (8.3%), and TP53 (3.9%). The most common loci altered at the copy number level were CDKN1B (6.3%), CHD1 (4.4%), and PTEN (3.4%). TP53 mutations and deep deletions in CDKN1B were associated with increased risks of metastasis on multivariable analyses (TP53: HR = 9.5; 95% CI, 2.2–40.6; P = 0.002; CDKN1B: HR = 6.7; 95% CI, 1.3–35.2; P = 0.026). Conclusions: Overall, PGA, somatic TP53 mutations, and a novel finding of deep deletions in CDKN1B were associated with poor prognosis in AA men. These findings require confirmation in additional AA cohorts.

Background: Tuberous Sclerosis Complex (TSC) is characterized by TSC1/2 loss, dysregulated mTORC1 signaling & renal tumors (angiomyolipomas (AML) & renal cell carcinoma (RCC)). The MiT/TFE transcription factors (MITF/TFE3/TFEB) drive autophagy/lysosomal biogenesis & are negatively regulated by mTORC1. However, this model raises a paradox in cancer, where elevated lysosomal activity must persist with mTORC1 activity. We recently showed that epidermal Tsc1 loss paradoxically increases MiT/TFE-activity (https://www.jci.org/articles/view/128287). Intriguingly, TFE3/TFEB gene rearrangements/amplifications and TSC1/2 loss are mutually exclusive drivers in PEComas & RCC. This raises the possibility that TSC1/2 loss & TFE3/TFEB gene rearrangements have overlapping cellular consequences. Here, we address the hypothesis that MiT/TFE-driven lysosomal biogenesis drives tumorigenesis in TSC. Design: We used HEK293T cells +/- CRISPR deletion (KO) of TSC1, 2 or both, to examine: a) Lysosomal gene enrichment by RNA-seq/GSEA, b) Expression of MiT/TFE & lysosomal markers (immunoblotting, qRT-PCR & IF), c) MiT/TFE localization (IF & nuclear fraction immunoblots), d) MiT/TFE activity (4X-CLEAR luciferase assays, qRT-PCR, cathepsin processing, autophagic flux, LC3 puncta). We analysed spontaneous renal tumors in Tsc2 +/- mice for MIT/TFE protein/gene expression (IHC, IF & qRT-PCR analyses of laser capture micro-dissected (LCM) renal tumors). We examined MIT/TFE proteins/lysosomal markers in FFPE samples of renal PEComas & eosinophilic solid & cystic (ESC) RCC & normal kidney. Results: RNA-seq/GSEA showed enrichment of lysosomal gene sets in TSC1/2 KO cells compared to controls. TSC2 KO cells had increased expression of lysosomal transcripts & proteins in cellular lysates & lysosomal fractions. TSC1, 2 & 1/2 KO cells showed increased nuclear TFEB/TFE3 (IF/nuclear-fraction immunoblots), compared to controls. MiT/TFE activity in 4X-CLEAR luciferase reporter assays was increased in TSC2 KO cells compared to controls. Treatment of TSC2 KO cells with chloroquine increased lipidated LC3-II, indicating increased autophagic flux. TSC2 KO cells also showed increased LC3-labelled puncta by IF. We analyzed renal tumors in Tsc2 +/- mice, where elevated mTORC1 signaling was confirmed by p-S6 IHC. Expression of lysosomal proteins (LAMP1, Lamtor 1, Rag C, Cathepsin B) & nuclear localization of TFE3 & TFEB was increased in these lesions by IF/IHC, compared to normal kidney. We performed LCM on renal tumors from Tsc2 +/- mice & found levels of MiT/TFE transcriptional targets to be significantly enriched in tumors compared to normal kidney. We analyzed TFE3 expression in 10 cases of ESC- RCC with sporadic bi-allelic TSC1/2 mutations & 2 cases of TSC-associated RCC; 8/10 cases showed elevated nuclear TFE3. Conclusions: Elevated MiT/TFE levels & activity may represent oncogenic drivers in human & murine renal tumors in TSC. Citation Format: Kaushal V. Asrani, Daniela Salles, Juhyung Woo, Adrianna Mendes, Sanjana Murali, Thiago Vidotto, Pedram Argani, Edward Gabrielson, Tamara Lotan. mTORC1 paradoxically drives MiT/TFE activity and lysosomal biogenesis in tuberous sclerosis complex [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2431.

Parenchymal brain metastases from prostate cancer are unusual and are associated with poor prognosis. Given the rarity of this entity, little is known about its molecular and histologic characteristics. Here we describe a patient with metastatic castration-resistant, mismatch repair–deficient (dMMR) prostate cancer with parenchymal brain metastases. Analysis of a brain metastasis revealed MLH1 loss consistent with dMMR, yet few tumor-infiltrating lymphocytes (TILs). He was treated with immune checkpoint blockade (ICB) and exhibited an extra–central nervous system (CNS) systemic response but CNS progression. Subsequent assessment of a brain metastasis following ICB treatment surprisingly showed increased TIL density and depletion of macrophages, suggestive of an enhanced antitumor immune response. Post-treatment tumoral DNA sequencing did not reveal acquired mutations that might confer resistance to ICB. This is the first description of ICB therapy for a patient with prostate cancer with parenchymal brain metastases, with pre- and post-treatment immunogenomic analyses.

5579 Background: IDC histology in PC has been suggested to associate with germline BRCA2 mutations (gBRCA2) in small series, leading to the potential recommendation of genetic testing for all PC patients with IDC in the primary tumor. Methods: We conducted a case-control study in which primary PC from 58 germline BRCA2 mutation carriers ( gBRCA2) and 116 from non-carriers (NC) were matched 1:2 by Gleason score and specimen type (core biopsy/prostatectomy). Samples were independently reviewed by two expert pathologists blinded to genetic status who established the presence of IDC and/or CRIB morphology with supportive immunohistochemical stains in a subset of cases. Next-generation sequencing, aCGH and/or FISH were used to assess for somatic mono-/bi-allelic BRCA2 alterations. PTEN protein loss was determined by IHC, and TMPRSS2-ERG was detected by FISH/qRT-PCR. Chi-square tests were used to compare the frequency of IDC and cribriform histology in gBRCA2 vs NC, as well as to assess the associations with other variables. Logistic regression models were built to identify independent factors associated with IDC and CRIB histology. Results: gBRCA2 cases were younger at diagnosis (median 61.3 vs 64, p < 0.01) and had T3-4 disease more often than NC cases (31% vs 10.5%, p < 0.01), but the two groups did not differ in any other clinical-pathologic characteristics. After independent histopathological review, 79 cases demonstrated IDC histology and 81 had CRIB histology. No differences in the prevalence of IDC (50% NC vs 36% gBRCA2, p = 0.09) or CRIB (43% NC vs 53% gBRCA2, p = 0.20) patterns were observed. The probability of IDC was higher in PC with bi-allelic BRCA2 alterations (OR 5.1, 95%CI 2.1-12.6), PTEN loss (OR 5.1, 95%CI 1.9-13.5) or both (OR 23.0, 95%CI 4.9-107.2) compared to those without these alterations. Bi-allelic BRCA2 alteration was also associated with higher probability of CRIB histology (OR 7.2, 95%CI 3.1-16.4). TMPRSS2-ERG fusions were not associated with IDC or CRIB histology. MVA confirmed the independent association of bi-allelic BRCA2 alteration (p < 0.01) and PTEN loss (p < 0.01) with IDC histology. Bi-allelic BRCA2 alteration (p < 0.01) and Gleason >8 (p < 0.01) were independent risk factors for CRIB histology. Conclusions: Primary PC with bi-allelic BRCA2 alterations was significantly associated with IDC and CRIB histology, independent of other clinical-pathologic factors (while gBRCA2 status alone was not). PTEN loss in primary PC was also independently associated with IDC, but not CRIB, histology.

Single-agent immunotherapeutic approaches are promising for treating castrate-resistant prostate cancer (CRPC) patients; however, to date no significant improvement in survival has been reported. Mixed response to therapy occurs because metastatic lesions are highly heterogeneous. These lesions exhibit distinct genomic changes that are linked to a tumor microenvironment that is often poorly populated by immune cells. In other tumor types, a high percent genome altered (e.g., aneuploidy) associates with an overall reduction in immune response in the primary tumor microenvironment. However, the association between genomic changes and immune cell composition is poorly understood in metastatic prostate cancers. Thus, we quantified the abundance of 22 immune cell types in 330 tumors from two independent public domain CRPC cohorts and 491 primary prostate tumors from TCGA using the Absolute Mode from CIBERSORT. The estimation in CRPC was performed using whole-transcriptome data from West Coast Dream Team (Abida et al., 2019; n=212) and East Coast Dream Team (Robinson et al., 2015; n=117) samples. We did not identify any significant correlations between mutational load and immune cell abundance in the three investigated cohorts. In primary tumors, aneuploidy was weakly negatively correlated with CD8+ and CD4+ T-cell abundance (R=-0.14 and R=-0.09, P Citation Format: Thiago Vidotto, Daniela C. Salles, Tamara L. Lotan. High aneuploidy levels are linked to a reduced immune-cell abundance in metastatic castrate-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A31.

A new prostate cancer grading system was proposed in 2013 and endorsed by major journals and societies in 2014, in part because of anecdotal evidence that Gleason scores (GSs) were incorrectly combined in the literature.To examine how published studies categorized GSs in current practice.A PubMed search was conducted on articles published in 2016-2017 using the search terms "Gleason" and "prostate". This literature review included 1576 articles after exclusions.(1) Separating GS 7: pathology journals were more likely than non-pathology journals to grade GS 7 separately (56.9% vs 40.0%, p0.05). Articles co-authored by a pathologist separated GS 7 more than those without a pathologist (53.2% vs 32.9%, p0.001). North American and European studies separated GS 7 more than Asian studies (47.6% and 44.1% vs 24.1%, p0.001). Clinical articles separated GS 7 more than research articles (43.7% vs 32.9%, p0.001). (2) Separating GS 8 from GS 9-10: pathology journals separated GS 8 from GS 9-10 more than non-pathology journals (55.2% vs 34.4%, p=0.001). Articles co-authored by a pathologist separated GS 8 from GS 9-10 more often than those without a pathologist (44.9% vs 29.5%, p0.001). (3) Using grade groups as "ideal" with all other groupings "non-ideal": pathology journals used ideal more than non-pathology journals (32.2% vs 15.9%, p0.001). Ideal grouping is more likely in articles co-authored by a pathologist than in those without a pathologist (20.6% vs 11.0%, p0.001). North American and European studies used ideal grouping more than Asian studies (17.6% and 14.0% vs 9.1%, p0.05). (4) Arranging groupings in decreasing order from ideal to non-ideal: pathology journals were closer to ideal than non-pathology journals (p=0.002). Articles co-authored by a pathologist were classified closer to ideal than those without a pathologist (p0.001). North American (p0.001) and European (p=0.02) studies were closer to ideal than Asian studies.There is still wide variation in how GSs are grouped world-wide. Only a minority of published articles group GSs accurately.In this report, we looked at how GSs were grouped world-wide. We found that only a minority of published articles on prostate cancer were grouping GSs accurately, which could lead to inaccurate results and affect patient care with different prostate cancer grades. Our study calls for more widespread adoption of the new prostate cancer grading system composed of five grade groups to minimize incorrect grouping for future studies.

Insulin-like 3 (INSL3) is a hormone produced by Leydig cells (LCs) and leads to physiological testicular descent during embryonic development. We investigated the expression of INSL3 by immunohistochemistry in normal LCs, in Leydig cell tumor (LCT) (n=17 including 15 testes and 2 ovaries) and in Leydig cell hyperplasia (LCH) (n=10). Normally distributed LCs showed strong immunostaining in the cytoplasm in all cases. All 10 cases (100%) of LCH were strongly and diffusely positive in the intertubular areas. Six cases of LCH had nodules raging in size from 0.2 to 0.9 cm with variable INSL3 staining. Fifteen of 17 (88.2%) LCTs showed marked decrease INSL3 staining, 10/17 (58.8%) were completely negative, and 5/17 (29.4%) were only focally positive. Two cases with multifocal LCTs showed strong and diffuse cytoplasmic staining of LCs around seminiferous tubules while the LCTs were negative. Two cases diagnosed as LCT were strongly positive for INSL3. Other sex cord stromal tumors tested were consistently negative including Sertoli-cell tumor (n=4), granulosa cell tumor (n=2), and fibrothecoma (n=1). In conclusion, our results contrast with those of previously published studies, and show that the great majority of LCTs are negative or have decreased expression of INSL3 while its expression is retained in LCH. INSL3 negative nodules within LCH may represent early LCTs. INSL3 immunostaining could be helpful to highlight LCs in cases where it is difficult to identify them (ie, small testicular biopsies performed for infertility workup) and in the differential diagnosis between florid LCH and LCT.