Cheville, John C.

548 Background: PD-L1 and B7-H3 have been found to be overexpressed in urothelial carcinoma (UC) of the urinary bladder. Recent studies have also demonstrated that B7-H3 and PD-L1 can promote resistance to platinum-based drugs but the predictive value of B7-H3 expression in patients treated with platinum-based chemotherapy is unknown. This study aims to investigate the association of PD-L1 and B7-H3 tumor expression with oncological outcomes in patients who underwent radical cystectomy (RC) and received subsequent adjuvant chemotherapy. Methods: Immunohistochemistry was performed on paraffin-embedded sections from bladder and lymph node specimens of 81 patients who had RC for bladder cancer. PD-L1 and B7-H3 expression on tumor cells was assessed by immunohistochemistry in both primary tumors and lymph node specimens. Association with clinicopathologic outcomes was determined using Fisher’s exact test and postoperative survival using Kaplan–Meier survival curves and Cox regression model. Results: B7-H3 expression in cystectomy specimens was more common than PD-L1 expression (72.8% vs. 35.8%). For both markers, no association was found with pathologic tumor stage, lymph node (LN) status, and histological subtype. Similar findings were observed for double-positive tumors (PD-L1+B7-H3+). Concordance between the primary tumor and patient-matched lymph nodes was found in 76.2% and 54.1% of patients for PD-L1 and B7-H3, respectively. PD-L1 tumor expression was not associated with oncologic outcomes. However, B7-H3 expression was associated with recurrence-free survival (HR: 2.38, 95% CI 1.06–5.31, p = 0.035) and cancer-specific survival (HR: 2.67, 95% CI 1.18–6.04, p = 0.019). Conclusions: In our single institutional study, B7-H3 is highly expressed in patients with UC treated with adjuvant chemotherapy and it was associated with decreased recurrence-free survival and cancer-specific survival. Pending further validation in larger cohorts, B7-H3 expression may function as a predictor of response to adjuvant chemotherapy and thus be useful in patient and regimen selection.

AbstractRenal cell carcinoma (RCC) is among the top 10 most common cancers in both men and women with an estimated 75 000 cases each year in the US. Over the last decade, the therapeutic landscape for patients with metastatic RCC has significantly evolved, with immunotherapy emerging as the new front-line therapy. Despite significant improvement in toxicity profile and survival outcomes, key concerns such as patient selection, treatment sequencing, and intrinsic and acquired resistance remain unresolved. Emerging options such as antibody-based therapeutics (eg, anti-CD70, anti-CA9, and anti-ENPP3) are being explored in clinical trials for patients with cancer resistant or refractory to current immunotherapies. Despite positive results for hematological cancers, breast cancer, and more recently bladder cancer, most antibody-based therapies failed to improve the outcomes in patients with advanced RCC. This underscores the need to understand the underlying causes of failed responses to this treatment class, which will ultimately support the rational design of more effective and tolerable treatments. In this review, we summarize the evolving landscape of RCC therapeutics and describe recent clinical trials with emerging antibody-based therapeutics. We also describe the challenges that need to be overcome for the successful creation of therapeutic antibodies for treating RCC.

Abstract Diffuse gliomas are aggressive brain tumors that lack effective therapies. Despite advances in the molecular characterization of gliomas, targeted treatments have failed to improve patient outcomes to date. Here, we employed a functional genomics approach to rapidly identify therapeutic vulnerabilities and provide a strategy to individualize patient treatment. Our process combined comprehensive genomic and transcriptomic tumor profiling, custom algorithms and visualization software for data integration, and preclinical 3-dimensional ex vivo models (mcancers) for drug screening to assess response to therapeutic agents targeting specific molecular alterations. A total of 32 cases, including 5 oligodendrogliomas (1 primary and 4 recurrent), 5 astrocytomas (1 primary and 4 recurrent), and 22 glioblastomas (5 primary and 17 recurrent), were included in the study. Immunohistochemistry and integrated genomics confirmed that mcancers maintain key molecular alterations, including mutations, chromosomal rearrangements and focal amplifications, cellularity, and heterogeneity, faithfully recapitulating the original tumor. A total of 119 single drugs and 183 combinations were tested, including a panel of 26-brain penetrant targeted agents (18 single drugs and 8 combinations) that was curated to target common glioma oncogenic pathways and was tested across all cases. A strong correlation was observed between therapeutic vulnerabilities of glioma mcancers to targeted agents (EGFR, MET, PDGFR, HDAC inhibitors, etc.) and clinical experience. Single agent activity of panel compounds in the ex vivo setting was generally poor, with only 35% mCancers exhibiting > 70% growth inhibition at Cmax. In contrast, combination strategies, especially ones including epigenetic drugs, were significantly more effective (affecting 22 out of 32 mCancers), underscoring the molecular complexity of gliomas and the need for clinical trials testing combination treatments. Our data identified a number of novel combination treatments with significant efficacy across glioma subtypes and molecular profiles, suggesting that our functional genomics pipeline can guide treatment selection and inform clinical trial enrollment for gliomas.

Context.— Epithelioid angiomyolipomas (eAMLs) are rare tumors of the kidney that occur in patients with tuberous sclerosis complex or in a sporadic setting; a subset of these tumors exhibit metastatic behavior. Objective.— To analyze molecular profiling data to identify pathogenic alterations in rare cases of metastatic eAML, and to identify immunohistochemistry (IHC)-based surrogate markers. Design.— Molecular profiling data from the American Association for Cancer Research GENIE registry was accessed for 23 patients with angiomyolipomas, and 9 of 16 patients with eAMLs in our institutional registry were evaluated with next-generation sequencing. IHC was performed to screen for alterations of P53, RB, and ATRX for all 16 institutional cases. Results.— Combined alterations of 5 tumor-suppressor genes (TP53, ATRX, RB1, APC, and NF1) were identified using next-generation sequencing in 7 of 8 (88%) patients with metastatic disease compared to a single patient with nonmetastatic disease (RB1 variant of uncertain significance; 1 of 24, 4%). No cases with abnormal IHC results were identified in 11 patients with nonmetastatic disease compared to 3 of 5 patients with metastatic disease. Conclusions.— Our results show that the majority of metastatic eAMLs have mutations of 5 tumor-suppressor genes (TP53, ATRX, RB1, APC, and NF1), while these are rare in patients with nonmetastatic disease. Furthermore, IHC for P53, RB, and ATRX may serve as a screen for a subset of these alterations in resource-limited settings. These findings, if validated in larger data sets, have the potential to predict metastatic behavior in eAMLs.

AbstractObjectiveThe objective of this study was to evaluate the role of adrenal venous sampling (AVS) in guiding the management of patients with corticotropin (ACTH)‐independent glucocorticoid secretory autonomy and bilateral adrenal masses.Design and PatientsA cohort with 25 patients underwent AVS and surgical management.MeasurementsCortisol was measured from the adrenal veins (AVs) and inferior vena cava (IVC). AV/IVC cortisol ratio and cortisol lateralization ratio (CLR) (dominant AV cortisol concentration divided by the nondominant AV cortisol concentration) were calculated. Posthoc receiver‐operating characteristic curves were generated to determine the specificity of revised AV/IVC cortisol ratio and CLR in differentiating unilateral from bilateral disease.ResultsPatients underwent unilateral (n = 21) or bilateral (n = 4) adrenalectomy. The mean AV/IVC cortisol ratio for unilateral adrenalectomy was 12.1 ± 9.6 (dominant) and 4.7 ± 3.8 (contralateral) with a mean CLR of 3.6 ± 3.5. The mean AV/IVC cortisol ratio for bilateral adrenalectomy was 7.5 ± 2.1, with a mean CLR of 1.1 ± 0.6. At a mean follow‐up of 22 months, one patient who underwent unilateral adrenalectomy for the predicted bilateral disease developed recurrent mild autonomous cortisol secretion. Posthoc analyses demonstrated a specificity of 95%–100% for unilateral disease with AV/IVC cortisol ratio >9 for one side, <2.0 for the opposite side and a CLR > 2.3. The specificity was 80%–90% for bilateral disease with AV/IVC cortisol ratio >5.1 bilaterally and a CLR < 1.1.ConclusionsAmong patients with bilateral adrenal masses and ACTH‐independent autonomous cortisol secretion, AVS can distinguish between unilateral and bilateral disease with high specificity and may guide surgical management.

Abstract Introduction: Pediatric rhabdomyosarcoma (RMS) is the most common soft tissue tumor in children, with nearly 20% of children presenting with locally aggressive and/or metastatic disease. Despite aggressive chemotherapy and radiotherapy, the 5-year overall survival rate is still less than 40%. In addition, resistance to chemotherapy remains the major cause of cancer-related mortality. Therefore, there is an unmet need to develop more effective and tolerable therapeutic strategies for the treatment of RMS. Our overall objective is to leverage transcriptomic and proteomic profiling methods to identify RMS-specific surface proteins targetable with immune-based therapies. Methods: To identify therapeutic targets, we performed cell-surface capture and mass spectrometry on fusion-positive (FP), fusion-negative (FN) RMS and normal skeletal muscle. Transcriptomic datasets of RMS tumor specimens and normal tissue were employed to identify high-confidence RMS-specific cell-surface proteins. Expression of immune checkpoint molecules was confirmed by flow cytometry, western blot and PCR. Paraffin-embedded tissue sections were used to determine protein expression and prognostic value in RMS patients. We developed an in vitro T-cell killing assay to assess the functional role of immune checkpoint molecules on the antitumor immune response. Results: Surfaceome profiling of RMS cells revealed unique protein signatures specific for each tumor type and normal tissue. We have identified 5061 cell-surface proteins in RMS and 31.6% of proteins were correctly located at the plasma membrane. By integrating proteomic and transcriptomic data, we have identified 30 and 38 proteins significantly overexpressed in fusion-negative and fusion-positive RMS respectively compared to normal muscle. We have identified the immune checkpoint molecule B7-H3 as cell-surface protein overexpressed in both RMS subtypes. Strong expression of B7-H3 was also observed in patient’s tumor specimens. Genetic deletion of B7-H3 gene in RMS cells was associated with higher antitumor immune response in tumor cells co-cultured with CD8+-T cells. No difference was observed in tumor cell proliferation and radioresistance. Conclusions: Through integrated transcriptomic and proteomic profiling, we have discovered B7-H3 as a key regulator of tumor immune-evasion in RMS. B7-H3 is a suitable immunotherapy target for the treatment of RMS. Further studies are warranted to elucidate underlying mechanisms of B7-H3 function in RMS. Citation Format: Roxane R. Lavoie, Fabrice Lucien, Patricio C. Gargollo, Mohamed E. Ahmed, Yohan Kim, Emily Baer, Doris Phelps, Cristine M. Charlesworth, Benjamin J. Madden, Liguo Wang, Peter J. Hougthon, John Cheville, Haidong Dong, Candace F. Granberg. Surfaceome profiling of rhabdomyosarcoma reveals B7-H3 as a mediator of immune evasion [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A036.

Abstract Intratumoral heterogeneity arising from tumor evolution poses significant challenges biologically and clinically. Dissecting this complexity may benefit from deep learning (DL) algorithms, which can infer molecular features from ubiquitous hematoxylin and eosin (H&E)–stained tissue sections. Although DL algorithms have been developed to predict some driver mutations from H&E images, the ability of these DL algorithms to resolve intratumoral mutation heterogeneity at subclonal spatial resolution is unexplored. Here, we apply DL to a paradigm of intratumoral heterogeneity, clear cell renal cell carcinoma (ccRCC), the most common type of kidney cancer. Matched IHC and H&E images were leveraged to develop DL models for predicting intratumoral genetic heterogeneity of the three most frequently mutated ccRCC genes, BAP1, PBRM1, and SETD2. DL models were generated on a large cohort (N = 1,282) and tested on several independent cohorts, including a TCGA cohort (N = 363 patients) and two tissue microarray (TMA) cohorts (N = 118 and 365 patients). These models were also expanded to a patient-derived xenograft (PDX) TMA, affording analysis of homotopic and heterotopic interactions of tumor and stroma. The status of all three genes could be inferred by DL, with BAP1 showing the highest sensitivity and performance within and across tissue samples (AUC = 0.87–0.89 on holdout). BAP1 results were validated on independent human (AUC = 0.77–0.84) and PDX (AUC = 0.80) cohorts. Finally, BAP1 predictions correlated with clinical outputs such as disease-specific survival. Overall, these data show that DL models can resolve intratumoral heterogeneity in cancer with potential diagnostic, prognostic, and biological implications. Significance: This work demonstrates the potential for deep learning analysis of histopathologic images to serve as a fast, low-cost method to assess genetic intratumoral heterogeneity. See related commentary by Song et al., p. 2672

AbstractBackgroundWe hypothesized full‐thickness chest wall resection (FTCWR) with advanced surgical techniques and modern systemic therapy is safe, provides local control, and good overall survival.MethodsRetrospective review of FTCWR (including rib or part of sternum) for breast cancer between 2000 and 2020. Primary endpoints included 90‐day morbidities and all‐cause mortality. Secondary endpoints were loco‐regional and distant recurrence, DFS and overall survival (OS).ResultsA total of 35 patients met the criteria. 34 FTCWR were for recurrence and the median time to chest wall recurrence was 6 years. Tumor subtype was triple‐negative in 51% and the remainder HR+ Her2−. 58% were palliative resections. FTCWR included rib(s) in 89% and portion of sternum in 57%; 94% required reconstruction and 80% were R0 resections. There were no 90‐day mortalities. Overall morbidity was 10/35(28%). 17(49%) patients received neoadjuvant systemic therapy for their recurrence and three received neoadjuvant radiation. Adjuvant treatment included chemotherapy (8), endocrine therapy (3), and both (8). Ten patients (28%) received adjuvant radiation. The Median follow‐up was 31 months and there were 6 (17%) loco‐regional and 7 (20%) distant recurrences. OS was 86% and 67% at 1 and 3 years, respectively.ConclusionFTCWR was associated with low morbidity, mortality, recurrence rates, and good OS. Selective FTCWR is safe and has acceptable short‐term survival rates.

Abstract The outcome of patients with Muscle Invasive Bladder Cancer (MIBC) is highly variable with many patients experiencing progressive disease despite multimodal therapy. A key variable that contributes to poor patient outcomes lies in our inability to predict therapeutic response due to the molecular heterogeneity of MIBC tumors. There is, therefore, a significant unmet clinical need to accurately predict effective drug combinations and therapeutic doses in individual patients with MIBC. Patient-derived tumor organoids (PDOs) have the potential to serve as an ex vivo model of urothelial carcinoma (UC) capable of functionally predicting patient-specific treatment responses. However, current culture procedures typically rely on the expansion of UC organoids from resected tumors or biopsy specimens, which is inherently invasive and hence limits clinical application. We hypothesized that culture of MIBC PDOs can be achieved from urine samples, as is the case in dogs with MIBC, since histopathological features, tumor heterogeneity and subtypes, metastatic activity, and gene expression profiles of canine MIBC mimic those of human MIBC. Herein, we successfully cultured MIBC organoids from both human and canine patients using first-catch urine. Briefly, after centrifuging urine samples to pellet cells, the cells were washed with basal culture media containing FBS and Pen/Strep before being cultured in canine or human culture media containing essential growth factors based on their species of origin. Paraffin-embedded samples were used for molecular characterization by RNA in situ hybridization or immunohistochemistry. Organoids from canine MIBC patients were analyzed for expression of the urothelial differentiation marker (FOXA1) associated with high grade bladder cancer, CD44, a non-kinase transmembrane glycoprotein expressed in UC cancer stem cells, and CK7, an epithelial cell marker that is upregulated in neoplastic UC cells. FOXA1 expression was undetectable in canine MIBC, although CD44 expression was enhanced compared to healthy dog bladder organoids. All canine MIBC organoids had significantly enhanced CK7 expression. Similarly, in human-derived UC organoids, GATA binding protein 3 (GATA3), transcription factor paired box 8 (Pax8), and tumor protein p63 were utilized as markers, with a Pax8-/p63+/GATA3+ profile indicating UC origin of the organoids. Functional response to chemotherapy (cisplatin and gemcitabine) was further assessed using cell viability assay in canine and human UC organoids. Our data suggest that organoids cultured from the urine of patients with MIBC can recapitulate organ structures and maintain marker expression profiles of the original tumor tissues. 3D organoids may provide novel insights to predict therapeutic response to chemotherapy and serve as early diagnostic biomarkers of bladder cancer in both canine and human patients. Citation Format: Dipak Kumar Sahoo, Allison P. Mosichuk, Fabrice Lucien, Igor Frank, John C. Cheville, Karin Allenspach, Jonathan P. Mochel. Urine-derived urinary carcinoma organoids: A novel tool for providing new insights into human and canine bladder cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3092.