Molecular Characterization of Residual Bladder Cancer after Neoadjuvant Pembrolizumab.

BACKGROUND: In patients with muscle-invasive urothelial bladder cancer (MIBC), molecular alterations in immunotherapy-resistant tumors found at radical cystectomy (RC) remain largely unstudied. OBJECTIVE: To investigate the biology of pembrolizumab-resistant tumors in comparison to an RC cohort treated without any systemic therapy and a cohort of neoadjuvant chemotherapy (NAC)-treated tumors. DESIGN, SETTING, AND PARTICIPANTS: Transcriptome-wide expression profiling was performed on 26 RC samples from patients with ypT2-4 disease after pembrolizumab treatment, of which 22 had matched pretherapy samples. Unsupervised consensus clustering (CC) was performed to compare 26 post-pembrolizumab samples with 94 RC samples without neoadjuvant treatment and 21 samples collected from the former tumor bed of NAC-treated patients (scar tissue). Clusters were investigated for their biological and clinical characteristics and were compared to a cohort of post-NAC tumors (n = 133). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Patient and tumor characteristics were compared between subgroups using χ2 tests and two-sided Wilcoxon rank-sum tests. The primary endpoint was recurrence-free survival. RESULTS AND LIMITATIONS: Molecular subtyping of pre- and post-pembrolizumab samples revealed significant differences: only 36% of samples had a concordant subtype according to the consensus classifier. Unsupervised CC revealed three distinct post-pembrolizumab clusters (basal, luminal, and scar-like). A scar-like subtype was present in 50% of the post-pembrolizumab cases (n = 13) and expressed genes associated with wound healing/scarring. This subtype had higher luminal marker expression in the post-pembrolizumab setting compared to CC scar-like tumors from the other cohorts. Patients with the scar-like subtype showed favorable prognosis after systemic therapy, but not in the RC-only setting. The small numbers in each subgroup represents the major study limitation. CONCLUSIONS: This study expands our understanding of the biology of pembrolizumab-resistant MIBC and provides a framework for defining molecular subtypes after treatment. The results further support the hypothesis that luminal-type tumors may be resistant to immunotherapy or that this treatment may select for, or induce, a luminal phenotype. PATIENT SUMMARY: We carried out genetic analysis for bladder cancer tumors from patients who had received an immunotherapy agent called pembrolizumab and compared them to tumors treated with standard chemotherapy or just bladder removal. We found differences in gene expression between the treatment types and between tumor tissue from the same patient before and after treatment. These results may be helpful in personalizing therapy strategies for patients with bladder cancer.

Validation of a 22-Gene Genomic Classifier in Patients With Recurrent Prostate Cancer: An Ancillary Study of the NRG/RTOG 9601 Randomized Clinical Trial.

Importance: Decipher (Decipher Biosciences Inc) is a genomic classifier (GC) developed to estimate the risk of distant metastasis (DM) after radical prostatectomy (RP) in patients with prostate cancer. Objective: To validate the GC in the context of a randomized phase 3 trial. Design, Setting, and Participants: This ancillary study used RP specimens from the phase 3 placebo-controlled NRG/RTOG 9601 randomized clinical trial conducted from March 1998 to March 2003. The specimens were centrally reviewed, and RNA was extracted from the highest-grade tumor available in 2019 with a median follow-up of 13 years. Clinical-grade whole transcriptomes from samples passing quality control were assigned GC scores (scale, 0-1). A National Clinical Trials Network-approved prespecified statistical plan included the primary objective of validating the independent prognostic ability of GC for DM, with secondary end points of prostate cancer-specific mortality (PCSM) and overall survival (OS). Data were analyzed from September 2019 to December 2019. Intervention: Salvage radiotherapy (sRT) with or without 2 years of bicalutamide. Main Outcomes and Measures: The preplanned primary end point of this study was the independent association of the GC with the development of DM. Results: In this ancillary study of specimens from a phase 3 randomized clinical trial, GC scores were generated from 486 of 760 randomized patients with a median follow-up of 13 years; samples from a total of 352 men (median [interquartile range] age, 64.5 (60-70) years; 314 White [89.2%] participants) passed microarray quality control and comprised the final cohort for analysis. On multivariable analysis, the GC (continuous variable, per 0.1 unit) was independently associated with DM (hazard ratio [HR], 1.17; 95% CI, 1.05-1.32; P = .006), PCSM (HR, 1.39; 95% CI, 1.20-1.63; P < .001), and OS (HR, 1.17; 95% CI, 1.06-1.29; P = .002) after adjusting for age, race/ethnicity, Gleason score, T stage, margin status, entry prostate-specific antigen, and treatment arm. Although the original planned analysis was not powered to detect a treatment effect interaction by GC score, the estimated absolute effect of bicalutamide on 12-year OS was less when comparing patients with lower vs higher GC scores (2.4% vs 8.9%), which was further demonstrated in men receiving early sRT at a prostate-specific antigen level lower than 0.7 ng/mL (-7.8% vs 4.6%). Conclusions and Relevance: This ancillary validation study of the Decipher GC in a randomized trial cohort demonstrated association of the GC with DM, PCSM, and OS independent of standard clinicopathologic variables. These results suggest that not all men with biochemically recurrent prostate cancer after surgery benefit equally from the addition of hormone therapy to sRT. Trial Registration: ClinicalTrials.gov identifier: NCT00002874.

Validation of a neuroendocrine-like classifier confirms poor outcomes in patients with bladder cancer treated with cisplatin-based neoadjuvant chemotherapy.

PURPOSE: Neuroendocrine (NE)-like carcinoma is a newly recognized molecular subtype of conventional urothelial carcinoma of the bladder with transcriptomic profiles and clinical outcomes highly similar to histological NE carcinoma. The identification of NE-like tumors is challenging, as these tumors often appear histologically like urothelial carcinoma and can be missed by routine morphological criteria. We previously developed a single-sample classifier to identify NE-like tumors, which we aimed to validate in an independent cohort. MATERIALS AND METHODS: A single-sample genomic classifier was performed on transurethral specimens from a retrospective multicenter cohort of 234 patients who underwent cisplatin-based neoadjuvant chemotherapy and subsequent radical cystectomy. Outcomes were compared for NE-like vs. non-NE-like. RESULTS: We identified 10 patients with urothelial tumors of the NE-like subtype, all of which had robust gene expression of neuronal markers, but did not express markers associated with basal or luminal tumors. The cancer-specific mortality rates were significantly higher compared to non-NE-like tumors (P < 0.001), with 5 of the 10 patients dying within 12 months from surgery. CONCLUSIONS: The single-sample classifier was able to identify urothelial carcinomas with NE-like subtype. These NE-like tumors have demonstrated transcriptomic profiles and clinical behavior similar to histological NE tumors across multiple patient cohorts. We propose that NE-like tumors should be managed similarly to histological NE tumors, and that standard treatments for small cell lung cancer as well as novel strategies may be evaluated in these patients.

Prospective study to define the clinical utility and benefit of Decipher testing in men following prostatectomy.

BACKGROUND: Genomic classifiers (GC) have been shown to improve risk stratification post prostatectomy. However, their clinical benefit has not been prospectively demonstrated. We sought to determine the impact of GC testing on postoperative management in men with prostate cancer post prostatectomy. METHODS: Two prospective registries of prostate cancer patients treated between 2014 and 2019 were included. All men underwent Decipher tumor testing for adverse features post prostatectomy (Decipher Biosciences, San Diego, CA). The clinical utility cohort, which measured the change in treatment decision-making, captured pre- and postgenomic treatment recommendations from urologists across diverse practice settings (n = 3455). The clinical benefit cohort, which examined the difference in outcome, was from a single academic institution whose tumor board predefined "best practices" based on GC results (n = 135). RESULTS: In the clinical utility cohort, providers' recommendations pregenomic testing were primarily observation (69%). GC testing changed recommendations for 39% of patients, translating to a number needed to test of 3 to change one treatment decision. In the clinical benefit cohort, 61% of patients had genomic high-risk tumors; those who received the recommended adjuvant radiation therapy (ART) had 2-year PSA recurrence of 3 vs. 25% for those who did not (HR 0.1 [95% CI 0.0-0.6], p = 0.013). For the genomic low/intermediate-risk patients, 93% followed recommendations for observation, with similar 2-year PSA recurrence rates compared with those who received ART (p = 0.93). CONCLUSIONS: The use of GC substantially altered treatment decision-making, with a number needed to test of only 3. Implementing best practices to routinely recommend ART for genomic-high patients led to larger than expected improvements in early biochemical endpoints, without jeopardizing outcomes for genomic-low/intermediate-risk patients.

Quantitative Spatial Profiling of PD-1/PD-L1 Interaction and HLA-DR/IDO-1 Predicts Improved Outcomes of Anti-PD-1 Therapies in Metastatic Melanoma.

Purpose: PD-1/L1 axis-directed therapies produce clinical responses in a subset of patients; therefore, biomarkers of response are needed. We hypothesized that quantifying key immunosuppression mechanisms within the tumor microenvironment by multiparameter algorithms would identify strong predictors of anti-PD-1 response.Experimental Design: Pretreatment tumor biopsies from 166 patients treated with anti-PD-1 across 10 academic cancer centers were fluorescently stained with multiple markers in discovery (n = 24) and validation (n = 142) cohorts. Biomarker-positive cells and their colocalization were spatially profiled in pathologist-selected tumor regions using novel Automated Quantitative Analysis algorithms. Selected biomarker signatures, PD-1/PD-L1 interaction score, and IDO-1/HLA-DR coexpression were evaluated for anti-PD-1 treatment outcomes.Results: In the discovery cohort, PD-1/PD-L1 interaction score and/or IDO-1/HLA-DR coexpression was strongly associated with anti-PD-1 response (P = 0.0005). In contrast, individual biomarkers (PD-1, PD-L1, IDO-1, HLA-DR) were not associated with response or survival. This finding was replicated in an independent validation cohort: patients with high PD-1/PD-L1 and/or IDO-1/HLA-DR were more likely to respond (P = 0.0096). These patients also experienced significantly improved progression-free survival (HR = 0.36; P = 0.0004) and overall survival (HR = 0.39; P = 0.0011). In the combined cohort, 80% of patients exhibiting higher levels of PD-1/PD-L1 interaction scores and IDO-1/HLA-DR responded to PD-1 blockers (P = 0.000004). In contrast, PD-L1 expression was not predictive of survival.Conclusions: Quantitative spatial profiling of key tumor-immune suppression pathways by novel digital pathology algorithms could help more reliably select melanoma patients for PD-1 monotherapy. Clin Cancer Res; 24(21); 5250-60. ©2018 AACR.

Deep exploration of the immune infiltrate and outcome prediction in testicular cancer by quantitative multiplexed immunohistochemistry and gene expression profiling.

Platinum-based chemotherapy is usually curative for patients with testicular germ cell tumors (TGCT), but a subset of patients experience disease progression and poor clinical outcomes. Here, we tested whether immune profiling of TGCT could identify novel prognostic markers and therapeutic targets for this patient cohort. We obtained primary and metastatic TGCT samples from one center. We performed immune profiling using multiplexed fluorescence immunohistochemistry (FIHC) for T-cell subsets and immune checkpoints, and targeted gene expression profiling (Nanostring nCounter Immune panel). Publically available data sets were used to validate primary sample analyses. Nearly all samples had some degree of T-cell infiltration and immune checkpoint expression. Seminomas were associated with increased CD3+ T-cell infiltration, decreased Regulatory T-cells, increased PD-L1, and increased PD-1/PD-L1 spatial interaction compared with non-seminomas using FIHC. Gene expression profiling confirmed these findings and also demonstrated increased expression of T-cell markers (e.g., IFNγ, and LAG3) and cancer/testis antigens (e.g., PRAME) in seminomas, whereas non-seminomas demonstrated high neutrophil and macrophage gene signatures. Irrespective of histology, advanced TGCT stage was associated with decreased T-cell and NK-cell signatures, while Treg, neutrophil, mast cell and macrophage signatures increased with advanced stage. Importantly, cancer/testis antigen, neutrophil, and CD8+/regulatory T-cell signatures correlated with recurrence free survival. Thus, deep immune characterization of TGCT using IHC and gene expression profiling identified activated T-cell infiltration which correlated with seminoma histology and good prognosis. These results may provide a rationale for testing of anti-PD-1/PD-L1 agents and suggest prognostic markers.

MDS-associated somatic mutations and clonal hematopoiesis are common in idiopathic cytopenias of undetermined significance.

Establishing a diagnosis in patients suspected of having a myelodysplastic syndrome (MDS) can be challenging and could be informed by the identification of somatic mutations. We performed a prospective study to examine the frequency and types of mutations encountered in 144 patients with unexplained cytopenias. Based on bone marrow findings, 17% were diagnosed with MDS, 15% with idiopathic cytopenias of undetermined significance (ICUS) and some evidence of dysplasia, and 69% with ICUS and no dysplasia. Bone marrow DNA was sequenced for mutations in 22 frequently mutated myeloid malignancy genes. Somatic mutations were identified in 71% of MDS patients, 62% of patients with ICUS and some dysplasia, and 20% of ICUS patients and no dysplasia. In total, 35% of ICUS patients carried a somatic mutation or chromosomal abnormality indicative of clonal hematopoiesis. We validated these results in a cohort of 91 lower-risk MDS and 249 ICUS cases identified over a 6-month interval. Mutations were found in 79% of those with MDS, in 45% of those with ICUS with dysplasia, and in 17% of those with ICUS without dysplasia. The spectrum of mutated genes was similar with the exception of SF3B1 which was rarely mutated in patients without dysplasia. Variant allele fractions were comparable between clonal ICUS (CCUS) and MDS as were mean age and blood counts. We demonstrate that CCUS is a more frequent diagnosis than MDS in cytopenic patients. Clinical and mutational features are similar in these groups and may have diagnostic utility once outcomes in CCUS patients are better understood.

False-positive restaging PET scans involving the spleen in two patients with aggressive non-Hodgkin lymphoma.

We report 2 patients with aggressive non-Hodgkin lymphoma who had positive restaging PET scans limited to the spleen and no significant uptake in nodal areas of previously known disease. Examination of the resected spleens from both patients revealed extensive inflammation surrounding necrotic tumor with no evidence of viable lymphoma or active infection. It is suggested that close observation of such patients for evidence of progressive disease may be considered as opposed to immediate intervention.

Utility of histologic parameters in screening for antibody-mediated rejection of the cardiac allograft: a study of 3,170 biopsies.

BACKGROUND: Diagnostic criteria for antibody-mediated rejection (AMR) of the cardiac allograft have recently been proposed as part of the International Society for Heart and Lung Transplantation (ISHLT) biopsy grading scheme. Histologic features of vascular adherence of macrophages (VASC) and endothelial activation or swelling in capillaries (ENDO) are proposed as criteria to prompt the immunohistochemical investigation of biopsies for AMR. The aim of this study was to determine whether VASC and ENDO are adequate to act as screening parameters to trigger further AMR investigation. METHODS: We examined our database of biopsy findings where histologic vascular parameters as well as immunofluorescence (IF) to detect AMR were collected (n = 3,170). Histologic parameters were graded semi-quantitatively on a scale from 1 to 5, where 1 = absence and 5 = obvious and generalized presence of the finding. RESULTS: Seven hundred sixty-eight of 3,170 biopsies had IF findings diagnostic of AMR in the absence of cellular rejection (ISHLT = 0). ENDO had a sensitivity of 63% and a specificity of 80%. VASC had a sensitivity of 30% and specificity of 99%. Combining the interpretation of the 2 tests did not result in a significant improvement of test sensitivity. CONCLUSIONS: Neither ENDO, VASC nor the combination of the tests indicated sufficiently high sensitivity to serve as a screening tool before further diagnostic investigation for AMR. Immunohistochemical testing remains necessary in the majority of cases to identify AMR.

Histone deacetylase inhibitors induce G2-checkpoint arrest and apoptosis in cisplatinum-resistant ovarian cancer cells associated with overexpression of the Bcl-2-related protein Bad.

Trichostatin A produces predominantly G(1) cell-cycle blockade and differentiation of the cisplatinum-sensitive A2780 ovarian cancer cell line. Given the propensity of ovarian tumors to become resistant to cisplatinum, often leading to cross-resistance to other agents, we have extended these observations by examining how the emergence of resistant phenotypes in A2780 cells affects the actions of histone deacetylase (HDAC) inhibitors. Trichostatin A exposure (100 ng/mL, 24 hours) induced ultrastructural differentiation of the "intrinsically" cisplatinum-resistant A2780-9M subline, with the reappearance of intercellular junctions and lumina containing primitive microvilli. Similar trichostatin A exposure in the acquired resistance A2780CP cells produced minimal differentiation consisting of occasional weak intercellular junctions. Independent of the differences in trichostatin A-induced differentiation, in both resistant sublines trichostatin A produced a similar reduction in cell viability, by >90%, within 5 days of treatment. Diminished viability in both A2780-9M and CP cells was associated with the absence of cell cycle arrest in G1, resulting in predominant G2-checkpoint arrest accompanied by a 10- to 20-fold increase in Annexin V binding and the reemergence of apoptosis. Similar cell cycle arrests and apoptosis were also observed using other HDAC inhibitors and in other resistant ovarian cancer cell lines (OVCAR-3 and SK-OV-3). Trichostatin A-induced apoptosis in resistant cells is in sharp contrast to its effects on the parental cisplatinum-sensitive A2780 and normal MRC-5 fibroblast cell lines (predominant cycle arrest in G1 with no detectable apoptosis). Western immunoblot analysis indicated trichostatin A triggers apoptosis in resistant ovarian cancer cells via p53-independent activation of the intrinsic "mitochondrial" pathway, commensurate with induction of the Bcl-2-related protein Bad. These results suggest cisplatinum resistance alters the effects of HDAC inhibition through a shift in cell cycle arrest from the G1 to the G2 checkpoint and reactivation of the intrinsic mitochondrial apoptotic cascade.

Early failure of a tricuspid valve replacement with a mitral valve homograft in a heart transplant recipient.

A 24-year-old woman experienced severe tricuspid valve regurgitation 6 years after heart transplantation. Tricuspid valve replacement was performed using a cryopreserved mitral valve homograft. Severe tricuspid valve regurgitation recurred within 4 months, associated with an increase in the panel reactive antibody titers from zero to 72%. Tricuspid valve replacement was repeated with a porcine bioprosthesis with excellent recovery and function for >2 years. The mitral valve homograft displayed inflammatory features consistent with humoral immune-mediated destruction.

Cell type-dependent regulation of hMLH1 promoter activity is influenced by the presence of multiple redundant elements.

Immunohistochemical analysis confirmed the presence of MLH1 protein in A2780 ovarian cancer cells and its absence in this same cell line on acquired resistance to cisplatinum (A2780/CP). Transfection of a -1781-bp hMLH1 promoter construct into either A2780 or A2780/CP cells produced similar (30-fold) induction of luciferase, an indication that the transcriptional machinery for hMLH1 expression remains intact. hMLH1-luciferase activity was also unaffected by re-expression of hMLH1 following treatment of A2780/CP cells with the methylase inhibitor 2'-deoxy-5-azacytidine. Serial 5'-deletion studies of the hMLH1 promoter region in ovarian cancer cells localized transcriptional enhancers to a region (-250 to -151 bp) that excludes the previously identified CCAAT element (-282) active in HeLa cells. When these same deletion constructs were transfected into HeLa cells, deletion of the CCAAT-containing region caused a significant loss of promoter activity, an indication of cell-specific use of enhancer elements. Finally, a series of internal deletion and linker mutation studies of the -250 to -151 bp ovarian enhancer region revealed that the hMLH1 promoter contains multiple redundant enhancer elements capable of independent promoter activation and may explain the association of this region with methylation silencing of hMLH1.

Cell cycle blockade and differentiation of ovarian cancer cells by the histone deacetylase inhibitor trichostatin A are associated with changes in p21, Rb, and Id proteins.

Inhibitors of histone deacetylase activity are emerging as a potentially important new class of anticancer agents. In the current studies, exposing A2780 ovarian cancer cells to the histone deacetylase inhibitor trichostatin A (TSA) produced a marked change in cellular morphology, proliferation, and differentiation. Within 24 h of TSA treatment, there was a morphological transformation of the cells, with increased cytoplasm, a more epithelial-like columnar appearance, and the emergence of distinct cellular boundaries. Commensurate with the morphological transformation, TSA also inhibited cell proliferation; cells treated with TSA for 72 h increased to 110% of the initial cell numbers versus control cell numbers of 622%, with a corresponding reduction in mitotic activity and a flow cytometry S-phase fraction of 3.9% in TSA-treated cells versus 28.8% for control. TSA also induced epithelial-like differentiation with increased cytokeratin expression from 2% of controls to 22-25% of TSA-treated cells and the reappearance of intercellular plasma membrane junctions and primitive microvilli. Immunocytochemical analyses indicate the molecular mechanism underlying the actions of TSA on A2780 cell cycle progression and differentiation involves reexpression of the CDK inhibitor p21. Elevated levels of p21, in TSA-treated cells, were associated with a reduction in the phosphorylation of the cell cycle regulator retinoblastoma protein (Rb). TSA also caused a decrease in the helix-loop-helix inhibitor of differentiation/DNA binding protein Id1, with no change in Id2 levels. In conclusion, the observed TSA-induced changes in p21, Rb, and Id1 are consistent with cell cycle senescence and differentiation of A2780 ovarian cancer cells.