Morphologic patterns observed in prostate biopsy cases with discrepant grade group and molecular risk classification.

BACKGROUND: Molecular-based risk classifier tests are increasingly being utilized by urologists and radiation oncologists to guide clinical decision making. The Decipher prostate biopsy test is a 22-gene RNA biomarker assay designed to predict likelihood of high-grade disease at radical prostatectomy and risk of metastasis and mortality. The test provides a risk category of low, intermediate, or high. We investigated histologic features of biopsies in which the Grade Group (GG) and Decipher risk category (molecular risk) were discrepant. METHODS: Our institutional urologic outcomes database was searched for men who underwent prostate biopsies with subsequent Decipher testing from 2016 to 2020. We defined discrepant GG and molecular risk as either GG1-2 with high Decipher risk category or GG ≥ 3 with low Decipher risk category. The biopsy slide on which Decipher testing was performed was re-reviewed for GG and various histologic features, including % Gleason pattern 4, types of Gleason pattern 4 and 5, other "high risk" features (e.g., complex papillary, ductal carcinoma, intraductal carcinoma [IDC]), and other unusual and often "difficult to grade" patterns (e.g., atrophic carcinoma, mucin rupture, pseudohyperplastic carcinoma, collagenous fibroplasia, foamy gland carcinoma, carcinoma with basal cell marker expression, carcinoma with prominent vacuoles, and stromal reaction). Follow-up data was also obtained from the electronic medical record. RESULTS: Of 178 men who underwent prostate biopsies and had Decipher testing performed, 41 (23%) had discrepant GG and molecular risk. Slides were available for review for 33/41 (80%). Of these 33 patients, 23 (70%) had GG1-2 (GG1 n = 5, GG2 n = 18) with high Decipher risk, and 10 (30%) had GG ≥ 3 with low Decipher risk. Of the 5 GG1 cases, one case was considered GG2 on re-review; no other high risk features were identified but each case showed at least one of the following "difficult to grade" patterns: 3 atrophic carcinoma, 1 collagenous fibroplasia, 1 carcinoma with mucin rupture, and 1 carcinoma with basal cell marker expression. Of the 18 GG2 high Decipher risk cases, 2 showed GG3 on re-review, 5 showed large cribriform and/or other high risk features, and 10 showed a "difficult to grade" pattern. Of the 10 GG ≥ 3 low Decipher risk cases, 5 had known high risk features including 2 with large cribriform, 1 with IDC, and 1 with Gleason pattern 5. CONCLUSIONS: In GG1-2 high Decipher risk cases, difficult to grade patterns were frequently seen in the absence of other known high risk morphologic features; whether these constitute true high risk cases requires further study. In the GG ≥ 3 low Decipher risk cases, aggressive histologic patterns such as large cribriform and IDC were observed in half (50%) of cases; therefore, the molecular classifier may not capture all high risk histologic patterns.

Multicolor fluorescence microscopy for surgical guidance using a chip-scale imager with a low-NA fiber optic plate and a multi-bandpass interference filter.

In curative-intent cancer surgery, intraoperative fluorescence imaging of both diseased and healthy tissue can help to ensure the successful removal of all gross and microscopic diseases with minimal damage to neighboring critical structures, such as nerves. Current fluorescence-guided surgery (FGS) systems, however, rely on bulky and rigid optics that incur performance-limiting trade-offs between sensitivity and maneuverability. Moreover, many FGS systems are incapable of multiplexed imaging. As a result, clinical FGS is currently limited to millimeter-scale detection of a single fluorescent target. Here, we present a scalable, lens-less fluorescence imaging chip, VISION, capable of sensitive and multiplexed detection within a compact form factor. Central to VISION is a novel optical frontend design combining a low-numerical-aperture fiber optic plate (LNA-FOP) and a multi-bandpass interference filter, which is affixed to a custom CMOS image sensor. The LNA-FOP acts as a planar collimator to improve resolution and compensate for the angle-sensitivity of the interference filter, enabling high-resolution and multiplexed fluorescence imaging without lenses. We show VISION is capable of detecting tumor foci of less than 100 cells at near video framerates and, as proof of principle, can simultaneously visualize both tumors and nerves in ex vivo prostate tissue.

Clinicopathologic and Molecular Characterization of High-Risk Human Papillomavirus-Positive Carcinomas of the Urinary Tract.

CONTEXT.­: Human papillomavirus (HPV) is a well-known cause of squamous cell carcinomas of anogenital and oropharyngeal regions, where treatment strategies and prognosis depend on HPV status. The significance of HPV status in tumors arising along the urinary tract is not well established. OBJECTIVE.­: To provide detailed clinical, morphologic, immunohistochemical, and molecular analysis of HPV+ urinary tract carcinomas (UTCs). DESIGN.­: We identified and retrospectively examined 12 HPV+ UTCs, confirmed by high-risk HPV in situ hybridization. RESULTS.­: The HPV+ UTCs originated from the urethra (9) and urinary bladder (3); 5 of 12 (42%) presented with nodal metastasis. On morphology, HPV+ UTCs were predominantly basaloid; well-differentiated squamous areas were focally seen. Available immunohistochemistry (IHC) showed strong staining for p16 (11 of 11), p63 (12 of 12), cytokeratin (CK) 903 (11 of 11), and CK5/6 (11 of 11); variable staining for GATA3 (8 of 12) and CK7 (4 of 11); and rare uroplakin II staining (1 of 12). Molecular analysis revealed the most frequently altered genes: KMT2C (42%), PIK3CA (42%), and KMT2D (25%). In contrast to published conventional urothelial and squamous cell carcinoma molecular data, TERTp mutation was rare (8%), and no TP53 or CDKN2A aberrations were identified. During available follow-up (11 of 12; median, 39 months), 6 patients required treatment for recurrence; ultimately, 1 died of disease, 2 were alive with disease, and 8 had no evidence of disease. Finally, we provide 11 HPV- squamous predominant UTCs for IHC and molecular comparisons; notably, a subset of HPV- UTCs was positive for p16 IHC (27%), making p16 IHC a less-specific surrogate marker for HPV status at this site. CONCLUSIONS.­: HPV+ UTCs show distinct clinical, morphologic, and molecular characteristics, suggesting important roles for HPV in UTC.

Large cribriform glands (> 0.25 mm diameter) as a predictor of adverse pathology in men with Grade Group 2 prostate cancer.

AIMS: A recent outcome-based, radical prostatectomy study defined > 0.25 mm diameter to distinguish large versus small cribriform glands, with > 0.25 mm associated with worse recurrence-free survival. This study evaluates whether identification of > 0.25 mm cribriform glands in Grade Group 2 patients at biopsy is associated with adverse pathology at radical prostatectomy. METHODS AND RESULTS: Tumours containing biopsy slides for 133 patients with Grade Group 2 prostate cancer with subsequent radical prostatectomy were re-reviewed for large cribriform glands (diameter > 0.25 mm). The primary outcome was adverse pathology (Grade Groups 3-5; stage pT3a or greater, or pN1). The secondary outcome was recurrence-free survival. Cribriform pattern was present in 52 of 133 (39%) patients; of these, 16 of 52 (31%) had large cribriform glands and 36 of 52 (69%) had only small cribriform glands. Patients with large cribriform glands had significantly more adverse pathology at radical prostatectomy compared to patients with small cribriform glands and no cribriform glands (large = 11 of 16, 69%; small = 12 of 36, 33%; no cribriform = 25 of 81, 31%; χ2 P-value 0.01). On multivariate analysis, large cribriform glands were also associated with adverse pathology, independent of age, prostate-specific antigen (PSA)/PSA density at diagnosis, year of diagnosis and biopsy cores percentage positive (global P-value 0.02). Large cribriform glands were also associated with increased CAPRA-S surgical risk score (Kruskal-Wallis P-value 0.02). CONCLUSIONS: Large cribriform glands using a diameter > 0.25 mm definition in Grade Group 2 patients on biopsy are associated with increased risk of adverse pathology at radical prostatectomy. The presence of large cribriform histology should be considered when offering active surveillance for those with Grade Group 2 disease.

Transcriptomic Heterogeneity of Expansile Cribriform and Other Gleason Pattern 4 Prostate Cancer Subtypes.

BACKGROUND: Prostate cancers featuring an expansile cribriform (EC) pattern are associated with worse clinical outcomes following radical prostatectomy (RP). However, studies of the genomic characteristics of Gleason pattern 4 subtypes are limited. OBJECTIVE: To explore transcriptomic characteristics and heterogeneity within Gleason pattern 4 subtypes (fused/poorly formed, glomeruloid, small cribriform, EC/intraductal carcinoma [IDC]) and the association with biochemical recurrence (BCR)-free survival. DESIGN, SETTING, AND PARTICIPANTS: This was a retrospective cohort study including 165 men with grade group 2-4 prostate cancer who underwent RP at a single academic institution (2016-2020) and Decipher testing of the RP specimen. Patients with Gleason pattern 5 were excluded. IDC and EC patterns were grouped. Median follow-up was 2.5 yr after RP for patients without BCR. OUTCOMES MEASUREMENTS AND STATISTICAL ANALYSIS: Prompted by heterogeneity within pattern 4 subtypes identified via exploratory analyses, we investigated transcriptomic consensus clusters using partitioning around medoids and hallmark gene set scores. The primary clinical outcome was BCR, defined as two consecutive prostate-specific antigen measurements >0.2 ng/ml at least 8 wk after RP, or any additional treatment. Multivariable Cox proportional-hazards models were used to determine factors associated with BCR-free survival. RESULTS AND LIMITATIONS: In this cohort, 99/165 patients (60%) had EC and 67 experienced BCR. Exploratory analyses and clustering demonstrated transcriptomic heterogeneity within each Gleason pattern 4 subtype. In the multivariable model controlled for pattern 4 subtype, margin status, Cancer of the Prostate Risk Assessment Post-Surgical score, and Decipher score, a newly identified steroid hormone-driven cluster (hazard ratio 2.35 95% confidence interval 1.01-5.47) was associated with worse BCR-free survival. The study is limited by intermediate follow-up, no validation cohort, and lack of accounting for intratumoral and intraprostatic heterogeneity. CONCLUSIONS: Transcriptomic heterogeneity was present within and across each Gleason pattern 4 subtype, demonstrating there is additional biologic diversity not captured by histologic subtypes. This heterogeneity can be used to develop novel signatures and to classify transcriptomic subtypes, which may help in refining risk stratification following RP to further guide decision-making on adjuvant and salvage treatments. PATIENT SUMMARY: We studied prostatectomy specimens and found that tumors with similar microscopic appearance can have genetic differences that may help to predict outcomes after prostatectomy for prostate cancer. Our results demonstrate that further gene expression analysis of prostate cancer subtypes may improve risk stratification after prostatectomy. Future studies are needed to develop novel gene expression signatures and validate these findings in independent sets of patients.

Multicolor fluorescence microscopy for surgical guidance using a chip-scale imager with a low-NA fiber optic plate and a multi-bandpass interference filter.

In curative-intent cancer surgery, intraoperative fluorescence imaging of both diseased and healthy tissue can help to ensure successful removal of all gross and microscopic disease with minimal damage to neighboring critical structures, such as nerves. Current fluorescence-guided surgery (FGS) systems, however, rely on bulky and rigid optics that incur performance-limiting trade-offs between sensitivity and maneuverability. Moreover, many FGS systems are incapable of multiplexed imaging. As a result, clinical FGS is currently limited to millimeter-scale detection of a single fluorescent target. Here we present a scalable, lens-less fluorescence imaging chip, VISION, capable of sensitive and multiplexed detection within a compact form factor. Central to VISION is a novel optical frontend design combining a low-numerical-aperture fiber optic plate (LNA-FOP) and a multi-bandpass interference filter, which is affixed to a custom CMOS image sensor. The LNA-FOP acts as a planar collimator to improve resolution and compensate for the angle-sensitivity of the interference filter, enabling high-resolution and multiplexed fluorescence imaging without lenses. We show VISION is capable of detecting tumor foci of less than 100 cells at near video framerates and, as proof of principle, can simultaneously visualize both tumor and nerves in ex vivo prostate tissue.

Wilms Tumor: An Unexpected Diagnosis in Adult Patients.

CONTEXT.­: Wilms tumor (WT) in adult patients is rare and has historically been a diagnostic and therapeutic conundrum, with limited data available in the literature. OBJECTIVE.­: To provide detailed diagnostic features, molecular profiling, and patient outcomes in a multi-institutional cohort of adult WT patients. DESIGN.­: We identified and retrospectively examined 4 adult WT cases. RESULTS.­: Two patients presented with metastatic disease, and diagnoses were made on fine-needle aspiration of their renal masses. The aspirates included malignant primitive-appearing epithelioid cells forming tubular rosettes and necrosis, and cell blocks demonstrated triphasic histology. In the remaining 2 cases, patients presented with localized disease and received a diagnosis on resection, with both patients demonstrating an epithelial-predominant morphology. Tumor cells in all cases were patchy variable positive for PAX8 and WT1 immunohistochemistry. Next-generation sequencing identified alterations previously reported in pediatric WT in 3 of 4 cases, including mutations in ASXL1 (2 of 4), WT1 (1 of 4), and the TERT promoter (1 of 4), as well as 1q gains (1 of 4); 1 case showed no alterations. Three patients were treated with pediatric chemotherapy protocols; during follow up (range, 26-60 months), 1 patient died of disease. CONCLUSIONS.­: WT is an unexpected and difficult entity to diagnose in adults and should be considered when faced with a primitive-appearing renal or metastatic tumor. Molecular testing may help exclude other possibilities but may not be sensitive or specific because of the relatively large number of driver mutations reported in WT.

FGFR1 gene fusions in a subset of pediatric mesenchymal tumors: Expanding the genetic spectrum of tumors sharing histologic overlap with infantile fibrosarcoma and "NTRK-rearranged" spindle cell neoplasms.

As the classification of kinase-driven spindle cell tumors continues to evolve, we describe the first series of pediatric mesenchymal tumors harboring FGFR1 gene fusions that share histologic overlap with infantile fibrosarcoma and "NTRK-rearranged" spindle cell neoplasms. Herein, we present three cases of FGFR1-rearranged pediatric mesenchymal tumors, including one case with FGFR1::PARD6B gene fusion and two cases with FGFR1::EBF2 gene fusion. The tumors involved infants ranging from 3 to 9 months in age with a male-to-female ratio of 2:1. All tumors involved the deep soft tissue of the gluteal, pelvic, or perirectal region. Histologically, the tumors comprised a cellular spindle cell neoplasm with primitive stellate cells, focal myxoid stroma, focal epithelioid features, no necrosis, and occasional mitotic figures (2-6 per 10 high-power field). By immunohistochemistry, the neoplastic cells focally expressed CD34 but lacked expression of S100 protein, SMA, desmin, myogenin, MyoD1, pan-TRK, and ALK. These three cases, including a case with long-term clinical follow-up, demonstrate that FGFR1 fusions occur in a subset of newly described pediatric kinase-driven mesenchymal tumors with locally aggressive behavior. Importantly, knowledge of these genetic alterations in this spectrum of pediatric tumors is key for diagnostic and targeted therapeutic purposes.

Urothelial carcinoma in situ with "overriding" features can evade detection by mimicking umbrella cells.

Urothelial carcinoma in situ (uCIS) is typically recognized by overtly malignant cells with characteristic nuclear features; multiple histologic patterns have been described. A rare "overriding" pattern, in which uCIS tumor cells extend on top of normal urothelium, has previously been mentioned in the literature, but not well described. Herein, we report 3 cases of uCIS with "overriding" features. Detailed morphologic evaluation revealed somewhat subtle cytologic atypia: variably enlarged hyperchromatic nuclei and scattered mitotic figures but with abundant cytoplasm and limited to superficial urothelium. Immunohistochemical (IHC) analysis showed a distinctive diffuse positive aberrant p53 pattern, limited to the atypical surface urothelial cells; these cells also showed CK20+, CD44-, and increased Ki-67. In 2 cases, there was a history of urothelial carcinoma and adjacent conventional uCIS. In the third case, the "overriding" pattern was the first presentation of urothelial carcinoma; therefore, next-generation sequencing molecular testing was also performed, revealing pathogenic mutations in TERTp, TP53, and CDKN1a to further support neoplasia. Notably, the "overriding" pattern mimicked umbrella cells, which normally line surface urothelium, can have abundant cytoplasm and more variation in nuclear and cell size and shape, and show CK20+ IHC. We therefore also evaluated umbrella cell IHC patterns in adjacent benign/reactive urothelium, which showed CK20+, CD44-, p53 wild-type, and very low Ki-67 (3/3). We also reviewed 32 cases of normal/reactive urothelium: all showed p53 wild-type IHC in the umbrella cell layer (32/32). In conclusion, caution is warranted to avoid overdiagnosis of usual umbrella cells as CIS; however, "overriding" uCIS should be recognized, may have morphologic features that fall short of the diagnostic threshold of conventional CIS, and requires further study.

Immunotherapeutic Targeting and PET Imaging of DLL3 in Small-Cell Neuroendocrine Prostate Cancer.

Effective treatments for de novo and treatment-emergent small-cell/neuroendocrine (t-SCNC) prostate cancer represent an unmet need for this disease. Using metastatic biopsies from patients with advanced cancer, we demonstrate that delta-like ligand 3 (DLL3) is expressed in de novo and t-SCNC and is associated with reduced survival. We develop a PET agent, [89Zr]-DFO-DLL3-scFv, that detects DLL3 levels in mouse SCNC models. In multiple patient-derived xenograft models, AMG 757 (tarlatamab), a half-life-extended bispecific T-cell engager (BiTE) immunotherapy that redirects CD3-positive T cells to kill DLL3-expressing cells, exhibited potent and durable antitumor activity. Late relapsing tumors after AMG 757 treatment exhibited lower DLL3 levels, suggesting antigen loss as a resistance mechanism, particularly in tumors with heterogeneous DLL3 expression. These findings have been translated into an ongoing clinical trial of AMG 757 in de novo and t-SCNC, with a confirmed objective partial response in a patient with histologically confirmed SCNC. Overall, these results identify DLL3 as a therapeutic target in SCNC and demonstrate that DLL3-targeted BiTE immunotherapy has significant antitumor activity in this aggressive prostate cancer subtype. SIGNIFICANCE: The preclinical and clinical evaluation of DLL3-directed immunotherapy, AMG 757, and development of a PET radiotracer for noninvasive DLL3 detection demonstrate the potential of targeting DLL3 in SCNC prostate cancer.

Molecular characterization of adenocarcinomas arising in the urinary bladder following augmentation cystoplasty: a multi-institutional study.

Development of malignancy is a rare complication following augmentation cystoplasty, and the majority of tumors observed in this setting are adenocarcinomas. Here, we sought to genetically profile these tumors by targeted DNA sequencing of a multi-institutional cohort of adenocarcinomas that developed in the urinary bladder following augmentation cystoplasty. Carcinomas arising in the urinary bladder of patients with history of augmentation cystoplasty were obtained from 4 major academic institutions, with cases diagnosed as urothelial carcinoma excluded from the study. The cases were analyzed using a DNA sequencing panel that includes 529 genes and genome-wide copy number assessment. The most frequently altered genes included TP53, KRAS, and MYC, and the vast majority of cases demonstrated mutational profiles consistent with gastrointestinal adenocarcinomas. One case demonstrated an EML4::ALK fusion together with an MSH3 frameshift mutation and hypermutated phenotype, characteristic of a rare but aggressive subtype of colorectal adenocarcinoma that may benefit from targeted ALK inhibition therapy. Importantly, six other tumors in the cohort also had potentially targetable molecular alterations, involving ATM (2 cases), BRCA1 (2 cases), EGFR (1 case), and ERBB2 (1 case). To our knowledge, this study represents the most comprehensive molecular characterization to date of adenocarcinomas arising in the urinary bladder following augmentation cystoplasty. Despite the unique environment of the augmented tissue, the resulting tumors demonstrate a spectrum of driver mutations similar to that of primary gastrointestinal adenocarcinomas. Importantly, molecular alterations potentially amenable to targeted therapy were identified in the majority of cases.

p53 null phenotype is a "positive result" in urothelial carcinoma in situ.

The concept of a "p53 null phenotype" (complete loss of staining) is well-recognized in the gynecologic pathology literature, implicitly reflecting that this staining pattern represents a TP53 mutation. However, in the genitourinary pathology literature, a p53 null phenotype has only been addressed regarding the prognosis of invasive urothelial carcinoma, and not as a diagnostic biomarker for urothelial carcinoma in situ (CIS). Herein, 25 cases of urothelial carcinoma in situ [diagnoses made on hematoxylin and eosin (H&E) stained sections] showing null pattern p53 staining were retrieved from 22 different patients (16 males and 6 females, age range 52-85 years; average 69.6 years), most commonly showing large cell pleomorphic pattern morphology. One representative tissue block per case was selected for next-generation DNA sequencing (NGS). All 21 cases (100%) passing quality control for NGS showed at least 1 TP53 mutation (majority nonsense or frameshift mutations), including 3 cases with 2 mutations and 3 cases with 3 mutations. Three patients with multiple available samples harbored 1 or more shared TP53 mutations at 2 different time points, indicating clonality of the temporally distinct lesions. Additionally, 2 patients had an additional unique TP53 mutation at a later time point, suggesting intratumoral heterogeneity and/or temporal clonal evolution. While urothelial CIS remains an H&E diagnosis in most cases, a p53 immunostain may be useful in a subset of challenging cases. This study demonstrates that a p53 null phenotype represents an aberrant result in urothelial CIS with supportive molecular analysis showing a previously unknown level of complexity for TP53 mutations among these noninvasive lesions. Adequate recognition of the p53 null phenotype as a "biologically supportive result", similar to strong and diffuse staining with p53, is important and may warrant a formal consensus statement for recommended p53 reporting (i.e., "wild type" versus "aberrant or mutant").

LZTS2: A novel and independent prognostic biomarker for clear cell renal cell carcinoma.

PURPOSE: Leucine zipper tumor suppressor 2 (LZTS2), a putative tumor suppressor gene, has been demonstrated to be a negative regulator of microtubule severing during cytokinesis and a negative regulator of the Wnt signaling pathway. In a genetically modified mouse model, deletion of Lzts2 altered normal ureteric bud branching morphogenesis and caused cystogenesis in mice. Cyst-lining cells demonstrated atypical features, closely resembling those observed in mouse models of human clear cell renal cell carcinoma (ccRCC), which could represent a preneoplastic lesion. These findings suggest that LZTS2 may play a role in ccRCC tumorigenesis. The aim of this study was to establish an association between LZTS2 differential expression and clinicopathological features of ccRCC and to investigate its prognostic value as well as the underlying mechanisms in ccRCC progression. MATERIALS AND METHODS: Gene expression data by RNA-sequencing for cohorts of 510 ccRCC cases with clinical outcome data were extracted from The Cancer Genome Atlas (TCGA) using cBioPortal. Chi-square test of independence, Kaplan-Meier curves, and Cox regression models were used to investigate the possible relationship between LZTS2 mRNA expression levels and clinicopathological parameters as well as patient survival to establish its prognostic values. To examine its cellular localization, we performed LZTS2 antibody staining and scored the expression levels in a pilot study on a tissue microarray (TMA) containing 31 clear cell RCCs, 32 chromophobe RCCs, 12 papillary RCCs, and 20 adjacent benign renal tissue, as well as placental tissue diagnosed between 2001 and 2007 at the University of California San Francisco Medical Center. Staining was subsequently repeated in 15 ccRCCs on whole section slides to confirm the results. RESULTS: Our analysis of TCGA data demonstrated that LZTS2 expression levels were associated with tumor grade (p = 0.005), T stage (p < 0.001), metastasis status (p < 0.001), and overall clinical stage (p < 0.001). High level of expression was correlated with worse overall survival (p < 0.001), disease-specific survival (p < 0.001), progression-free survival (p < 0.001), and disease-free survival (p < 0.001) compared to low level of expression. Multivariate Cox regression analysis revealed that high LZTS2 expression was an independent poor prognostic factor for overall survival (HR = 2.083, p < 0.001), disease-specific survival (HR = 2.298, p < 0.001), and progression-free survival (HR = 1.896, p < 0.001) in patients with ccRCC. A few known driver mutations in ccRCC pathogenesis, including BAP1, NF2, and RELN, were enriched in LZTS2 high expression tumors. In particular, LZTS2 expression level could be a biomarker for risk stratification of the prognosis of BAP1-mutated ccRCCs. Immunohistochemical staining with anti-LZTS2 antibody was performed to examine its cellular localization in ccRCC and demonstrated centrosomal and acentrosomal distribution in tumors of various Fuhrman nuclear grades. Furthermore, high LZTS2 cytoplasmic expression was associated with centrosomal amplification (p = 0.030) in this small-scale study. CONCLUSIONS: The current study established an independent prognostic value of LZTS2 expression in ccRCC and explored the molecular mechanisms of LZTS2 in predicting the prognosis of ccRCC. Further studies are needed to validate our analysis and to provide a precise understanding of the function of LZTS2 in ccRCC.

TROP2 Expression Across Molecular Subtypes of Urothelial Carcinoma and Enfortumab Vedotin-resistant Cells.

Sacituzumab govitecan (SG) is an antibody-drug conjugate (ADC) targeting TROP2, which has recently been approved for treatment-refractory metastatic urothelial cancer (UC). However, the variability of TROP2 expression across different bladder cancer (BC) subtypes, as well as after enfortumab vedotin (EV) exposure, remains unknown. Using gene expression data from four clinical cohorts with >1400 patient samples of muscle-invasive BC and a BC tissue microarray, we found that TROP2 mRNA and protein are highly expressed across basal, luminal, and stroma-rich subtypes, but depleted in the neuroendocrine subtype. In addition, TROP2 mRNA levels are correlated with NECTIN4 mRNA but are more highly expressed than NECTIN4 mRNA in patient cohorts and BC cell lines. Moreover, CRISPR/Cas9-mediated knockdown of TROP2 demonstrates that its expression is one factor governing SG sensitivity. After prolonged EV exposure, cells can downregulate NECTIN4, leading to EV resistance, but retain TROP2 expression and remain sensitive to SG, suggesting nonoverlapping resistance mechanisms to these ADCs. While our findings warrant further validation, they have significant implications for biomarker development, patient selection, and treatment sequencing in the clinic as well as clinical trial design and stratification for metastatic BC patients. PATIENT SUMMARY: In this report, we investigated the expression levels of the drug target TROP2 across different molecular subtypes of bladder cancer in multiple patient cohorts and cell lines. We found high levels of TROP2 in most subtypes except in the neuroendocrine subtype. Overall, TROP2 gene expression is higher than NECTIN4 gene expression, and cells resistant to enfortumab vedotin (EV), a NECTIN4-targeting antibody-drug conjugate, remain sensitive to sacituzumab govitecan (SG). Our findings suggest that SG may be effective across most bladder cancer subtypes, including the bladder cancers previously treated with EV.

Single-cell analysis of human primary prostate cancer reveals the heterogeneity of tumor-associated epithelial cell states.

Prostate cancer is the second most common malignancy in men worldwide and consists of a mixture of tumor and non-tumor cell types. To characterize the prostate cancer tumor microenvironment, we perform single-cell RNA-sequencing on prostate biopsies, prostatectomy specimens, and patient-derived organoids from localized prostate cancer patients. We uncover heterogeneous cellular states in prostate epithelial cells marked by high androgen signaling states that are enriched in prostate cancer and identify a population of tumor-associated club cells that may be associated with prostate carcinogenesis. ERG-negative tumor cells, compared to ERG-positive cells, demonstrate shared heterogeneity with surrounding luminal epithelial cells and appear to give rise to common tumor microenvironment responses. Finally, we show that prostate epithelial organoids harbor tumor-associated epithelial cell states and are enriched with distinct cell types and states from their parent tissues. Our results provide diagnostically relevant insights and advance our understanding of the cellular states associated with prostate carcinogenesis.

Molecular risk classifier score and biochemical recurrence risk are associated with cribriform pattern type in Gleason 3+4=7 prostate cancer.

PURPOSE: Among Gleason pattern 4 types, cribriform pattern is associated with the worst outcomes. We hypothesized that larger cribriform patterns would be associated with increased Decipher scores and higher biochemical recurrence (BCR) risk in Gleason 3+4=7 prostatectomy patients. MATERIALS AND METHODS: The slide from patients who underwent prostatectomy from January 2016 to March 2020 on which Decipher was performed was re-reviewed for Gleason score and cribriform patterns, with large cribriform defined as cribriform acini with greater than 12 lumens and simple cribriform as 12 or fewer lumens. Differences in Decipher score were analyzed in a generalized linear model controlling for pathology stage and tumor margin status. A multivariable Cox proportional hazards model was performed for BCR-free survival. RESULTS: Of 337 cases, 118 were Gleason 3+4=7. The mean Decipher scores in 3+4=7 cases without cribriform, with simple cribriform, and with large cribriform were 0.41, 0.54, and 0.62, respectively. In a multivariable model with pathology stage, margin tumor length, and percentage pattern 4 as covariates, compared to cases without cribriform, simple cribriform was associated with 0.10 increase in Decipher (p=0.03) and 4.7-fold hazard ratio of BCR (95% confidence interval [CI], 0.4-56.5; p=0.22) and large cribriform was associated with 0.17 increase in Decipher (p<0.001) and 16.0-fold hazard ratio of BCR (95% CI, 1.4-181.2; p=0.02). CONCLUSIONS: Among Gleason 3+4=7 carcinomas, large cribriform was associated with higher Decipher scores and greater BCR risk. Our results support that large cribriform is an aggressive pattern 4 subtype and should be considered a contraindication for active surveillance.

Papillary Renal Cell Carcinoma With Microcystic Architecture Is Strongly Associated With Extrarenal Invasion and Metastatic Disease.

Papillary renal cell carcinoma (PRCC) is well-recognized as a morphologically and molecularly heterogenous group of kidney tumors with variable clinical behavior. Our goal was to analyze a unique histologic pattern of PRCC we have observed in routine practice to evaluate for potential clinical significance or distinct molecular signature. We identified 42 cases of PRCC showing a morphologically distinct architecture characterized by numerous epithelial-lined cysts containing the papillary tumor (herein called "microcysts"), which are typically separated by fibrous stroma. Of the initial 42 case test set with microcystic features, 23 (55%) were stage pT3a or higher. Most tumors had strong and diffuse cytoplasmic immunoreactivity for CK7 (93%, 37/40) and AMACR (100%, 40/40). Fumarate hydratase staining was retained in all cases tested (39/39). We performed next-generation sequencing on 15 of these cases with available tissue and identified chromosomal alterations commonly reported in historically "type 1" PRCC, notably multiple chromosomal gains, particularly of chromosomes 7 and 17, and MET alterations. However, alterations in pathways associated with more aggressive behavior (including SETD2, CDKN2A, and members of the NRF pathway) were also identified in 6 of 15 cases tested (40%). Given this molecular and immunophenotypic data, we subsequently reviewed an additional group of 60 consecutive pT2b-pT3 PRCCs to allow for comparisons between cases with and without microcysts, to assess for potential associations with other recently described histologic patterns (ie, "unfavorable architecture": micropapillary, solid, and hobnail), and to assess interobserver reproducibility for diagnosing architectural patterns and grade. Of the total combined 102 PRCCs, 67 (66%) had microcystic architecture within the intrarenal component but were commonly admixed with other patterns (39% had micropapillary, 31% solid, and 31% hobnail). Twenty-seven cases (26%) had metastatic disease, and 24 of these 27 (89%) had microcystic architecture in the intrarenal tumor. Within the pT3 subset, 21 of 22 cases with metastases (95%) had extrarenal invasion as either individual microcysts in renal sinus fat or aggregates of microcysts bulging beyond the confines of the capsule. Backward elimination and stepwise regression methods to detect features significantly associated with adverse outcome identified solid architecture (hazard ratio [HR]: 6.3; confidence interval [CI]: 2.1-18.8; P=0.001), hobnail architecture (HR: 5.3; CI: 1.7-16.7; P=0.004), and microcystic architecture at the tumor-stromal interface (HR: 4.2; CI: 1.1-16.7; P=0.036) as strongest. Of architectural patterns and grade, the microcystic pattern had a substantial interobserver agreement (κ score=0.795) that was highest among the 6 observers. In summary, PRCCs with microcystic architecture represents a subset of historically "type 1" PRCC with a predilection for morphologically distinctive extrarenal involvement and metastatic disease. Microcysts co-vary with other "unfavorable" architectural patterns also associated with higher risk for aggressive disease (ie, micropapillary, hobnail, and solid), but microcysts were more common and have superior interobserver reproducibility. These findings suggest that microcystic PRCC should be recognized as a potentially aggressive histologic pattern of growth in PRCC.

Differential immunohistochemical and molecular profiling of conventional and aggressive components of chromophobe renal cell carcinoma: pitfalls for diagnosis.

Chromophobe renal cell carcinoma (ChRCC) is a relatively rare subtype of RCC with a characteristic histologic appearance. Most ChRCCs are slow growing, but sarcomatoid differentiation and metastases can occur, indicative of aggressive behavior and poor prognosis. Herein, we characterize ten ChRCCs with aggressive components, defined as sarcomatoid change and/or metastasis. Immunohistochemistry (IHC) and next-generation sequencing were performed on available formalin-fixed paraffin-embedded tissue, with differential profiling of conventional and aggressive components. All ten cases showed a conventional component of renal tumor morphologically consistent with ChRCC: three had sarcomatoid change, four had metastases, and three had both sarcomatoid change and metastases. In the primary conventional components, a typical ChRCC IHC pattern (CK7+, CD117+, and CAIX-) was observed in 8 of 10 cases; 2 cases had rare CK7 staining. In the aggressive components, CD117 and/or CK7 was lost in 7 of 10 cases; 3 cases showed loss of both. Two of 10 cases showed significant CAIX staining in the aggressive component. All 7 cases that had molecular profiling performed showed characteristic chromosomal losses reported for ChRCC, with the aggressive components generally demonstrating more copy number complexity. Recurrent TP53 mutations (TP53m) were also seen; however, surprisingly, the conventional and aggressive components had no shared TP53m: a TP53m was private to aggressive components in 2 cases and to the conventional component in 1 case, and in 4 cases, components demonstrated different TP53m. Of the 21 pathogenic alterations identified in 7 tumors, only a PTEN splicing alteration was shared between both components in one case. In conclusion, ChRCC can have IHC staining patterns and molecular profile that differ between conventional and aggressive components. Interpretation of stains on metastases or small biopsies to determine histologic subtype can be misleading. The lack of shared pathogenic mutations between the two components supports a model in which aggressive ChRCC can have convergent subclones with different TP53m.

Heterogeneity in NECTIN4 Expression Across Molecular Subtypes of Urothelial Cancer Mediates Sensitivity to Enfortumab Vedotin.

PURPOSE: Enfortumab vedotin (EV) is an antibody-drug conjugate (ADC) targeting NECTIN4 (encoded by the PVRL4/NECTIN4 gene) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In this study, we sought to (i) examine heterogeneity of NECTIN4 gene expression across molecular subtypes of bladder cancer and (ii) determine whether NECTIN4 expression mediates EV sensitivity or resistance. EXPERIMENTAL DESIGN: Molecular subtyping and NECTIN4 expression data from seven muscle-invasive bladder cancer clinical cohorts (n = 1,915 total specimens) were used to assess NECTIN4 expression across molecular subtypes. The outcome of the transcriptomic analysis was relative NECTIN4 expression in the consensus molecular subtypes of bladder cancer. Expression of NECTIN4 was validated in bladder cancer cell lines. NECTIN4 was stably overexpressed or knocked down in basal and luminal bladder cancer cell lines and EV drug sensitivity assays were performed, as measured by cell proliferation and clonogenic assays. RESULTS: NECTIN4 expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes. NECTIN4 expression is positively correlated with luminal markers GATA3, FOXA1, and PPARG across all cohorts. NECTIN4 expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of NECTIN4 leads to EV resistance. CONCLUSIONS: Sensitivity to EV is mediated by expression of NECTIN4, which is enriched in luminal subtypes of bladder cancer. These findings may have implications for biomarker development, patient selection, and the inclusion of molecular subtyping in ongoing and future EV clinical trials.See related commentary by Teo and Rosenberg, p. 4950.

Metabolic imaging with hyperpolarized 13 C pyruvate magnetic resonance imaging in patients with renal tumors-Initial experience.

BACKGROUND: Optimal treatment selection for localized renal tumors is challenging because of their variable biologic behavior and limitations in the preoperative assessment of tumor aggressiveness. The authors investigated the emerging hyperpolarized (HP) 13 C magnetic resonance imaging (MRI) technique to noninvasively assess tumor lactate production, which is strongly associated with tumor aggressiveness. METHODS: Eleven patients with renal tumors underwent HP 13 C pyruvate MRI before surgical resection. Tumor 13 C pyruvate and 13 C lactate images were acquired dynamically. Five patients underwent 2 scans on the same day to assess the intrapatient reproducibility of HP 13 C pyruvate MRI. Tumor metabolic data were compared with histopathology findings. RESULTS: Eight patients had tumors with a sufficient metabolite signal-to-noise ratio for analysis; an insufficient tumor signal-to-noise ratio was noted in 2 patients, likely caused by poor tumor perfusion and, in 1 patient, because of technical errors. Of the 8 patients, 3 had high-grade clear cell renal cell carcinoma (ccRCC), 3 had low-grade ccRCC, and 2 had chromophobe RCC. There was a trend toward a higher lactate-to-pyruvate ratio in high-grade ccRCCs compared with low-grade ccRCCs. Both chromophobe RCCs had relatively high lactate-to-pyruvate ratios. Good reproducibility was noted across the 5 patients who underwent 2 HP 13 C pyruvate MRI scans on the same day. CONCLUSIONS: The current results demonstrate the feasibility of HP 13 C pyruvate MRI for investigating the metabolic phenotype of localized renal tumors. The initial data indicate good reproducibility of metabolite measurements. In addition, the metabolic data indicate a trend toward differentiating low-grade and high-grade ccRCCs, the most common subtype of renal cancer. LAY SUMMARY: Renal tumors are frequently discovered incidentally because of the increased use of medical imaging, but it is challenging to identify which aggressive tumors should be treated. A new metabolic imaging technique was applied to noninvasively predict renal tumor aggressiveness. The imaging results were compared with tumor samples taken during surgery and showed a trend toward differentiating between low-grade and high-grade clear cell renal cell carcinomas, which are the most common type of renal cancers.