Metabolic reprogramming of tumor-associated macrophages using glutamine antagonist JHU083 drives tumor immunity in myeloid-rich prostate and bladder cancer tumors.

Glutamine metabolism in tumor microenvironments critically regulates anti-tumor immunity. Using glutamine-antagonist prodrug JHU083, we report potent tumor growth inhibition in urologic tumors by JHU083-reprogrammed tumor-associated macrophages (TAMs) and tumor-infiltrating monocytes (TIMs). We show JHU083-mediated glutamine antagonism in tumor microenvironments induces TNF, pro-inflammatory, and mTORC1 signaling in intratumoral TAM clusters. JHU083-reprogrammed TAMs also exhibit increased tumor cell phagocytosis and diminished pro-angiogenic capacities. In vivo inhibition of TAM glutamine consumption resulted in increased glycolysis, a broken TCA cycle, and purine metabolism disruption. Although the anti-tumor effect of glutamine antagonism on tumor-infiltrating T cells was moderate, JHU083 promoted a stem cell-like phenotype in CD8+ T cells and decreased Treg abundance. Finally, JHU083 caused a ubiquitous shutdown in glutamine utilizing metabolic pathways in tumor cells, leading to reduced HIF-1alpha, c-MYC phosphorylation, and induction of tumor cell apoptosis, all key anti-tumor features.

Development and Validation of a Multivariable Nomogram Predictive of Post-Nephroureterectomy Renal Function.

BACKGROUND AND OBJECTIVE: The timing of perioperative nephrotoxic chemotherapy for upper tract urothelial carcinoma (UTUC) remains controversial and strongly depends on predicted platinum eligibility after radical nephroureterectomy (RNU). The study objective was to develop and validate a multivariable nomogram to predict estimated glomerular filtration rate (eGFR) following RNU. METHODS: This was a multi-institutional retrospective study of patients with UTUC treated with RNU from 2000 to 2020 at seven high-volume referral centers. Use of adjuvant chemotherapy was risk-stratified. Patients were retrospectively randomly allocated 2:1 to discovery and validation cohorts. Discovery data were used to identify independent factors associated with GFR at 1-3 mo after RNU on linear regression, and backward selection was applied for model construction. Accuracy was defined as the percentage of predicted eGFR results within 30% of the corresponding observed eGFR. KEY FINDINGS AND LIMITATIONS: We included 1100 patients, of whom 733 were in the discovery and 367 were in the validation cohort. Multivariable predictors of postoperative eGFR decline included advanced age (odds ratio [OR] -0.18, 95% confidence interval [CI] -0.28 to -0.08), diabetes (OR -2.38, 95% CI -4.64 to -0.11), and hypertension (OR -2.24, 95% CI -4.16 to -0.32). Factors associated with favorable postoperative eGFR included larger tumor size (OR 10.57, 95% CI 7.4-13.74 for tumors >5 cm vs ≤2 cm) and preoperative eGFR (OR 0.44, 95% CI 0.39-0.49). A composite nomogram predicted postoperative eGFR with good accuracy in both the discovery (80.5%) and validation (78.6%) cohorts. Limitations include exclusion of patients who received neoadjuvant chemotherapy. CONCLUSIONS: A nomogram that incorporates ubiquitous preoperative clinical variables can predict post-RNU eGFR and was validated with an independent cohort. PATIENT SUMMARY: We developed a tool that uses patient data to predict eligibility for chemotherapy after surgery to remove the kidney and ureter in patients with cancer in the upper urinary tract.

Immunomodulatory response to neoadjuvant nivolumab in non-metastatic clear cell renal cell carcinoma.

Novel perioperative strategies are needed to reduce recurrence rates in patients undergoing nephrectomy for high-risk, non-metastatic clear cell renal cell carcinoma (ccRCC). We conducted a prospective, phase I trial of neoadjuvant nivolumab prior to nephrectomy in 15 evaluable patients with non-metastatic ccRCC. We leveraged tissue from that cohort to elucidate the effects of PD-1 inhibition on immune cell populations in ccRCC and correlate the evolving immune milieu with anti-PD-1 response. We found that nivolumab durably induces a pro-inflammatory state within the primary tumor, and baseline immune infiltration within the primary tumor correlates with nivolumab responsiveness. Nivolumab increases CTLA-4 expression in the primary tumor, and subsequent nephrectomy increases circulating concentrations of sPD-L1, sPD-L3 (sB7-H3), and s4-1BB. These findings form the basis to consider neoadjuvant immune checkpoint inhibition (ICI) for high-risk ccRCC while the tumor remains in situ and provide the rationale for perioperative strategies of novel ICI combinations.

Oncologic Outcomes in Patients with Residual Upper Tract Urothelial Carcinoma Following Neoadjuvant Chemotherapy.

BACKGROUND AND OBJECTIVE: Growing evidence supports the use of neoadjuvant chemotherapy (NAC) for upper tract urothelial carcinoma (UTUC). However, the implications of residual UTUC at radical nephroureterectomy (RNU) after NAC are not well characterized. Our objective was to compare oncologic outcomes for pathologic risk-matched patients who underwent RNU for UTUC who either received NAC or were chemotherapy-naïve. METHODS: We retrospectively identified 1993 patients (including 112 NAC recipients) who underwent RNU for nonmetastatic, high-grade UTUC between 1985 and 2022 in a large, international, multicenter cohort. We divided the cohort into low-risk and high-risk groups defined according to pathologic findings of muscle invasion and lymph node involvement at RNU. Recurrence-free survival (RFS), overall survival (OS), and cancer-specific survival (CSS) estimates were calculated using the Kaplan-Meier method. Multivariable analyses were performed to determine clinical and demographic factors associated with these outcomes. KEY FINDINGS AND LIMITATIONS: Among patients with low-risk pathology at RNU, RFS, OS, and CSS were similar between the NAC and chemotherapy-naïve groups. Among patients with high-risk pathology at RNU, the NAC group had poorer RFS (hazard ratio [HR] 3.07, 95% confidence interval [CI] 2.10-4.48), OS (HR 2.06, 95% CI 1.33-3.20), and CSS (subdistribution HR 2.54, 95% CI 1.37-4.69) in comparison to the pathologic risk-matched, chemotherapy-naïve group. Limitations include the lack of centralized pathologic review. CONCLUSIONS AND CLINICAL IMPLICATIONS: Patients with residual invasive disease at RNU after NAC represent a uniquely high-risk population with respect to oncologic outcomes. There is a critical need to determine an optimal adjuvant approach for these patients. PATIENT SUMMARY: We studied a large, international group of patients with cancer of the upper urinary tract who underwent surgery either with or without receiving chemotherapy beforehand. We identified a high-risk subgroup of patients with residual aggressive cancer after chemotherapy and surgery who should be prioritized for clinical trials and drug development.

Tumour tissue-derived small extracellular vesicles reflect molecular subtypes of bladder cancer.

mRNA-based molecular subtypes have implications for bladder cancer prognosis and clinical benefit from certain therapies. Whether small extracellular vesicles (sEVs) can reflect bladder cancer molecular subtypes is unknown. We performed whole transcriptome RNA sequencing for formalin fixed paraffin embedded (FFPE) tumour tissues and sEVs separated from matched tissue explants, urine and plasma in patients with bladder cancer. sEVs were separated using size-exclusion chromatography, and characterized by transmission electron microscopy, nano flow cytometry and western blots, respectively. High yield of sEVs were obtained using approximately 1 g of tissue, incubated with media for 30 min. FFPE tumour tissue and tumour tissue-derived sEVs demonstrated good concordance in molecular subtype classification. All urinary sEVs were classified as luminal subtype, while all plasma sEVs were classified as Ba/Sq subtype, regardless of the molecular subtypes indicated by their matched FFPE tumour tissue. The comparison within urine sEVs, which may exclude the sample type specific background, could pick up the different biology between NMIBC and MIBC, as well as the signature genes related to molecular subtypes. Four candidate sEV-related bladder cancer-specific mRNA biomarkers, FAM71E2, OR4K5, FAM138F and KRTAP26-1, were identified by analysing matched urine sEVs, tumour tissue derived sEVs, and adjacent normal tissue derived sEVs. Compared to sEVs separated from biofluids, tissue-derived sEVs may reflect more tissue- or disease-specific biological features. Urine sEVs are promising biomarkers to be used for liquid biopsy-based molecular subtype classification, but the current algorithm needs to be modified/adjusted. Future work is needed to validate the four new bladder cancer-specific biomarkers in large cohorts.

Intravenous BCG vaccination reduces SARS-CoV-2 severity and promotes extensive reprogramming of lung immune cells.

Bacillus Calmette-Guérin (BCG) confers heterologous immune protection against viral infections and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here, we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model. BCG vaccination conferred a modest reduction on lung SCV2 viral load, bronchopneumonia scores, and weight loss, accompanied by a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. BCG uniquely recruited immunoglobulin-producing plasma cells to the lung suggesting accelerated local antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, with a transcriptional shift away from exhaustion markers and toward antigen presentation and repair. Similarly, BCG enhanced recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, that show reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

Novel urinary tract obstruction marker discovery by multi-marker profiling of urinary extracellular vesicles derived from a rat UTO model.

INTRODUCTION: Congenital urinary obstruction is a common cause of end-stage renal disease in the pediatric population. However, non-invasive diagnostics to predict which patients will benefit from early intervention are lacking. METHODS: Using a rat model of upper and lower urinary tract partial obstruction and the Nanostring nCounter Fibrosis V2 Panel, we evaluated the mRNA cargo of urinary small extracellular vesicles (sEVs) and mRNA expression patterns of kidney and bladder tissues from rats with lower tract urinary obstruction and upper tract urinary obstruction. RESULTS: While mRNA hierarchical clustering of urinary sEVs was unable to differentiate upper compared to lower tract urinary obstruction, clustering was able to detect overall disease state (UUTO or LUTO) versus healthy controls. Further, urinary sEVs carried genes unique to each treatment group (UUTO: 59 genes, LUTO: 17 genes), while only one gene was uniquely carried in the control group. Notable genes of interest found in urinary sEVs were VCAM-1 and NOS1 for UUTO, Egfr for LUTO, and Pck1 for healthy controls. CONCLUSION: This study provides support that differential gene expression of urinary sEV mRNA has potential to act as biomarkers in the diagnosis and prognosis of UTO. Urinary sEVs demonstrated higher numbers of unique genes representative of injury to the kidney than that of injury to the bladder. Importantly, there were genes unique to UUTO sEVs, indicating the extent and reversibility of renal damage can be independent of the function, damage, and architecture of the bladder.

Comparison of clinicopathological characteristics, gene expression profiles, mutational analysis, and clinical outcomes of pure and mixed small-cell carcinoma of the bladder.

AIMS: Small cell bladder carcinoma (SCBC) is a rare, divergent form of urothelial carcinoma (UC). We aimed to determine whether pure (n = 16) and mixed (SCBC and UC; n = 30) tumours differed in pathology, gene expression characteristics, genetic alterations, and clinical outcomes. METHODS AND RESULTS: Forty (87%) patients received first-line chemotherapy. Twenty-nine patients had no metastatic disease at diagnosis and underwent radical cystectomy. There were no differences in age, sex, race distribution, tumour size, stage at presentation, therapy response with pathological downstaging to ≤ypT1N0, or overall or progression-free survival (PFS) between pure and mixed tumours. There was a longer PFS among downstaged chemotherapy-responding tumours ≤ypT2N0M0 than among unresponsive tumours ≥ypT2 ≥ yN1M1 (P = 0.001). Patients who achieved pathological downstaging with neoadjuvant chemotherapy (n = 10) were stage cT2N0M0 at the time of diagnosis and were alive at the last follow-up (median 37 months), while 46% of patients who failed to achieve pathological downstaging were alive at the last follow-up (median 38 months; P = 0.008). RNA sequencing showed that the UC of mixed SCBC had similar neural expression signatures to pure SCBC. DNA sequencing revealed alterations in TERT (83%), P53 (56%), ARID1A (28%), RB1 (22%), and BRCA2 (11%). Immunohistochemistry for RB1 showed loss of expression in 18/19 (95%) patients, suggesting frequent pathway downregulation despite a low prevalence of RB1 mutation. CONCLUSION: Patients with pure and mixed SCBC have similar outcomes and these outcomes are determined by the pathological stage at RC and are best among patients who have pathological downstaging after NAC.

Improved bladder cancer antitumor efficacy with a recombinant BCG that releases a STING agonist.

Despite the introduction of several new agents for the treatment of bladder cancer (BC), intravesical BCG remains a first line agent for the management of non-muscle invasive bladder cancer. In this study we evaluated the antitumor efficacy in animal models of BC of a recombinant BCG known as BCG-disA-OE that releases the small molecule STING agonist c-di-AMP. We found that compared to wild-type BCG (BCG-WT), in both the orthotopic, carcinogen-induced rat MNU model and the heterotopic syngeneic mouse MB-49 model BCG-disA-OE afforded improved antitumor efficacy. A mouse safety evaluation further revealed that BCG-disA-OE proliferated to lesser degree than BCG-WT in BALB/c mice and displayed reduced lethality in SCID mice. To probe the mechanisms that may underlie these effects, we found that BCG-disA-OE was more potent than BCG-WT in eliciting IFN-ß release by exposed macrophages, in reprogramming myeloid cell subsets towards an M1-like proinflammatory phenotypes, inducing epigenetic activation marks in proinflammatory cytokine promoters, and in shifting monocyte metabolomic profiles towards glycolysis. Many of the parameters elevated in cells exposed to BCG-disA-OE are associated with BCG-mediated trained innate immunity suggesting that STING agonist overexpression may enhance trained immunity. These results indicate that modifying BCG to release high levels of proinflammatory PAMP molecules such as the STING agonist c-di-AMP can enhance antitumor efficacy in bladder cancer.

Stem cell phenotype predicts therapeutic response in glioblastomas with MGMT promoter methylation.

A growing body of evidence supports the presence of a population of cells in glioblastoma (GBM) with a stem cell-like phenotype which shares certain biological markers with adult neural stem cells, including expression of SOX2, CD133 (PROM1), and NES (nestin). This study was designed to determine the relationship between the expression of these stem cell markers and the clinical outcome in GBM patients. We quantified the intensity of expression of the proteins CD133 and SOX2 by immunohistochemistry (IHC) in a cohort of 86 patients with IDH-wildtype GBM, and evaluated patient outcomes using Kaplan-Meier and Cox proportional hazards analysis. In our patients, MGMT promoter methylation status and age were predictors of overall survival and progression free survival. The levels of SOX2 and CD133 were not associated with outcome in univariate analysis; however, stratification of tumors based on low or high levels of CD133 or SOX2 expression revealed that MGMT methylation was a predictor of progression-free survival and overall survival only for tumors with high levels of expression of CD133 or SOX2. Tumors with low levels of expression of CD133 or SOX2 did not show any relationship between MGMT methylation and survival. This relationship between MGMT and stem cell markers was confirmed in a second patient cohort, the TCGA dataset. Our results show that stratification of GBM by the level of expression of CD133 and SOX2 improved the prognostic power of MGMT promoter methylation status, identifying a low-expressing group in which the clinical outcome is not associated with MGMT promoter methylation status, and a high-expressing group in which the outcome was strongly associated with MGMT promoter methylation status. These findings support the concept that the presence of a high stem cell phenotype in GBM, as marked by expression of SOX2 or CD133, may be associated with the clinical response to treatment.

Dynamic single-cell RNA sequencing reveals BCG vaccination curtails SARS-CoV-2 induced disease severity and lung inflammation.

COVID-19 continues to exact a toll on human health despite the availability of several vaccines. Bacillus Calmette Guérin (BCG) has been shown to confer heterologous immune protection against viral infections including COVID-19 and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model together with immune profiling and single cell RNA sequencing (scRNAseq). We observed that BCG reduced both lung SCV2 viral load and bronchopneumonia. This was accompanied by an increase in lung alveolar macrophages, a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. Single cell transcriptome profiling showed that BCG uniquely recruits immunoglobulin-producing plasma cells to the lung suggesting accelerated antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, and differentially expressed gene (DEG) analysis showed a transcriptional shift away from exhaustion markers and towards antigen presentation and repair. Similarly, BCG enhanced lung recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, with both cell-types also showing reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

Re-engineered BCG overexpressing cyclic di-AMP augments trained immunity and exhibits improved efficacy against bladder cancer.

In addition to its role as a TB vaccine, BCG has been shown to elicit heterologous protection against many other pathogens including viruses through a process termed trained immunity. Despite its potential as a broadly protective vaccine, little has been done to determine if BCG-mediated trained immunity levels can be optimized. Here we re-engineer BCG to express high levels of c-di-AMP, a PAMP recognized by stimulator of interferon genes (STING). We find that BCG overexpressing c-di-AMP elicits more potent signatures of trained immunity including higher pro-inflammatory cytokine responses, greater myeloid cell reprogramming toward inflammatory and activated states, and enhances epigenetic and metabolomic changes. In a model of bladder cancer, we also show that re-engineered BCG induces trained immunity and improved functionality. These results indicate that trained immunity levels and antitumor efficacy may be increased by modifying BCG to express higher levels of key PAMP molecules.

A Molecular Inquiry into the Role of Antibody-Drug Conjugates in Bacillus Calmette-Guérin-exposed Non-muscle-invasive Bladder Cancer.

The treatment landscape for advanced urothelial cancer has changed dramatically owing to the US Food and Drug Administration approval and introduction of antibody-drug conjugates (ADCs), including enfortumab vedotin and sacituzumab govitecan. Efforts have begun to use these therapies in earlier disease states, specifically bacillus Calmette-Guérin (BCG)-unresponsive non-muscle-invasive bladder cancer (NMIBC). We assessed gene expression associated with these newly approved therapies in a novel cohort of treatment-naïve NMIBC tumors before and after BCG therapy. Multiple genes, including Nectin-4, Trop-2, and Her-2, exhibited increased expression after BCG therapy compared to baseline. However, few of the tumors with increased expression of ADC targets also exhibited increased PD-L1/PD-1 expression. Taken together, these data demonstrate the heterogeneous genomic landscape of BCG-exposed NMIBC, and provide evidence supporting the evaluation of ADCs in NMIBC. PATIENT SUMMARY: We evaluated the potential role of targeted therapies that have been approved in the USA for advanced non-muscle-invasive bladder cancer (NMIBC) that has recurred after treatment with bacillus Calmette-Guérin (BCG). By assessing levels of specific genes and proteins linked to the targeted therapies, we demonstrate that there is rationale for further evaluation of these therapies in NMIBC.

BCG invokes superior STING-mediated innate immune response over radiotherapy in a carcinogen murine model of urothelial cancer.

Radiation and bacillus Calmette-Guérin (BCG) instillations are used clinically for treatment of urothelial carcinoma, but the precise mechanisms by which they activate an immune response remain elusive. The role of the cGAS-STING pathway has been implicated in both BCG and radiation-induced immune response; however, comparison of STING pathway molecules and the immune landscape following treatment in urothelial carcinoma has not been performed. We therefore comprehensively analyzed the local immune response in the bladder tumor microenvironment following radiotherapy and BCG instillations in a well-established spontaneous murine model of urothelial carcinoma to provide insight into activation of STING-mediated immune response. Mice were exposed to the oral carcinogen, BBN, for 12 weeks prior to treatment with a single 15 Gy dose of radiation or three intravesical instillations of BCG (1 × 108 CFU). At sacrifice, tumors were staged by a urologic pathologist and effects of therapy on the immune microenvironment were measured using the NanoString Myeloid Innate Immunity Panel and immunohistochemistry. Clinical relevance was established by measuring immune biomarker expression of cGAS and STING on a human tissue microarray consisting of BCG-treated non-muscle-invasive urothelial carcinomas. BCG instillations in the murine model elevated STING and downstream STING-induced interferon and pro-inflammatory molecules, intratumoral M1 macrophage and T-cell accumulation, and complete tumor eradication. In contrast, radiotherapy caused no changes in STING pathway or innate immune gene expression; rather, it induced M2 macrophage accumulation and elevated FoxP3 expression characteristic of immunosuppression. In human non-muscle-invasive bladder cancer, STING protein expression was elevated at baseline in patients who responded to BCG therapy and increased further after BCG therapy. Overall, these results show that STING pathway activation plays a key role in effective BCG-induced immune response and strongly indicate that the effects of BCG on the bladder cancer immune microenvironment are more beneficial than those induced by radiation. © 2021 The Pathological Society of Great Britain and Ireland.

New and topics: enfortumab vedotin mechanisms of response and resistance in urothelial cancer - What do we understand so far?

Enfortumab vedotin (EV) was FDA approved in December 2019 for platinum- and checkpoint-refractory urothelial cancer based on an exceptional 44% response rate, and is currently approved for use after platinum and checkpoint inhibitor therapy. Enfortumab is an antibody-drug conjugate that targets Nectin-4, which is widely expressed in urothelial cancer. Despite this ample target, clinical benefit is not achieved by all patients, and mechanisms of treatment resistance are undescribed. Herein we summarize what is known to date regarding coorelative findings and subgroup analysis and EV response, including novel biopsy data in patients with tumor progression post EV.

Molecular subtypes of upper tract urothelial cancer: Setting the stage for precision therapy.

A comprehensive genomic characterization of a large, high-quality cohort of upper tract urothelial carcinomas (UTUCs) in this issue of Cancer Cell reveals that UTUCs can be divided into five DNA-based molecular subtypes. Feasibility data establish that molecular subtyping can be performed non-invasively by sequencing tumor DNA in urine.

Expression of Nectin-4 in Bladder Urothelial Carcinoma, in Morphologic Variants, and Nonurothelial Histotypes.

The antibody-drug conjugate enfortumab-vedotin acts by targeting nectin-4, a protein that is nearly ubiquitously expressed in conventional urothelial cancer. However, expression of nectin-4 in morphologic variants of urothelial carcinoma and nonurothelial histotypes was unknown. Immunohistochemistry for nectin-4 using was performed on 169 patients including 83 with nonmuscle invasive bladder cancer and 86 patients with muscle invasive bladder cancer. Staining was scored for intensity (0 to 3) and extent (% positive cells) using the histological score system, where >15 was considered positive. Overall, 72/83 (87%) samples of nonmuscle invasive urothelial carcinoma were positive, including 29/30 (97%) noninvasive papillary urothelial carcinomas, 7/8 (87.5%) carcinomas in situ, 36/45 (80%) papillary urothelial carcinomas invading the lamina propria. Overall, 50/86 muscle invasive tumors were positive, including 15/22 (68.2%) urothelial carcinomas, 7/10 (70%) squamous cell carcinomas, 3/11 (28%) micropapillary tumors, 4/6 (66%) adenocarcinomas, 2/4 (50%) nested carcinomas, 5/8 (63%) plasmacytoid, 1/10 (10%) sarcomatoid carcinomas, and 0/15 (0%) small cell carcinomas. Whole transcriptome RNA sequencing revealed that compared with conventional urothelial carcinomas, most sarcomatoid carcinomas and all but 2 small cell carcinomas expressed very low levels of nectin-4 mRNA but expressed significant levels of either trop2 or ERBB2, which are the molecular targets of 2 other antibody-drug conjugates-sacituzumab gavitecan (trop2) or trastuzumab deruxtecan (ERBB2/HER2). In summary, our study demonstrates that there is heterogeneity of expression of nectin-4 in morphologic variants of urothelial cancer and nonurothelial histotypes, and suggests that testing expression of nectin-4 should be considered in morphologic variants or nonurothelial histotypes found to have lower expression.

Immune Microenvironment in Gallbladder Adenocarcinomas.

Programmed death-1 (PD1) expression has not been reported in gallbladder adenocarcinoma. In this study we examined PD1 expression in gallbladder cancer to explore the correlation between PD1 expression and the clinicopathologic parameters. We found that 98% (46/47) cases expressed programmed death-ligand 1 (PD-L1) with 85% cases being PD-L1 3+. PD1+ tumor-infiltrating lymphocytes (TILs) were present in 78.7% cases (37/47). The tumor size was significantly smaller and the stromal CD3+ TILs were significantly higher in tumors with PD1+ TILs than those with PD1- TILs. In the tumors with size of <3 cm, stromal CD3+ TILs >115/HPF or stromal CD8+ TILs >45/HPF were associated with much better survival than those with stromal CD3+ TILs ≤115/HPF or stromal CD8+ TILs ≤45/HPF. In tumors with the size of 3 cm or larger, PD1+ TILs or stromal CD8+ TILs >45/HPF carried a significantly poorer survival than PD1- tumors or stromal CD8+ TILs <=45/HPF. No correlation was identified between PD1 expression and lymphovascular invasion, distant metastasis, pathologic tumor stage or prognostic stage. Multivariate survival analysis showed that tumor TNM stage and age were independent prognostic factors in gallbladder adenocarcinomas. We conclude that gallbladder adenocarcinomas may have high PD-L1 expression and PD1+ TILs. Smaller tumor size and greater amount of stromal CD3+ T cells were found in tumors with PD1+ TILs. In small tumors (<3 cm), high stromal CD3+ TILs or high stromal CD8+ TILs were associated with better survival. However, in large tumors (≥3 cm), PD1+ TILs or high stromal CD8+ TILs carried a poorer survival. Our study implied that immune-based therapy including PD1/PD-L1 checkpoint blockade might be useful in gallbladder adenocarcinomas.

Urothelial Carcinoma In Situ of the Bladder: Correlation of CK20 Expression With Adaptive Immune Resistance, Response to BCG Therapy, and Clinical Outcome.

Immunohistochemical stains have been suggested to aid in diagnostically challenging cases of urothelial carcinoma in-situ (CIS). Although full thickness immunostaining for CK20 is supportive of CIS, a subset of CIS cases is CK20(-), the clinical significance of which was unknown. This study included 43 patients with primary diagnosis of bladder CIS including 32 with only CIS, 5 with CIS and separate noninvasive high-grade papillary urothelial carcinoma, and 6 with CIS and separate high-grade urothelial carcinoma with lamina propria invasion. Digital morphometric image analysis showed that the average nuclear areas of enlarged nuclei were similar in CK20(+) and CK20(-) CIS (26.9 vs. 24.5 µM2; P=0.31). Average Ki67 index for CK20(+) CIS was higher than CK20(-) CIS (31.1% vs. 18.3%; P=0.03). Patients with CK20(+) CIS [28 (65%)] and patients with CK20(-) CIS [15 (35%)] had the same rates of Bacillus Calmete-Guerin (BCG) failure but patients with CK20(-) CIS had higher stage progression [3 CK20(+) (11%) vs. 6 CK20(-) (40%); P=0.02]. Given recent approval of immune checkpoint inhibitors in patients with CIS refractory to BCG, programmed death ligand-1 expression and colocalization with CD8(+) lymphocytes was investigated as signature of adaptive immune response and was seen in 8 patients regardless of CK20 status and exclusively among patients who failed BCG. Our results confirm that negative CK20 IHC does not exclude CIS and that those patients have similar clinical outcomes as patients with CK20(+) CIS. Programmed death ligand-1 and CD8 colocalization seen among patients who failed BCG therapy is an easy assay to perform to identify patients who could potentially benefit from combined BCG therapy and immune checkpoint inhibition.

Contemporary Characterization and Recategorization of Adult Unclassified Renal Cell Carcinoma.

Our recent study of early-onset unclassified eosinophilic renal cell carcinoma (RCC) demonstrated that two third of cases could be reclassified by performing a limited number of immunohistochemistry stains. Following the same approach, we aimed to investigate what proportion of adult unclassified RCC could be reclassified. We identified 79 cases. The mean age at presentation was 58 years (range, 29 to 84 y). Tumors were grouped based on their predominant morphologic features as oncocytic (n=23); papillary (n=22); clear cell (n=22); mucinous tubular and spindle cell (MTSC; n=5); rhabdoid (n=4); or lacking a dominant pattern (n=3). By reviewing the morphologic features and performing ancillary studies, we were able to reclassify 10 cases (13%). Four cases were positive for CK20 and showed morphologic features consistent with eosinophilic solid and cystic RCC. Four cases were reclassified as MTSC based on VSTM2A expression by RNA in situ hybridization. One case was negative for SDHB and reclassified as succinate dehydrogenase-deficient RCC. None of the cases showed loss of expression of fumarate hydratase. One case was diffusely positive for CK7 and negative for CD117 and reclassified as a low-grade oncocytic tumor. Four cases were positive for both cathepsin-K and TFE3 by immunohistochemistry, although fluorescence in situ hybridization failed to identify rearrangement in either TFE3 or TFEB genes. Of the tumors that remained unclassified, those with oncocytic features were less likely to be a high grade (odds ratio [OR]=0.22, P=0.013) or advanced stage (OR=0.19, P=0.039) and were more common in women (OR=3.4, P=0.05) compared with those without oncocytic features. Tumors with rhabdoid morphology were associated with advanced stage (relative risk=3.6, P=0.009), while tumors with clear cell or papillary features had a wide range of grades and stages at presentation. In summary, the most frequent reclassified entity is eosinophilic solid and cystic RCC. Investigation of expression of succinate dehydrogenase or fumarate hydratase in individuals older than 35 years with unclassifiable tumors is low yield in the absence of specific morphologic features. A subset of MTSC without well-developed morphologic features can be reclassified by using RNA-ISH for VSTM2A. Recognition of more-recently described RCC subtypes allows for their distinction from the unclassified subtype and improves the prognostic information provided.