Variant PNLDC1, Defective piRNA Processing, and Azoospermia.

BACKGROUND: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility. METHODS: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing. RESULTS: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in PNLDC1: the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying PNLDC1 mutations. CONCLUSIONS: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.).

Pilot study of the diagnostic utility of 89 Zr-df-IAB2M and 68 Ga-PSMA-11 PET imaging and multiparametric MRI in localized prostate cancer.

BACKGROUND: Accurate diagnosis of localized prostate cancer (PCa) is limited by inadequacy of multiparametric (mp) MRI to fully identify and differentiate localized malignant tissue from benign pathologies. Prostate-specific membrane antigen (PSMA) represents an excellent target for molecular imaging. IAB2M, an 85-kD minibody derived from a de-immunized monoclonal antibody directed at the extracellular domain of human PSMA (huJ591), and PSMA-11, a small molecule ligand have been previously tested as probes for visualization of recurrent/metastatic PCa with PET/CT. This pilot, non-randomized trial studied their diagnostic utility in patients (pts) with localized PCa. METHODS: Pts planned for radical prostatectomy (RP) were enrolled and underwent mpMRI and PET/CT imaging with 89 Zr-df-IAB2M and/or 68 Ga-PSMA-PET/CT. Image results were read by a radiologist blinded to clinical information and pathology results, mapped and compared to corresponding histopathology findings from all lesions, both clinically significant and nonsignificant. The detection rates of all three imaging modalities were measured and correlated. RESULTS: 20 pts with median age of 64.5 (46-79) years and PSA level of 7.5 (1.6-36.56) ng/ml were enrolled. 19 pts underwent RP and were imaged pre-operatively with 89 Zr-Df-IAB2M PET/CT and mpMRI. Nine of those were imaged using 68 Ga-PSMA-11 as well. Out of 48 intraprostatic lesions verified on surgical pathology, IAB2M PET/CT was able to detect 36 (75%). A similar proportion of pathologically confirmed, clinically significant lesions (22/29, 76%) was detected. IAB2M PET/CT was also able to identify 14/19 (74%) extraprostatic lesions. The performance of mpMRI was inferior, with 24/48 detectable lesions (50%) and 18/29 clinically significant intraprostatic lesions (62%). Compared to the current standard (mpMRI), IAB2M PET/CT had a sensitivity of 88%, specificity 38%, positive predictive value 58%, and accuracy 63%. In 9 pts who underwent Ga-PSMA-11 as well, the latter yielded a detection rate of 70% (14/20), which was also seen in clinically significant lesions (10/14, 71%). Ga-PSMA-11 PET/CT also detected 4/6 (67%) extraprostatic lesions. CONCLUSIONS: In this pilot study, the performance of 89 Zr-df-IAB2M was superior to mpMRI and similar to 68 Ga-PSMA-11 PET/CT. The higher detection rate of PSMA-PET supports its use as a diagnostic tool with consequent management change implications in men with localized PCa.

Transcriptional and metabolic remodeling in clear cell renal cell carcinoma caused by ATF4 activation and the integrated stress response (ISR).

Research has shown extensive metabolic remodeling in clear cell renal cell carcinoma (ccRCC), with increased glutathione (GSH) levels. We hypothesized that activating transcription factor-4 (ATF4) and the integrated stress response (ISR) induce a metabolic shift, including increased GSH accumulation, and that Vitamin A deficiency (VAD), found in ccRCCs, can also activate ATF4 signaling in the kidney. To determine the role of ATF4, we used publicly available RNA sequencing (RNA-seq) data sets from The Cancer Genomics Atlas. Subsequently, we performed RNA-seq and liquid chromatography-mass spectrometry-based metabolomics analysis of the murine TRAnsgenic Cancer of the Kidney (TRACK) model for early-stage ccRCC. To validate our findings, we generated RCC4 cell lines with ATF4 gene edits (ATF4-knockout [KO]) and subjected these cells to metabolic isotope tracing. Analysis of variance, the two-sided Student's t test, and gene set enrichment analysis were used (p < 0.05) to determine statistical significance. Here we show that most human ccRCC tumors exhibit activation of the transcription factor ATF4. Activation of ATF4 is concomitant with enrichment of the ATF4 gene set and elevated expression of ATF4 target genes ASNS, ALDH1L2, MTHFD2, DDIT3 (CHOP), DDIT4, TRIB3, EIF4EBP1, SLC7A11, and PPP1R15A (GADD34). Transcript profiling and metabolomics analyses show that activated hypoxia-inducible factor-1α (HIF1α) signaling in our TRACK ccRCC murine model also induces an ATF4-mediated ISR. Notably, both normoxic HIF1α signaling in TRACK kidneys and VAD in wild-type kidneys diminish amino acid levels, increase ASNS, TRIB3, and MTHFD2 messenger RNA levels, and increase levels of lipids and GSH. By metabolic isotope tracing in human RCC4 kidney cancer parental and ATF4 gene-edited (ATF4-KO) cell lines, we show that ATF4 increases GSH accumulation in part via activation of the mitochondrial one-carbon metabolism pathway. Our results demonstrate for the first time that activation of ATF4 enhances GSH accumulation, increases purine and pyrimidine biosynthesis, and contributes to transcriptional and metabolic remodeling in ccRCC. Moreover, constitutive HIF1α expressed only in murine kidney proximal tubules activates ATF4, leading to the metabolic changes associated with the ISR. Our data indicate that HIF1α can promote ccRCC via ATF4 activation. Moreover, lack of Vitamin A in the kidney recapitulates aspects of the ISR.

Gender distribution of editors and authors of reference textbooks in anatomic pathology: further edits are required.

Compared to other medical specialties, pathology has a significant number of women in the academic workforce (43%). Gender disparities, particularly those disadvantaging women, are a reality in academic medicine with documented inequalities in salary, leadership opportunities, and faculty promotion. One important element of academic advancement is the recognition obtained when serving as editor or main author of reference textbooks. We aimed to document the gender distribution of editors/authors in anatomic pathology by surveying 205 subspecialty publications over a 20-year period. Gender of each editor/author was recorded after surveying their institutional or other professional biographies. When biography was non-contributory, gender was extracted from the National Provider Identifier Database. A total of 462 editors/authors were identified: 275 (59.5%) men and 187 (40.5%) women. This distribution was similar to the 2015 (39% women) and 2019 (43.4% women) Association of American Medical Colleges (AAMC) benchmark for US academic pathologists. The gender distribution in each of the main anatomic pathology subspecialties was estimated by surveying the websites of 20 North American academic pathology departments (totaling 1893 listed individuals). Compared to this benchmark, some subspecialties had more men in editor/author roles than their representation in academic departments including Dermatopathology (observed vs expected difference, ∆ = 41.3%), Genitourinary Pathology (∆ = 29.4%), Renal & Transplant Pathology (∆ = 22.4%) and Head & Neck Pathology (∆ = 21.6%). Other subspecialties had more women in editor/author roles than their representation in academic departments including Molecular Pathology (∆ = 31.4%), Gastrointestinal Pathology (∆ = 21.4%), and Bone & Soft Tissue Pathology (∆ = 19.4%). Editors/authors of multiple (>1) publications were frequent and skewed gender representation in most specialties. The overall gender distribution of editor/author roles is similar to that of the US pathology workforce. However, significant disparities exist in certain subspecialties affecting both women and men. This landscape can guide efforts by editors, publishers, and academic institutions to bring equity to the academic field by providing fair editorial and authorship opportunities to academic pathologists.

Testicular Germ-Cell Tumors with Spermatic Cord Involvement: A Retrospective International Multi-Institutional Experience.

The 8th Edition of the American Joint Committee on Cancer (AJCC) Staging Manual designates discontinuous involvement of spermatic cord soft tissue by testicular germ cell tumors as a metastatic deposit. We conducted a retrospective international multi-institutional study to validate the current recommendations. Thirty-three (72%) nonseminomatous and 13 (28%) seminomatous testicular germ cell tumors were collected from 15 institutions in America, Europe, and Asia. Testicular tumor size ranged from 1.3 to 18.0 cm (mean: 6.1). Cases were classified as discontinuous involvement of spermatic cord soft tissue (n = 26), continuous cord involvement (n = 17), or cord lymphovascular invasion (n = 3). The mean follow-up was 39 months. Clinical stage for discontinuous involvement of spermatic cord soft-tissue patients was I (local disease) in 2/24 (8%), II (regional disease) in 6/24 (25%), and III (distant disease) in 16/24 (67%) cases; 16 (67%) patients presented with distant metastasis. Clinical stage for continuous cord involvement patients was I in 9/17 (53%), II in 4/17 (23%), and III in 4/17 (23%); 4 (23%) patients presented with distant metastasis. Disease progression was seen in 4 patients with discontinuous involvement of spermatic cord soft tissue and 5 with continuous cord-involvement (p = 0.699). When comparing discontinuous and continuous cord involvement, a significant difference was found in cord margin status (p = 0.044), spermatic cord tumor size (p = 0.016), lymph-node involvement (p = 0.037), distant metastasis (p = 0.010), individual clinical stage (p = 0.003), and nonadvanced vs. advanced disease (p = 0.003) at presentation. In multivariate analysis, after adjusting for age, histology, testicular tumor size, percent of embryonal carcinoma, lymphovascular invasion, and cord margin status, discontinuous involvement of spermatic cord soft tissue was significantly associated (p = 0.011) with advanced clinical stage at presentation. Our findings support the designation of metastatic disease for discontinuous involvement of spermatic cord soft tissue, as introduced by the 8th edition of the AJCC staging.

Quantifying Heterogeneity of Testicular Histopathology in Men with Nonobstructive Azoospermia.

PURPOSE: We sought to determine if testicular histopathological heterogeneity is associated with sperm retrieval rates (SRRs) in men with nonobstructive azoospermia (NOA) who are undergoing microdissection testicular sperm extraction (mTESE). MATERIALS AND METHODS: All patients undergoing mTESE by a single, high-volume surgeon at a tertiary infertility referral center between 2010 and 2020 were evaluated. Pathology reports from testis biopsy at the time of mTESE reported by fellowship-trained genitourinary pathologists were reviewed. Testicular heterogeneity was correlated to absolute SRRs. Logistic regression was used to determine if heterogeneity was associated with sperm retrieval. RESULTS: A total of 918 men with mTESE were included. Of these, 391 men (43%) had 1 pathology, 388 men (42%) had 2, 108 (12%) had 3, and 31 (3.4%) had 4. Overall, the most common histopathology was Sertoli-cell only, followed by maturation arrest. The overall SRR was 42% with a clinical intrauterine gestation rate of 30%. Increasing histopathological variety was associated with higher SRRs (p <0.01); a SRR of 33% was observed when one histopathological subtype was present vs 94% with 4 subtypes. Furthermore, men with any foci of spermatogenesis had higher SRRs. CONCLUSIONS: In men with NOA, increasing testicular histopathological heterogeneity is correlated with higher SRRs driven by the identification of focal areas of spermatogenesis. This is an important, although predictable, observation. While diagnostic biopsy is not routinely required, these findings emphasize the value of having histology to perhaps predict the chance of sperm retrieval for future mTESE procedures.

A novel prognostic two-gene signature for triple negative breast cancer.

The absence of a robust risk stratification tool for triple negative breast cancer (TNBC) underlies imprecise and nonselective treatment of these patients with cytotoxic chemotherapy. This study aimed to interrogate transcriptomes of TNBC resected samples using next generation sequencing to identify novel biomarkers associated with disease outcomes. A subset of cases (n = 112) from a large, well-characterized cohort of primary TNBC (n = 333) were subjected to RNA-sequencing. Reads were aligned to the human reference genome (GRCH38.83) using the STAR aligner and gene expression quantified using HTSEQ. We identified genes associated with distant metastasis-free survival and breast cancer-specific survival by applying supervised artificial neural network analysis with gene selection to the RNA-sequencing data. The prognostic ability of these genes was validated using the Breast Cancer Gene-Expression Miner v4. 0 and Genotype 2 outcome datasets. Multivariate Cox regression analysis identified a prognostic gene signature that was independently associated with poor prognosis. Finally, we corroborated our results from the two-gene prognostic signature by their protein expression using immunohistochemistry. Artificial neural network identified two gene panels that strongly predicted distant metastasis-free survival and breast cancer-specific survival. Univariate Cox regression analysis of 21 genes common to both panels revealed that the expression level of eight genes was independently associated with poor prognosis (p < 0.05). Adjusting for clinicopathological factors including patient's age, grade, nodal stage, tumor size, and lymphovascular invasion using multivariate Cox regression analysis yielded a two-gene prognostic signature (ACSM4 and SPDYC), which was associated with poor prognosis (p < 0.05) independent of other prognostic variables. We validated the protein expression of these two genes, and it was significantly associated with patient outcome in both independent and combined manner (p < 0.05). Our study identifies a prognostic gene signature that can predict prognosis in TNBC patients and could potentially be used to guide the clinical management of TNBC patients.

Intraductal carcinoma of the prostate in the absence of high-grade invasive carcinoma represents a molecularly distinct type of in situ carcinoma enriched with oncogenic driver mutations.

Intraductal carcinoma of the prostate (IDC-P) most often appears associated with high-grade invasive prostate carcinoma (PCa), where it is believed to represent retrograde spread. However, IDC-P rarely occurs as an isolated finding at radical prostatectomy or with concurrent low-grade (Grade Group 1) invasive carcinoma. We hypothesized that isolated IDC-P (iIDC-P) in these unusual cases may represent a distinct in situ lesion and molecularly profiled 15 cases. iIDC-P was characterized by copy number alteration (CNA) profiling and targeted next generation sequencing in cases with sufficient tissue (n = 7). Immunohistochemistry for PTEN and ERG was performed on the total cohort (n = 15), where areas of iIDC-P and associated invasive disease were evaluated separately (n = 9). By copy number profiling, iIDC-P alterations were similar to those previously described in high-grade invasive PCa (PTEN, RB1, and CHD1 loss; MYC gain). However, in four cases, targeted sequencing revealed a striking number of activating oncogenic driver mutations in MAPK and PI3K pathway genes, which are extraordinarily rare in conventional PCa. In addition, pathogenic mutations in DNA repair genes were found in two cases of iIDC-P (BRCA2, CHEK2, CDK12) and other known PCa-associated mutations (FOXA1, SPOP) in two cases. Overall, ERG was expressed in 7% (1/15) of the iIDC-P lesions and PTEN was lost in 53% (8/15). Discordance for ERG or PTEN status between IDC-P and the low-grade PCa was observed in five of nine cases, with intact PTEN in the invasive tumor and PTEN loss in IDC-P in four. Despite a CNA profile similar to conventional PCa, iIDC-P is enriched with potentially targetable oncogenic driver mutations in MAPK/PI3K genes. Based on PTEN and ERG status, iIDC-P is not likely a precursor to the associated low-grade invasive PCa, but represents a molecularly unique in situ tumor of unclear clinical significance. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The Clinical Utility of the Genomic Prostate Score in Men with Very Low to Intermediate Risk Prostate Cancer.

PURPOSE: We retrospectively investigated the Genomic Prostate Score® assay in clinical practice at an urban tertiary care academic center. MATERIALS AND METHODS: We reviewed all Genomic Prostate Score results acquired during a 3-year period. Changes in patient NCCN® (National Comprehensive Cancer Network®) risk group, including very low, low, intermediate or high risk, and ultimate management decisions were recorded. RESULTS: Genomic Prostate Score risk stratification was performed in 134 men. According to the NCCN Guidelines®, 31 of the 134 men (23.1%) were at very low risk, 45 (33.6%) were at low risk and 58 (43.3%) were at intermediate risk. After adding the score the risk group changed in 32 of 134 patients (23.9%). The risk group did not change in the 31 men at very low risk. However, in the low risk group the risk changed in 19 of the 45 men (42.2%), including in 15 to very low and in 4 to intermediate risk. Also, in the intermediate risk group the risk changed in 13 of the 58 men (22.4%), including to low in 12 and to high risk in 1. Nine of the 15 men (60%) in whom risk changed from low to very low elected active surveillance. Nine of the 12 patients (75%) at intermediate risk in whom risk changed to low risk elected active surveillance, 2 (16.7%) elected definitive therapy and in 1 (8.3%) the choice was unknown. Of the 45 men at intermediate risk in whom risk was unchanged 28 (62.2%) elected definitive therapy, 12 (26.0%) elected active surveillance and in 5 (11.1%) the choice was unknown. Of the 4 men upgraded from low to intermediate risk after adding the genomic prostate score 2 elected definitive therapy and 2 chose active surveillance. CONCLUSIONS: The Genomic Prostate Score has limited clinical usefulness in patients at very low risk since the NCCN risk group did not change. While it may be more useful for men at low and intermediate risk, for 32 (31%) of whose risk group was reclassified, clinical management decisions did not always appear to reflect these changes.

Oncogenic Addiction to ERBB2 Signaling Predicts Response to Trastuzumab in Urothelial Cancer.

Urothelial carcinoma (UC) is a common and frequently lethal cancer. Despite the presence of genomic alterations creating dependency on particular signaling pathways, the use of targeted therapies in advanced and metastatic UC has been limited. We performed an integrated analysis of whole-exome and RNA sequencing of primary and metastatic tumors in a patient with platinum-resistant UC. We found a strikingly high ERBB2 mRNA expression and enrichment of downstream oncogenic ERBB2 signaling in this patient's tumors compared with tumors from an unselected group of patients with UC (N=17). This patient had an exceptional sustained response to trastuzumab. Our findings show that oncogenic addiction to ERBB2 signaling potentially predicts response to ERBB2-directed therapy of UC.

Accuracy of Grading Gleason Score 7 Prostatic Adenocarcinoma on Needle Biopsy: Influence of Percent Pattern 4 and Other Histological Factors.

BACKGROUND: Recognition of Gleason pattern 4 in prostatic needle biopsies is crucial for both prognosis and therapy. Recently, it has been recommended to record percent pattern 4 when Gleason score 7 cancer is the highest grade in a case. METHODS: Four hundred and five prostate needle core biopsies received for a second opinion at our institution from February-June, 2015 were prospectively diagnosed with prostatic adenocarcinoma Gleason score 7 as the highest score on review by a consultant urological pathologist. Percentage of core involvement by cancer, percentage of Gleason pattern 4 per core, distribution of Gleason pattern 4 (clustered, scattered), morphology of pattern 4 (cribriform, non-cribriform), and whether the cancer was continuous or discontinuous were recorded. RESULTS: Better agreement was noted between the consultant and referring pathologists when pattern 4 was clustered as opposed to dispersed in biopsies (P = 0.009). The percentage of core involvement by cancer, morphology of pattern 4, and continuity of cancer did not affect the agreement between the consultant and referring pathologists. There was a trend (P = 0.06) for better agreement based on the percent of pattern 4. CONCLUSIONS: When pattern 4 is scattered amongst pattern 3 as opposed to being discrete foci, there is less interobserver reproducibility in grading Gleason score 7 cancer, and in this setting pathologists should consider obtaining second opinions either internally within their group or externally. Prostate 77: 681-685, 2017. © 2017 Wiley Periodicals, Inc.

Frequency and Prognostic Value of PTEN Loss in Patients with Upper Tract Urothelial Carcinoma Treated with Radical Nephroureterectomy.

PURPOSE: To our knowledge the frequency and prognostic significance of PTEN protein expression in upper tract urothelial carcinoma have not yet been investigated in large studies. We analyzed PTEN protein status and its association with disease recurrence and survival outcomes in a large, multi-institutional upper tract urothelial carcinoma cohort. MATERIALS AND METHODS: We retrospectively analyzed the records of 611 patients with upper tract urothelial carcinoma treated with radical nephroureterectomy between 1991 and 2008 at a total of 7 institutions. Median followup was 23 months. Tissue microarrays and immunohistochemical PTEN staining (monoclonal antibody) were performed. Univariable and multivariable Cox regression models were created to address the association of PTEN protein expression with disease recurrence, and cancer specific and overall mortality. RESULTS: PTEN staining was absent in 45 cases (7.4%). Patients with PTEN loss had significantly advanced pathological tumor stage and grade (p <0.001), and higher rates of lymph node metastasis (p <0.01) and lymphovascular invasion (p <0.001) compared to patients with PTEN expression. PTEN loss was associated with disease recurrence, and cancer specific and overall mortality on univariable Cox regression analyses. However, on multivariable Cox regression analyses adjusted for the effect of standard clinicopathological features PTEN loss was only associated with overall mortality (HR 1.69, 95% CI 1.09-2.61, p = 0.02). CONCLUSIONS: In patients undergoing radical nephroureterectomy for upper tract urothelial carcinoma loss of PTEN protein expression is rare but associated with features of biologically aggressive disease such as higher grade and stage as well as lymph node metastasis. Loss of PTEN expression was associated with overall mortality. PTEN loss seemed to promote worse outcomes in this relatively small group of patients.

Association of oncofetal protein expression with clinical outcomes in patients with urothelial carcinoma of the bladder.

PURPOSE: Oncofetal proteins are expressed in the developing embryo. Oncofetal protein expression correlates with the clinical outcome of nonmuscle invasive bladder urothelial carcinoma. IMP3, MAGE-A, glypican-3 and TPBG are oncofetal proteins that have not been well characterized in urothelial carcinoma of the bladder. MATERIALS AND METHODS: We investigated the expression of these 4 proteins and their association with clinical outcomes using tissue microarrays from 384 consecutive patients treated with radical cystectomy between 1988 and 2003 at 1 academic center. We stained for IMP3, MAGE-A, glypican-3 and TPBG. Univariable and multivariable Cox regression analyses were done to evaluate the association of oncofetal protein expression with disease recurrence and cancer specific mortality. RESULTS: IMP3, MAGE-A, glypican-3 and TPBG were expressed in 39.5%, 45%, 6% and 85% of urothelial bladder carcinomas, respectively. Expression was tumor specific and did not correlate with pathological features except for TPBG. At a median followup of 128 months 176 patients (46%) experienced disease recurrence, 175 (45.5%) had died of the disease and 96 (27.5%) had died of another cause. On univariable analysis IMP3 and MAGE-A expression was associated with an increased risk of disease recurrence (p <0.001 and 0.03) and cancer specific mortality (p = 0.004 and 0.03, respectively). On multivariable Cox regression analysis adjusted for the effects of standard clinicopathological features IMP3 and MAGE-A expression was independently associated with disease recurrence (p = 0.004, HR 1.55, 95% CI 1.15-2.11 and p = 0.02, HR 1.44, 95% CI 1.05-1.99, respectively) but not with cancer specific mortality. CONCLUSIONS: Oncofetal proteins are commonly and differentially expressed in urothelial carcinoma of the bladder compared to normal urothelium. IMP3 and MAGE-A expression was associated with disease recurrence and cancer specific mortality but glypican-3 and TPBG expression was not.

Current gross examination and reporting patterns of post-neoadjuvant chemotherapy cystectomy specimens: Is it time for a standardized approach?

OBJECTIVES: Neoadjuvant chemotherapy (NACT) is recommended for muscle-invasive bladder cancer, and robust treatment response may result in lack of grossly identifiable tumor in the cystectomy specimen. Current gross examination and reporting protocols, however, do not include specific guidance on the approach to these specimens. METHODS: A Qualtrics survey was disseminated by email and X (formerly Twitter). Responses from pathologists and pathologists' assistants (PAs) were included. The survey interrogated demographics, practice settings, prevalence of NACT use, approach to gross examination, and reporting practices in the setting of both grossly visible tumor or ulcer bed and the complete absence of a gross lesion. RESULTS: Based on 55 respondents' experience, identifying gross tumor occurred less frequently than tumor or ulcer bed (40% vs 71%). Lack of identification of any gross lesions was estimated to occur in 29% of cases. Gross examination practices were relatively consistent in cases with residual gross tumor or gross tumor bed, with agreement that gross tumor should be submitted as 1 block per centimeter (66%), and tumor or ulcer bed should be submitted in its entirety (97%). Gross examination practices appeared more varied when no gross lesions were identified. Overall, most responders stated they "definitely" or "maybe" support a standardized gross examination (89%) and reporting (96%) protocol. CONCLUSIONS: With the increased use of NACT, lack of any gross lesion leads to inconsistent gross examination techniques. This study provides insight into the current approach to examination of post-NACT cystectomies and suggests that a desire exists among pathologists and pathologists' assistants for more standardized practice.

Canonical AREs are tumor suppressive regulatory elements in the prostate.

The androgen receptor (AR) is the central determinant of prostate tissue identity and differentiation, controlling normal, growth-suppressive prostate-specific gene expression 1 . It is also a key driver of prostate tumorigenesis, becoming "hijacked" to drive oncogenic transcription 2-5 . However, the regulatory elements determining the execution of the growth suppressive AR transcriptional program, and whether this can be reactivated in prostate cancer (PCa) cells remains unclear. Canonical androgen response element (ARE) motifs are the classic DNA binding element for AR 6 . Here, we used a genome-wide strategy to modulate regulatory elements containing AREs to define distinct AR transcriptional programs. We find that activation of these AREs is specifically associated with differentiation and growth suppressive transcription, and this can be reactivated to cause death in AR + PCa cells. In contrast, repression of AREs is well tolerated by PCa cells, but deleterious to normal prostate cells. Finally, gene expression signatures driven by ARE activity are associated with improved prognosis and luminal phenotypes in human PCa patients. This study demonstrates that canonical AREs are responsible for a normal, growth-suppressive, lineage-specific transcriptional program, that this can be reengaged in PCa cells for potential therapeutic benefit, and genes controlled by this mechanism are clinically relevant in human PCa patients.

The evolution of metastatic upper tract urothelial carcinoma through genomic-transcriptomic and single-cell protein markers analysis.

The molecular characteristics of metastatic upper tract urothelial carcinoma (UTUC) are not well understood, and there is a lack of knowledge regarding the genomic and transcriptomic differences between primary and metastatic UTUC. To address these gaps, we integrate whole-exome sequencing, RNA sequencing, and Imaging Mass Cytometry using lanthanide metal-conjugated antibodies of 44 tumor samples from 28 patients with high-grade primary and metastatic UTUC. We perform a spatially-resolved single-cell analysis of cancer, immune, and stromal cells to understand the evolution of primary to metastatic UTUC. We discover that actionable genomic alterations are frequently discordant between primary and metastatic UTUC tumors in the same patient. In contrast, molecular subtype membership and immune depletion signature are stable across primary and matched metastatic UTUC. Molecular and immune subtypes are consistent between bulk RNA-sequencing and mass cytometry of protein markers from 340,798 single cells. Molecular subtypes at the single-cell level are highly conserved between primary and metastatic UTUC tumors within the same patient.

Distinct mesenchymal cell states mediate prostate cancer progression.

In the complex tumor microenvironment (TME), mesenchymal cells are key players, yet their specific roles in prostate cancer (PCa) progression remain to be fully deciphered. This study employs single-cell RNA sequencing to delineate molecular changes in tumor stroma that influence PCa progression and metastasis. Analyzing mesenchymal cells from four genetically engineered mouse models (GEMMs) and correlating these findings with human tumors, we identify eight stromal cell populations with distinct transcriptional identities consistent across both species. Notably, stromal signatures in advanced mouse disease reflect those in human bone metastases, highlighting periostin's role in invasion and differentiation. From these insights, we derive a gene signature that predicts metastatic progression in localized disease beyond traditional Gleason scores. Our results illuminate the critical influence of stromal dynamics on PCa progression, suggesting new prognostic tools and therapeutic targets.

Functional outcomes following robotic prostatectomy using athermal, traction free risk-stratified grades of nerve sparing.

OBJECTIVE: To report our unique approach for individualizing robotic prostate cancer surgery by risk stratification and sub classification of the periprostatic space into 4 distinct compartments, and thus performing 4 precise different grades of nerve sparing based on neurosurgical principles and to present updated potency and continence outcomes data of patients undergoing robotic-assisted laparoscopic prostatectomy (RALP) using our risk-stratified approach based on layers of periprostatic fascial dissection. PATIENTS AND METHODS: (1) Between January 2005 and December 2010, 2,536 men underwent RALP by a single surgeon at our institution. (2) Included patients were those with ≥ 1-year follow-up and were preoperatively continent and potent, defined as having a SHIM questionnaire score of >21; thus, the final number of patient in the study cohort was 1,335. (3) Postoperative potency was defined as the ability to have successful intercourse (score of ≥ 4 on question 2 of the SHIM); continence was defined as the use of no pads per 24 h. RESULTS: (1) The potency and continence for NS grades 1, 2, 3, and 4 were found to be 90.6, 76.2, 60.5, and 57.1 % (P < 0.001) and 98, 93.2, 90.1, and 88.9 % (P < 0.001), respectively. (2) The overall PSM rates for patients with NS grades 1, 2, 3, and 4 were 10.5, 7, 5.8, and 4.8 %, respectively (P = 0.064). CONCLUSIONS: The study found a correlation between risk-stratified grades of NS technique and continence and potency. Patients with lesser grades of NS had higher rates of potency and continence.

The Cytidine Deaminase APOBEC3G Contributes to Cancer Mutagenesis and Clonal Evolution in Bladder Cancer.

Mutagenic processes leave distinct signatures in cancer genomes. The mutational signatures attributed to APOBEC3 cytidine deaminases are pervasive in human cancers. However, data linking individual APOBEC3 proteins to cancer mutagenesis in vivo are limited. Here, we showed that transgenic expression of human APOBEC3G promotes mutagenesis, genomic instability, and kataegis, leading to shorter survival in a murine bladder cancer model. Acting as mutagenic fuel, APOBEC3G increased the clonal diversity of bladder cancer, driving divergent cancer evolution. Characterization of the single-base substitution signature induced by APOBEC3G in vivo established the induction of a mutational signature distinct from those caused by APOBEC3A and APOBEC3B. Analysis of thousands of human cancers revealed the contribution of APOBEC3G to the mutational profiles of multiple cancer types, including bladder cancer. Overall, this study dissects the mutagenic impact of APOBEC3G on the bladder cancer genome, identifying that it contributes to genomic instability, tumor mutational burden, copy-number loss events, and clonal diversity. SIGNIFICANCE: APOBEC3G plays a role in cancer mutagenesis and clonal heterogeneity, which can potentially inform future therapeutic efforts that restrict tumor evolution. See related commentary by Caswell and Swanton, p. 487.

Novel genetically engineered mouse models for clear cell renal cell carcinoma.

Genetically engineered mouse models (GEMMs) are important immunocompetent models for research into the roles of individual genes in cancer and the development of novel therapies. Here we use inducible CRISPR-Cas9 systems to develop two GEMMs which aim to model the extensive chromosome p3 deletion frequently observed in clear cell renal cell carcinoma (ccRCC). We cloned paired guide RNAs targeting early exons of Bap1, Pbrm1, and Setd2 in a construct containing a Cas9D10A (nickase, hSpCsn1n) driven by tetracycline (tet)-responsive elements (TRE3G) to develop our first GEMM. The founder mouse was crossed with two previously established transgenic lines, one carrying the tet-transactivator (tTA, Tet-Off) and one with a triple-mutant stabilized HIF1A-M3 (TRAnsgenic Cancer of the Kidney, TRACK), both driven by a truncated, proximal tubule-specific γ-glutamyltransferase 1 (ggt or γGT) promoter, to create triple-transgenic animals. Our results indicate that this model (BPS-TA) induces low numbers of somatic mutations in Bap1 and Pbrm1 (but not in Setd2), known tumor suppressor genes in human ccRCC. These mutations, largely restricted to kidneys and testis, induced no detectable tissue transformation in a cohort of 13 month old mice (N = 10). To gain insights into the low frequencies of insertions and deletions (indels) in BPS-TA mice we analyzed wild type (WT, N = 7) and BPS-TA (N = 4) kidneys by RNAseq. This showed activation of both DNA damage and immune response, suggesting activation of tumor suppressive mechanisms in response to genome editing. We then modified our approach by generating a second model in which a ggt-driven, cre-regulated Cas9WT(hSpCsn1) was employed to introduce Bap1, Pbrm1, and Setd2 genome edits in the TRACK line (BPS-Cre). The BPS-TA and BPS-Cre lines are both tightly controlled in a spatiotemporal manner with doxycycline (dox) and tamoxifen (tam), respectively. In addition, whereas the BPS-TA line relies on paired guide RNAs (gRNAs), the BPS-Cre line requires only single gRNAs for gene perturbation. In the BPS-Cre we identified increased Pbrm1 gene-editing frequencies compared to the BPS-TA model. Whereas we did not detect Setd2 edits in the BPS-TA kidneys, we found extensive editing of Setd2 in the BPS-Cre model. Bap1 editing efficiencies were comparable between the two models. Although no gross malignancies were observed in our study, this is the first reported GEMM which models the extensive chromosome 3p deletion frequently observed in kidney cancer patients. Further studies are required (1) to model more extensive 3p deletions, e.g. impacting additional genes, and (2) to increase the cellular resolution, e.g. by employing single-cell RNAseq to ascertain the effects of specific combinatorial gene inactivation.