BAP1 Mutations Research Articles

Tumor evolution of brain-specific tropism in metastatic renal cell carcinoma.

4530 Background: Brain metastases (BMets) pose a clinical challenge in the management of patients (pts) with metastatic renal cell carcinoma (RCC), leading to significant morbidity and mortality. Herein, we sought to comprehensively characterize the molecular landscape of BMets in RCC. Methods: We performed panel-based DNA (DNAseq) and whole transcriptome (RNAseq) sequencing to analyze BMets, matched non-Brain metastases (nBMets), and matched primary renal tumors (PRT) from pts who underwent surgical resection of BMet(s) from RCC at our institution. Results: Our cohort consisted of 95 samples from 53 patients (BMets = 54, nBMets = 14, PRT = 27) with clear cell histology in 45 pts (84.9%). DNAseq was available 85 samples (50 pts). Patient-level mutations revealed recurring mutations in VHL(18 pts, 36%), TP53 (n = 12 pts, 24%), PBRM1(12 pts, 24%), SETD2(12 pts, 24%), and BAP1(4 pts, 8%). Mutations within the MTOR signaling pathway were enriched, with 25 pts (50%) having at least one mutation in PTEN (12 pts, 24%), TSC1 (4 pts, 8%), TSC2 (5 pts, 10%), or MTOR (9 pts, 18%). Copy number analyses revealed frequent deletions in chr14q21 (34 pts, 68%) and chr9p21 (35 pts, 70%) and gains in chr20q13 (n = 31, 62%) and chr7p15 (n = 31, 62%). At the sample-level, PTEN mutations were more common in BMets (11 pts, 22.9%) vs nBMets (0 pts) and PRTs (3 pts, 12%, p = 0.124), as were deletions in chr13q22 (BMets = 13pts [26.5%], nBMet = 1pts [8.3%], PRT = 1 [4%]; p = 0.036) and gains in chr20q13 (BMets = 30pts [61.2%], nBMet = 4pts [33.3%], PRT = 8 [32%]; p = 0.03). Deletions in chr9p21 were enriched in BMets (32, 65.3%) and nBMets (8, 66.7%) relative to PRTs (7, 28%, p = 0.006), whereas other copy number alterations and somatic mutations exhibited similar proportions across specimen sites. Differential expression analysis performed on 86 samples (51 pts) identified 806 differentially expressed genes (DEGs) between BMets and PRT (adjusted [adj.] p < 0.05) and 399 DEGs between BMet and nBMets (adj. p < 0.05). Gene set enrichment analysis of MSigDB Hallmark gene sets revealed upregulation of MTORC1 signaling, glycolysis, and MYC targets in BMets comparted to PRT and nBMETs (adj. p <0.0001). In contrast, immune-related gene sets, such as interferon-alpha, interferon-gamma, and tumor necrosis factor-alpha, were enriched in PRT relative BMets (adj. p <0.0001), but not nBMETs (adj. p > 0.05). CIBERSORT cellular deconvolution analysis comparing BMets with PRT revealed decreased proportions of M1 macrophages (p = 0.0003) and CD8+ T-cells (p = 0.0106), but an increase proportion of M2 macrophages (p = 0.0009). Conclusions: RCC with brain metastases are characterized by distinct copy number alterations, enrichment of MTOR pathway mutations, MTOR pathway hyperactivation, and an immunosuppressive tumor milieu. These findings may hold therapeutic implications of MTOR pathway inhibition and immune modulators in treating RCC BMets.

Uncovering actionable genetic alterations and immune predictive biomarkers for anal squamous cell carcinomas in the era of immunotherapy: PD-L1 and beyond.

3518 Background: Squamous cell carcinoma of the anal canal (SCAC) is a rare cancer with limited treatments. Immune checkpoint inhibitors (ICIs) use is routine for recurrent/refractory disease, but predictive biomarkers remain elusive. We analyzed the largest dataset of SCAC to date, focusing on PD-L1 expression. Methods: Next-generation sequencing of DNA (592 genes or WES) and RNA (WTS) was tested at Caris Life Sciences (Phoenix, AZ). PD-L1 was tested by immunohistochemistry (IHC) (SP142) and grouped as high (2+ and ≥5%), low (1-2 and 1-5%), and negative (0) CPS. dMMR/MSI-H was tested by IHC/NGS and tumor mutation burden (TMB)-High was defined as >10 mt/MB. RNA expression data was used to estimate the tumor microenvironment (TME) using QuantiSEQ. RNA signatures predictive of ICI response (interferon gamma [IFNγ]; T-cell inflamed signature [TIS]) were tested. X2/Fisher-Exact was used with significance shown as P-value adjusted for multiple comparisons (Q<0.05). Real-world overall survival (rwOS) was from insurance claims and calculated from tissue collection to last contact; time-on-treatment (TOT) was from start to finish of ICI. Results: Of 1242 SCAC samples, 64.3% (798) expressed PD-L1, 41.7% (518) showed high PD-L1 expression, 1.2% were dMMR/MSI-H and 14.5% were TMB-high. TMB-H trended higher in PD-L1 low vs PD-L1 negative (16.3% vs 10.2%, p=0.036). No trend was seen for dMMR/MSI-H. Mutations in PIK3CA (38.3% vs 21.7%) and CASP8(5.3% vs 0.3%) were significantly higher in PD-L1 high vs negative. Similar trends were seen in NF1 (2.4% vs 0.4%) and ARID1A (2.7% vs 0.6%). The opposite was noted for JAK2 (0% vs 2.6%), BAP1 (1.1% vs 3.6%) mutations, and FGFR3 amplifications (0% vs 2.2%). For PD-L1 high vs negative, infiltration of Tregs, M1 macrophages, neutrophils, CD8+ cells, and cancer-associated fibroblasts decreased with PD-L1 expression (fold change [FC]: 0.67-0.80, Q<0.05). Expression of immune-oncology (IO) markers IDO1, PDCD1, IFNG, CD274, HVACR2, CTLA4, LAG3, CD80, CD86 and PDCD1LG2 decreased with PD-L1 expression (FC:0.35-0.73, Q<0.05). Similarly, TIS and IFNg scores decreased with PD-L1 IHC (Q<0.05). In 316 SCAC patients treated with ICIs, PD-L1 high had longer TOT compared to PD-L1 negative (N=168 vs 80, 5.5m vs 2.9m, HR 0.758, 95% CI 0.579 to 0.992, p=0.044) while PD-L1 low (N=68) showed a TOT in between (3.2m) but not significantly different compared to high or negative. No differences in rwOS were observed. Conclusions: PD-L1 is expressed in over 50% of SCACs, while dMMR/MSI-H is rare. High PD-L1-expressing tumors have higher PIK3CAand CASP8 mutations and overexpress IO markers, are inflamed, and have prolonged treatment times with ICIs. This is the largest study to date reporting SCAC genomic profiles and identifies the utility of PD-L1 as a predictive biomarker of IO efficacy. This discovery needs to be confirmed in prospective trials.

Clinical and Genomic Features of Patients with Renal Cell Carcinoma and Advanced Chronic Kidney Disease: Analysis of a Multi-Institutional Database

Background: Patients with advanced chronic kidney disease (ACKD) are at an increased risk of developing renal cell carcinoma (RCC), but molecular alterations in RCC specimens arising from ACKD and overall survival (OS) in affected patients are not well defined. Patients and Methods: Using the Oncology Research Information Exchange Network (ORIEN) Total Cancer Care® protocol, 296 consented adult patients with RCC and somatic tumor whole exome sequencing were included. Patients with ACKD were defined as those with serum creatinine ≥1.5 mg/dL prior to RCC diagnosis. Results: Of 296 patients with RCC, 61 met the criteria for ACKD. The most common somatic mutations in the overall cohort were in VHL (126, 42.6%), PBRM1 (102, 34.5%), and SETD2 (54, 18.2%). BAP1 had a decreased mutational frequency in RCC specimens from patients without ACKD as compared to those with ACKD (10.6% versus 1.6%), but this was not statistically significant in univariable (OR 0.14, p = 0.056) or multivariable (OR 0.15, p = 0.067) analysis. Median OS was not reached in either cohort. Conclusions: Using the clinicogenomic ORIEN database, our study found lower rates of BAP1 mutations in RCC specimens from patients with ACKD, which may reflect a BAP1-independent mutational driver of RCC in patients with ACKD.

Primary leptomeningeal melanocytic neoplasms: A clinicopathologic, immunohistochemical, and molecular study of 12 cases

This study investigated the clinicopathological, immunohistochemical, and molecular features of primary leptomeningeal melanocytic neoplasms (LMNs). Twelve LMN cases were retrospectively reviewed. We performed Fluorescence in-situ hybridization (including a 4-probe FISH assay with CDKN2A and MYC assay) and Next-Generation sequencing analyses on available cases. Histologically, 2 tumours were classified as melanocytomas (MC), 2 as intermediate-grade melanocytomas (IMC), and 8 as leptomeningeal melanomas (LMM). Two rare cases of LMM were associated with large plaque-like blue nevus. One MC case was associated with Ota. Ten cases (83.3%) showed melanocytic cells with benign features diffusely proliferating within the meninges. The Ki-67 in three categories differed (MC 0–1%, IMC 0–3%, LMM 3–10%). 57.1% of LMM cases (4/7) were positive for FISH. Nine of 10 tumours harboured activating hotspot mutations in GNAQ, GNA11, or PLCB4. Additional mutations of EIF1AX, SF3B1, or BAP1 were found in 40%, 30%, and 10% of tumours, respectively. During the follow-up (median = 43 months), 5 LMM patients experienced recurrence and/or metastasis, 3 of them died of the disease and the other 2 are alive with the tumour. Our study is by far the first cohort of LMN cases tested by FISH. In addition to morphological indicators including necrosis and mitotic figures, using a combination of Ki-67 and FISH helps to differentiate between IMC and LMM, especially in LMM cases with less pleomorphic features. SF3B1 mutation is first described in 2 cases of plaque-type blue nevus associated with LMM. Patients with SF3B1 mutation might be related to poor prognosis in LMN.

MOICS, a novel classier deciphering immune heterogeneity and aid precise management of clear cell renal cell carcinoma at multiomics level

ABSTRACT Recent studies have indicated that the tumor immune microenvironment plays a pivotal role in the initiation and progression of clear cell renal cell carcinoma (ccRCC). However, the characteristics and heterogeneity of tumor immunity in ccRCC, particularly at the multiomics level, remain poorly understood. We analyzed immune multiomics datasets to perform a consensus cluster analysis and validate the clustering results across multiple internal and external ccRCC datasets; and identified two distinctive immune phenotypes of ccRCC, which we named multiomics immune-based cancer subtype 1 (MOICS1) and subtype 2 (MOICS2). The former, MOICS1, is characterized by an immune-hot phenotype with poor clinical outcomes, marked by significant proliferation of CD4+ and CD8+ T cells, fibroblasts, and high levels of immune inhibitory signatures; the latter, MOICS2, exhibits an immune-cold phenotype with favorable clinical characteristics, characterized by robust immune activity and high infiltration of endothelial cells and immune stimulatory signatures. Besides, a significant negative correlation between immune infiltration and angiogenesis were identified. We further explored the mechanisms underlying these differences, revealing that negatively regulated endopeptidase activity, activated cornification, and neutrophil degranulation may promote an immune-deficient phenotype, whereas enhanced monocyte recruitment could ameliorate this deficiency. Additionally, significant differences were observed in the genomic landscapes between the subtypes: MOICS1 exhibited mutations in TTN, BAP1, SETD2, MTOR, MUC16, CSMD3, and AKAP9, while MOICS2 was characterized by notable alterations in the TGF-β pathway. Overall, our work demonstrates that multi-immune omics remodeling analysis enhances the understanding of the immune heterogeneity in ccRCC and supports precise patient management.

Recent Advances in Pathology of Intrahepatic Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICCA) is a malignant epithelial neoplasm characterized by biliary differentiation within the liver. ICCA is molecularly heterogeneous and exhibits a broad spectrum of histopathological features. It is a highly aggressive carcinoma with high mortality and poor survival rates. ICCAs are classified into two main subtypes: the small-duct type and large-duct types. These two tumor types have different cell origins and clinicopathological features. ICCAs are characterized by numerous molecular alterations, including mutations in KRAS, TP53, IDH1/2, ARID1A, BAP1, BRAF, SAMD4, and EGFR, and FGFR2 fusion. Two main molecular subtypes-inflammation and proliferation-have been proposed. Recent advances in high-throughput assays using next-generation sequencing have improved our understanding of ICCA pathogenesis and molecular genetics. The diagnosis of ICCA poses a significant challenge for pathologists because of its varied morphologies and phenotypes. Accurate diagnosis of ICCA is essential for effective patient management and prognostic determination. This article provides an updated overview of ICCA pathology, focusing particularly on molecular features, histological subtypes, and diagnostic approaches.

Abstract 5367: Single cell resolution spatial transcriptomics reveals genetic driver and TME interaction in clear cell renal cell carcinoma

Abstract BAP1 mutations are associated with increased T cell and tumor-associated macrophage infiltration in clear cell renal cell carcinoma (ccRCC) and are predictive of poor prognosis. BAP1 mutations have also been found to be associated with T-cell infiltration in peritoneal mesothelioma and uveal melanoma. Because BAP1 mutations are known to be associated with aggressive disease and poor survival, we hypothesize that these mutations lead to a more immunosuppressive microenvironment. Using single nucleus RNA sequencing and high-resolution spatial transcriptomics, we confirmed that BAP1 mutant tumors are immune hot with a high fraction of Treg, exhausted CD8+ T cells, CD4+ T cells, and macrophages. At the same time, these tumors have a lower proportion of endothelial cells and pericytes. Evident of pro-tumor microenvironment we have found BAP1 mutations to be associated with the upregulation of SPP1, CD163, and MARCO immunosuppressive genes in tumor-associated macrophages. Here we propose a mechanism by which BAP1-mutant cancer cells induce an immunosuppressive microenvironment: through activation of the complement system and TGF-β signaling to attract macrophages. Taken together, our results suggest that BAP1 mutations contribute to poor ccRCC outcomes not only through cell-intrinsic effects, but also by promoting infiltration of Tregs and tumor-associated macrophages and polarizing infiltrated macrophages towards immunosuppressive phenotype. Citation Format: Ilya Strunilin, Wagma Caravan, Atieh Abedin-Do, Andrew Houston, Yize Li, Yizhe Song, Song Cao, Chia-Kuei Mo, Siqi Chen, Russel Pachynski, Feng Chen, Li Ding. Single cell resolution spatial transcriptomics reveals genetic driver and TME interaction in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5367.

Abstract 4581: Identifying class 1 HDACs as a vulnerability for aggressive uveal melanoma

Abstract Uveal melanoma (UM) is the most common primary intraocular cancer in adults, with a strong propensity to metastasize, primarily to the liver. Metastatic UM is notoriously unresponsive to existing therapies, rendering it almost uniformly lethal. Virtually all UM tumors have activating mutations in the Gq signaling pathway, predominantly in GNAQ/11, and around half of tumors have additional loss-of-function mutations in the tumor suppressor BAP1. Mutations in the epigenetic modulator BAP1 are the most significant clinical marker of metastatic risk. The limited efficacy of current treatment options necessitates an urgent exploration of novel therapeutic approaches to improve patient prognosis. Thus, we conducted a large epigenetic compound library screen with the currently most well characterized, focused epigenetic library available, which consisted of 960 potent, cell permeable, medically active, epigenetic-directed small molecule modulators (TargetMol, L1200). We screened BAP1-mutant as well as BAP1-wildtype uveal melanoma cell lines and followed up with comprehensive dose-response and synergy tests. Our lead compounds were highly efficient in reducing viability of UM cells. We find that inhibitors with high specificity for class 1 HDAC inhibition are especially cytotoxic to UM cells. Together, our results reveal a possible mechanism of interfering with UM progression and identify lead compounds to be tested in clinical trials. Citation Format: Gulum Yenisehirli, Ashley Zuniga, Sara Rodriguez, Emily V. Adis, Gabriela Quintana, Sebastian Borges, Sofia Lopez, Kassandra Lopez, Steffanie Braun, Claude H. Volmar, J. W. Harbour, Stefan Kurtenbach. Identifying class 1 HDACs as a vulnerability for aggressive uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4581.

Abstract 1758: Chromosome 3 copy number loss alone drives tumor evolution to aggressive disease in uveal melanoma

Abstract Uveal Melanoma (UM) is a rare tumor characterized by mutations in GNAQ or GNA11, followed by a mutation in BAP1, SF3B1 or EIF1AX. Notably, a large subset of BAP1 mutant patients present with a unique chromosome 3 copy-number (CN) loss and higher likelihood of metastases. The biology underlying the causes and consequences of Monosomy 3 (M3), and its role in metastases is largely unexplored. This can be partially attributed to the relatively few numbers of preclinical models of UM, and the inability to delineate M3 from BAP1 loss. To address this, we derived a preclinical isogenic model of chromosome 3 CN loss through CRISPR-based centromere targeting in a well characterized UM cell line lacking a BAP1 mutation. Clones with and without chromosome 3 loss were derived from these efforts. Recent studies have evaluated genomic events in primary UM and described five CN aberration-based subtypes expanding beyond M3, with the fifth subtype being called the “ultra-high-risk UM” based on poor survival and extremely high metastatic rate. Whole genome sequencing across our clones confirmed chromosome 3 CN loss, and in addition to this, recapitulated all of the core genomic features characteristic of the ultra-high-risk UM group. In comparison, our models without chromosome 3 CN loss maintained a genomic profile more characteristic of lower risk UM subtypes. The genomic landscape of our isogenic low risk (LR) and high risk (HR) clones suggest that chromosome 3 CN loss alone in the absence of BAP1 mutations can be the driver for acquisition of downstream genomic events seen in aggressive UM. Further, using single cell DNA sequencing on the parental model, LR and HR clones, we confirmed that chromosome 3 CN loss in our clones was engineered, and not a pre-existing subclone in the parental cell line. Bulk RNAseq on our LR and HR clones demonstrate concordance with genomic regions of losses and gains, while also highlighting upregulation of pathways such as cell cycle and DNA damage response in the HR clones. Next, we developed patient derived xenograft (PDX) models demonstrating differential behavior in paired in vivo settings, where HR clones had higher fitness advantage and metastatic potential, specifically to the mouse liver. This modeling has enabled us to employ spatial transcriptomics in a PDX setting, adapt existing methodology to infer chromosomal copy number events in our system and inform transcriptional and genomic phenotypes akin to the aggressive HR clones. Thus, using our engineered preclinical models, we demonstrate that chromosome 3 loss, in the absence of BAP1 mutations is capable of driving aggressive disease in UM, characterized by genomic evolution, transcriptional reprogramming and in vivo invasiveness. Citation Format: Sanjana Srinivasan, Johnathon Rose, Rosalba Minelli, Christopher Terranova, Parth Shah, Joseph R. Daniele, Michael Peoples, Meng He, Chiu-Yi Liu, Jason Gay, Melinda Soeung, Sanjay Soni, Fangyang Wang, Christopher Bristow, Khalida Wani, Luigi Perelli, Dmitry Loza, Ningping Feng, Maxime Tarabichi, Peter Van Loo, Andrew Futreal, Scott Woodman, Alexander Lazar, Christopher P. Vellano, Joseph Marszalek, Giulio F. Draetta, Timothy Heffernan, Alessandro Carugo, Giannicola Genovese, Virginia Giuliani. Chromosome 3 copy number loss alone drives tumor evolution to aggressive disease in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1758.

Abstract 3512: Joint probabilistic modeling of multimodal metabolic data with UnitedMet

Abstract Metabolites play a crucial role in the functioning of biological systems. Measurements of metabolite abundances by metabolomics and metabolic pathway activity by isotope-tracing are powerful approaches to study metabolic phenotypes. However, large-scale measurements of metabolite levels and pathway activity remain extremely scarce due to the technical challenges. Motivated by the strength of RNA-metabolite covariation, we present UnitedMet, a Bayesian probabilistic method for end-to-end joint modeling of RNA-sequencing and metabolic datasets that is capable of dimensionality reduction, data integration and metabolic modality prediction. UnitedMet demonstrates high accuracy predicting metabolite levels and isotopologue distributions from gene expression data in human tumor samples. To evaluate UnitedMet’s efficacy in clinical applications, we inferred metabolomic profiles and isotope labeling patterns from the RNA sequencing data of clear cell renal cell carcinoma (ccRCC) patients enrolled in TCGA and clinical trials. Correlation analysis between genetic mutations and predicted metabolic phenotypes revealed that BAP1 mutations were associated with increased contribution of glucose to TCA cycle in ccRCC. Increased TCA cycle activity was found to be associated with disease progression and poorer clinical outcome. UnitedMet, therefore, provides a solution that could be broadly applicable to enhance multimodal metabolomic datasets and facilitates comprehensive study of interplays between metabolic profiles, molecular alterations and human phenotypes. Citation Format: Amy X. Xie, Casey Bradshaw, Christopher Tosh, Wesley Tansey, Ed Reznik. Joint probabilistic modeling of multimodal metabolic data with UnitedMet [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3512.

Abstract 1468: Low exposures to amphibole or serpentine asbestos in germline Bap1-mutant mice induce mesotheliomacharacterized by an immunosuppressive tumor microenvironment

Abstract The purpose of this study was to determine if minimal exposures to amphibole (crocidolite) and serpentine (chrysotile) asbestos fibers significantly increase the incidence and rate of malignant mesothelioma (MM) onset in mice with a germline heterozygous Bap1 mutation, and to assess the effect of the resulting gene × environment interactions on the tumor immune microenvironment (TIME). The studies were carried out by chronically injecting intraperitoneally Bap1+/- and Bap1+/+ (wild-type, WT) mice with minimal doses of asbestos (in some experiments, as low as 0.0125 mg x 8 injections; total, 0.1 mg/mouse) and then monitoring the mice for tumor formation for up to 90 weeks. Pro-inflammatory and pro-tumorigenic factors in the TIME were evaluated by immunohistochemistry (IHC) and immunofluorescence (IF) for immune cell markers. With either type of asbestos and at each dose tested, MMs occurred at a significantly higher rate and earlier onset time in Bap1-mutant mice than in WT littermates. MMs from mice injected with chrysotile, but less so with crocidolite, had widespread adhesions in the peritoneal cavity owing to an overwhelming immune response, particularly at the higher dose used. IHC staining demonstrated an increased number of immune cells in granulomas and MMs. In particular, pro-inflammatory M1 macrophages were found mainly in granulomas, whereas pro-tumorigenic M2 macrophages were predominant in MMs. IF staining revealed that the relative number of M2 macrophages was strikingly greater in chrysotile-induced than crocidolite-induced MMs, suggesting that chrysotile induces a more profound immune response. MMs from Bap1-mutant mice showed upregulation of both CD39/CD73-adenosine and Ccl2/Ccr2 pathways, which collectively are known to mechanistically create favorable conditions for neoplastic transformation, invasion, and the development of an immunosuppressive TIME, in part by attracting M2 macrophages. This implies that chemoprevention and immunotherapy strategies targeting these pro-tumorigenic immune pathways could be beneficial in BAP1 mutation carriers. In conclusion, we provide evidence that low-level exposures to either chrysotile or crocidolite in germline Bap1 heterozygous mice result in a markedly increased risk of MM characterized by an immunosuppressive tumor microenvironment. Drawing parallels to humans, these experimental findings suggest that exposure of BAP1 mutation carriers to even minimal doses of different forms of asbestos may be associated with significantly enhanced susceptibility to MM. Citation Format: Yuwaraj Kadariya, Eleonora Sementino, Maggie Ruan, Mitchell Cheung, Andres J. Klein-Szanto, Kathy Cai, Joseph R. Testa. Low exposures to amphibole or serpentine asbestos in germline Bap1-mutant mice induce mesotheliomacharacterized by an immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1468.

Abstract 5673: Transposable element expression patterns in epigenetic mutational subtypes of clear cell renal cell carcinoma

Abstract Background: Clear cell renal cell carcinoma (ccRCC) is characterized by mutations in epigenetic modulators and by aberrant transposable element (TE) expression, each of which have been separately associated with immunotherapy (IO) benefit in specific clinical contexts. However, the relationship between mutations in epigenetic regulators and TE expression, and the potential effect of both somatic mutations and TE expression on clinical outcomes, has not been fully investigated. Methods: We utilized RNA sequencing from ccRCC patients’ tumors and cell lines to quantify TE subfamily expression with TEtranscripts (n=123 metastatic IO-treated; CheckMate-009, 010, 025, n=355 all stage TCGA untreated, n=8 CCLE). Locus-specific endogenous retrovirus (ERV) expression was quantified using hervQuant. Differential TE and ERV expression was determined using DESeq2. Samples were stratified by mutations in PBRM1, SETD2, BAP1 and KDM5C, four of the top five mutated genes in ccRCC. Multivariable CoxPH models and the log-rank test were used to compare overall (OS) and progression-free survival (PFS). Two validation metastatic ccRCC cohorts were employed: the everolimus arm of CheckMate-025 (n=78, CM025e) and IMmotion150 (n=146, IM150). Results: Global TE expression (LTR, LINE, SINE and DNA transposons) did not differ significantly across mutational and clinical subtypes. However, 29 of 795 TE subfamilies were differentially expressed in >1 mutational subtype across datasets, and 22 of 29 were ERVs. At locus-specific resolution, 52 of 3173 ERVs were differentially expressed in >1 mutational subtype across datasets. Additionally, 6 of the 52 ERVs nominally associated with PFS, and 4 nominally associated with OS, in the IO cohort. Among them, only ERVs 544 and 2014 associated with improved PFS (multivariable CoxPH p = 0.03 and 0.02, respectively) and were expressed across all datasets. Both ERVs also exhibited increased expression in PBRM1 mutant and decreased expression in BAP1 mutant vs. wildtype samples, in discovery (IO, TCGA, CCLE) and validation (CM025e, IM150) cohorts. Lastly, PBRM1 mutant patients with high ERV 544 or 2014 expression had improved PFS in the IO (log-rank p = 0.04 and 0.03, multivariable CoxPH p = 0.008 and 0.009, respectively, HR = 0.43 both), but not in the CM025e cohort, compared to other patients regardless of PBRM1 mutations. Conclusion: ERVs 544 and 2014 were upregulated in PBRM1 mutant and downregulated in BAP1 mutant samples. Coupled with PBRM1 inactivation, their expression served as a candidate biomarker of improved PFS in IO-treated ccRCC that may have additional biological relevance. Expanded functional and clinicogenomic studies of epigenetic mutational subtype-specific patterns of TE expression, and their relation to selective IO response, may guide additional discovery and therapeutic development strategies in ccRCC. Citation Format: Kevin Meli, Cora A. Ricker, Sabrina Y. Camp, Christof C. Smith, Chris Labaki, Eddy Saad, Hanna Soulati, Brendan Reardon, Jihye Park, Natalie I. Vokes, Benjamin G. Vincent, Toni K. Choueiri, David A. Braun, Eliezer M. Van Allen. Transposable element expression patterns in epigenetic mutational subtypes of clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5673.

Abstract 4304: Chromosome 3 dosage-loss informs on the unique biology underlying ultra high-risk uveal melanoma

Abstract Uveal melanoma (UM) is a rare and lethal tumor characterized by mutually exclusive activating mutations in GNAQ and GNA11, secondary mutations in EIF1AX, SF3B1 and BAP1, and a late-stage chromosome 3 copy-number (CN) loss event. Importantly, chromosome 3 CN loss is associated with BAP1 loss-of-function (LOF) and corresponds with tumor metastasis and poor patient prognosis. Understanding the biology underlying this chromosome 3 CN loss event is critical to informing on metastatic UM. To this end, we leveraged CRISPR-based centromere targeting to engineer multiple clones exhibiting chromosome 3 CN loss. We determined that dosage-loss of chromosome 3 was sufficient to develop multiple isogenic clones exhibiting genomic features of “ultra high-risk” UM, independent of BAP1 LOF. Leveraging this isogenic system, we evaluated in vitro and in vivo profiling, demonstrating site-specific liver metastasis only in orthotopic HR tumors, along with a significantly increased fitness in the liver. Next, we sought to inform on differential signaling pathways and therapeutic sensitivity between LR and HR models. Characterization of established UM oncogenic pathways identified a clear disengagement from classical MAPK and non-canonical Hippo signaling in the HR clones. These findings also correlate with a significant decrease in sensitivity to MEK and FAK inhibitors. In addition, we observed a PKC isoform swap from PKCε and PKCδ to PKCα, a feature observed in late-stage UM patient tumors. Interestingly, we also observed an increase in the expression of PRAME and NBS1 in HR models, both markers for poor prognosis in patients. Genome-anchored transcriptomic analysis also captured distinct profiles between the LR and HR clones, highlighting a significant increase in MYC associated targets upon chromosome 3 dosage-loss. This increase in MYC signaling coincides with site-specific 8q amplifications across the HR clones, implicating MYC as a core fitness driver in high-risk UM and a potential contributor to poor patient prognosis in monosomy 3 patients. Leveraging engineered models of chromosome 3 CN loss, and the subsequent induction of “ultra high-risk” UM, provides a unique opportunity to inform on the underlying biology of UM evolution and potential therapeutic strategies. Citation Format: Johnathon Lynn Rose, Sanjana Srinivasan, Meng He, Rosalba Minelli, Chiu-Yi Liu, Christopher Terranova, Jason Gay, Joseph R. Daniele, Loza Dmitriy, Michael Peoples, Melinda Soeung, Vandhana Ramamoorthy, Parth Shah, Sanjay Soni, Christopher Bristow, Fangyang Wang, Anastasia Lopez, Charles Deckard, Brooke Meyers, Luigi Perelli, Khalida Wani, Ningping Feng, Christopher P. Vellano, Joe Marszalek, Alexander J. Lazar, Andrew Futreal, Giulio F. Draetta, Scott E. Woodman, Timothy Heffernan, Giannicola Genovese, Alessandro Carugo, Virginia Giuliani. Chromosome 3 dosage-loss informs on the unique biology underlying ultra high-risk uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4304.

Identification of Somatic Mutations in Thai Patients with Renal Cell Carcinoma Using Whole Exome Sequencing Analysis

Background: Whole exome sequencing (WES) is increasingly used to identify genetic alterations of renal cell carcinoma (RCC). However, in Thailand, there is no report that clarifies the common somatic mutations and tumor mutational burden (TMB) in RCC. Therefore, the understanding of the tumor somatic mutational landscape could improve RCC management in the authors’ country. Objective: To perform a descriptive study to identify common somatic mutated genes and TMB in clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (pRCC), and clear cell papillary renal cell carcinoma (ccpRCC). Materials and Methods: The authors enrolled 13 patients, which consisted of 10 cases of ccRCC, two cases of pRCC, and one case of ccpRCC. DNA was isolated from peripheral blood mononuclear cells and tumor tissues for WES to identify tumor somatic mutations. The results were analyzed for correlations with tumor-aggressive features and compared with the public database. Results: The authors identified common somatic mutations in VHL, SVIL, MUC16, CSMD3, CSMD1, and BAP1 in the study patients. In ccRCC cases, VHL mutation was detected in 90% of the cases corresponding to its high frequency in the TCGA’s ccRCC database. In pRCC cases, KDM6A was the only common mutated gene that overlapped with the top-ten most common genes in the TCGA’s pRCC database. Somatic mutations in BAP1, SETD2, and PBRM1 were significantly associated with tumor-aggressive features in the present study. The mean TMB of ccRCC, pRCC, and ccpRCC were 2.017, 2.143, and 6.61 mutations per megabase, respectively. Conclusion: Identification of common somatic mutations and TMB in all subtypes of RCC from the present study showed the diversity of genetic alterations between Thai patients and the public database. This leads to specific therapeutic approaches for Thai patients. Moreover, the authors also detected similar associations between significant mutations reported in prior studies with the tumor-aggressive features in the present cases. Keywords: Renal cell carcinoma; Somatic mutation; Tumor mutational burden; Whole exome sequencing

The Prevalence and Radiologic Features of Renal Cancers Associated with FLCN, BAP1, SDH, and MET Germline Mutations.

Purpose To investigate the prevalence of FLCN, BAP1, SDH, and MET mutations in an oncologic cohort and determine the prevalence, clinical features, and imaging features of renal cell carcinoma (RCC) associated with these mutations. Secondarily, to determine the prevalence of encountered benign renal lesions. Materials and Methods From 25 220 patients with cancer who prospectively underwent germline analysis with a panel of more than 70 cancer-predisposing genes from 2015 to 2021, patients with FLCN, BAP1, SDH, or MET mutations were retrospectively identified. Clinical records were reviewed for patient age, sex, race/ethnicity, and renal cancer diagnosis. If RCC was present, baseline CT and MRI examinations were independently assessed by two radiologists. Summary statistics were used to summarize continuous and categorical variables by mutation. Results A total of 79 of 25 220 (0.31%) patients had a germline mutation: FLCN, 17 of 25 220 (0.07%); BAP1, 22 of 25 220 (0.09%); SDH, 39 of 25 220 (0.15%); and MET, one of 25 220 (0.004%). Of these 79 patients, 18 (23%) were diagnosed with RCC (FLCN, four of 17 [24%]; BAP1, four of 22 [18%]; SDH, nine of 39 [23%]; MET, one of one [100%]). Most hereditary RCCs demonstrated ill-defined margins, central nonenhancing area (cystic or necrotic), heterogeneous enhancement, and various other CT and MR radiologic features, overlapping with the radiologic appearance of nonhereditary RCCs. The prevalence of other benign solid renal lesions (other than complex cysts) in patients was up to 11%. Conclusion FLCN, BAP1, SDH, and MET mutations were present in less than 1% of this oncologic cohort. Within the study sample size limits, imaging findings for hereditary RCC overlapped with those of nonhereditary RCC, and the prevalence of other associated benign solid renal lesions (other than complex cysts) was up to 11%. Keywords: Familial Renal Cell Carcinoma, Birt-Hogg-Dubé Syndrome, Carcinoma, Renal Cell, Paragangliomas, Urinary, Kidney © RSNA, 2024.

Integrated analyses of the genetic and clinicopathological features of cholangiolocarcinoma: cholangiolocarcinoma may be characterized by mismatch-repair deficiency.

Cholangiolocarcinoma (CLC) is a primary liver carcinoma that resembles the canals of Hering and that has been reported to be associated with stem cell features. Due to its rarity, the nature of CLC remains unclear, and its pathological classification remains controversial. To clarify the positioning of CLC in primary liver cancers and identify characteristics that could distinguish CLC from other liver cancers, we performed integrated analyses using whole-exome sequencing (WES), immunohistochemistry, and a retrospective review of clinical information on eight CLC cases and two cases of recurrent CLC. WES demonstrated that CLC includes IDH1 and BAP1 mutations, which are characteristic of intrahepatic cholangiocarcinoma (iCCA). A mutational signature analysis showed a pattern similar to that of iCCA, which was different from that of hepatocellular carcinoma (HCC). CLC cells, including CK7, CK19, and EpCAM, were positive for cholangiocytic differentiation markers. However, the hepatocytic differentiation marker AFP and stem cell marker SALL4 were completely negative. The immunostaining patterns of CLC with CD56 and epithelial membrane antigen were similar to those of the noncancerous bile ductules. In contrast, mutational signature cluster analyses revealed that CLC formed a cluster associated with mismatch-repair deficiency (dMMR), which was separate from iCCA. Therefore, to evaluate MMR status, we performed immunostaining of four MMR proteins (PMS2, MSH6, MLH1, and MSH2) and detected dMMR in almost all CLCs. In conclusion, CLC had highly similar characteristics to iCCA but not to HCC. CLC can be categorized as a subtype of iCCA. In contrast, CLC has characteristics of dMMR tumors that are not found in iCCA, suggesting that it should be treated distinctly from iCCA. © 2024 The Pathological Society of Great Britain and Ireland.

Quantification of eosinophilic area and its potential molecular feature in clear cell renal cell carcinoma.

Previous studies have acknowledged the presence of eosinophilic cytoplasm in clear cell renal cell carcinoma, yet the precise quantification method and potential molecular attributes in clear cell renal cell carcinoma remain elusive. This study endeavours to precisely quantify the eosinophilic attribute and probe into the molecular mechanisms governing its presence in clear cell renal cell carcinoma. Data from cohorts of clear cell renal cell carcinoma patients who underwent nephrectomy, comprising The Cancer Genome Atlas cohort (n=475) and Sun Yat-sen University Cancer Center cohort (n=480), were aggregated to assess the eosinophilic attribute. Additionally, Omics data from Clinical Proteomic Tumor Analysis Consortium (CPTAC) (n=58) were leveraged to explore the potential molecular features associated with eosinophilic clear cell renal cell carcinoma. Employing receiver operating characteristic curve analysis, the proportion of tumour cells with eosinophilic cytoplasm was determined, leading to the classification of each cohort into distinct groups: a clear group (<5%) and an eosinophilic group (≥5%). In both cohorts, the eosinophilic feature consistently correlated with higher International Society of Urological Pathology (ISUP) grade, elevated tumor stage, and the presence of necrosis. Furthermore, the Kaplan-Meier method demonstrated that patients in the eosinophilic group exhibited shorter overall survival or disease-free survival compared with those in the clear group, a pattern reaffirmed in various stratified survival analyses. Intriguingly, within The Cancer Genome Atlas cohort, the pathological characterization of cell cytoplasm (eosinophilic vs. clear) emerged as an independent risk factor for overall survival (hazard ratio = 2.507 [95% confidence interval: 1.328-4.733], P = 0.005) or disease-free survival (hazard ratio = 1.730 [95% confidence interval: 1.062-2.818], P = 0.028) via Cox regression analysis. Moreover, multi-Omics data unveiled frequent BAP1 mutations and down-regulation of Erythroblast Transformation-Specific-Related Gene associated with the eosinophilic feature in clear cell renal cell carcinoma. Additionally, patients with low expression of Erythroblast Transformation-Specific-Related Gene showed worse overall survival (P < 0.001). The quantification of the eosinophilic feature serves as a robust predictor of clinical prognosis in clear cell renal cell carcinoma. Furthermore, the manifestation of this feature may be linked to BAP1 mutations and the down-regulation of Erythroblast Transformation-Specific-Related Gene in clear cell renal cell carcinoma. Significantly, the expression levels of Erythroblast Transformation-Specific-Related Gene manifest as an exemplary prognostic marker, providing exceptional predictive accuracy for the clinical prognosis in clear cell renal cell carcinoma.

Mass Spectrometry-Based Profiling of Histone Post-Translational Modifications in Uveal Melanoma Tissues, Human Melanocytes, and Uveal Melanoma Cell Lines - A Pilot Study.

Epigenetic alterations in uveal melanoma (UM) are still neither well characterized, nor understood. In this pilot study, we sought to provide a deeper insight into the possible role of epigenetic alterations in the pathogenesis of UM and their potential prognostic relevance. To this aim, we comprehensively profiled histone post-translational modifications (PTMs), which represent epigenetic features regulating chromatin accessibility and gene transcription, in UM formalin-fixed paraffin-embedded (FFPE) tissues, control tissues, UM cell lines, and healthy melanocytes. FFPE tissues of UM (n = 24), normal choroid (n = 4), human UM cell lines (n = 7), skin melanocytes (n = 6), and uveal melanocytes (n = 2) were analyzed through a quantitative liquid chromatography-mass spectrometry (LC-MS) approach. Hierarchical clustering showed a clear separation with several histone PTMs that changed significantly in a tumor compared to normal samples, in both tissues and cell lines. In addition, several acetylations and H4K20me1 showed lower levels in BAP1 mutant tumors. Some of these changes were also observed when we compared GNA11 mutant tumors with GNAQ tumors. The epigenetic profiling of cell lines revealed that the UM cell lines MP65 and UPMM1 have a histone PTM pattern closer to the primary tissues than the other cell lines analyzed. Our results suggest the existence of different histone PTM patterns that may be important for diagnosis and prognosis in UM. However, further analyses are needed to confirm these findings in a larger cohort. The epigenetic characterization of a panel of UM cell lines suggested which cellular models are more suitable for epigenetic investigations.

The distribution of mutations across tumor size in ccRCC and their prognostic importance in small masses.

479 Background: Substantial efforts have been made to delineate mutational pathways to progression in ccRCC and the prognostic importance of key mutations, yet the distribution of these mutations across tumor size remains unknown. We hypothesized that VHL and PBRM, as more truncal mutations, would be roughly equally prevalent across tumor sizes, while mutations associated with aggressive disease - SETD2, BAP1, and CDKN2a copy-number loss - would be predominantly observed in larger tumors. We further hypothesized that SETD2, BAP1, and CDKN2a copy-number loss mutations, when present in smaller (≤7 cm) ccRCC tumors, would portend worse prognosis. Methods: We assessed a combined cohort of 333 ccRCC tumors from TCGA-KIRC and TRACERx Renal for the distribution of mutations across tumor size. Logistic regression was used to model the presence of each mutation against tumor size. We assessed a subset of 194 tumors ≤7cm for associations of key mutations with clinical outcomes while controlling for size. In small masses, logistic regression was used to model the presence of metastatic disease and invasive disease, and Cox proportional hazards was used to model overall survival, against SETD2, BAP1, CDKN2a copy-number loss, and tumor size. Results: On logistic regression an increase in one centimeter of tumor size was associated with SETD2, BAP1, and CDKN2a loss at odds ratios of 1.16, 1.11, 1.19 (p&lt;0.05); whereas no significant association was observed between tumor size and both VHL and PBRM1 (p=0.18, p=0.65). Among 194 tumors ≤7 cm, SETD2 and CDKN2a loss were associated with metastatic disease at odds ratios of 3.86 and 3.84 (p&lt;0.05); CDKN2a loss was associated with worse overall survival at hazard ratio 2.19 (p&lt;0.05), all while controlling for tumor size. Conclusions: SETD2 mutations, BAP1 mutations, and CDKN2a copy-number loss are rare in small ccRCC and are increasingly common in larger tumors, whereas VHL and PBRM1 are fairly evenly distributed across tumor sizes. In tumors≤7 cm, SETD2 mutation and CDKN2a loss were associated with metastatic disease and CDKN2a loss was associated with worse overall survival. SETD2 mutations and CDKN2a loss may help risk stratify ccRCC in biopsied and resected tumors. [Table: see text]

Uveal Melanoma: Molecular and Genetic Mechanisms of Development and Therapeutic Approaches

Uveal melanoma (UM) is a neuroectodermal tumor that results from malignant transformation of melanocytes in the eye uvea, including the iris, the ciliary body, and the choroid. UM accounts for 5% of all melanoma cases and is extremely aggressive with half of the UM patients developing metastases within the first 1-2 years after tumor development. Molecular mechanisms of UM carcinogenesis are poorly understood, but are known to differ from those of skin melanoma. Activating mutations of the GNAQ and GNA11 genes, which code for the large G protein subunits Gq and G11, respectively, are found in 90% of UM patients. The Gaq/PKC/MAPK signaling pathway is a main signaling cascade that leads to the transformation of melanocytes of the uveal tract, and major regulators of the cascade provide targets for the development of drugs. Metastatic UM (MUM) is most often associated with mutations of BAP1, EIF1AX, GNA11, GNAQ, and SF3B1. A combination of a commercial expression test panel of 15 genes and a mutation panel of 7 genes, supplemented with data on the size of the primary tumor, is highly efficient in predicting the risk of metastasis. The risk of metastasis determines the choice of therapy and the patient follow-up regimen. However, no systemic therapy for MUM has been developed to date. New drugs undergoing clinical trials are mostly targeted drugs designed to inhibit the protein products of mutant genes or immunotherapeutic agents designed to stimulate the immune response against specific antigens. In addition to these approaches, potential therapeutic targets of epigenetic regulation of UM development are considered in the review.