BAP1 Mutations Research Articles

Potential Role of VHL, PTEN, and BAP1 Mutations in Renal Tumors.

The genetic profiling of renal tumors has revealed genomic regions commonly affected by structural changes and a general genetic heterogeneity. The VHL, PTEN, and BAP1 genes are often mutated in renal tumors. The frequency and clinical relevance of these mutations in renal tumors are still being researched. In our study, we investigated VHL, PTEN, and BAP1 genes and the sequencing of 24 samples of patients with renal tumors, revealing that VHL was mutated at a noticeable frequency (25%). Six of the investigated samples showed mutations, and one genetic polymorphism (rs779805) was detected in both heterozygote and homozygote forms. PTEN gene mutation was observed in only one sample, and one specimen showed genetic polymorphism. In the case of the BAP1 gene, all of the samples were wild types. Interestingly, VHL mutation was detected in two female patients diagnosed with AML and in one with oncocytoma. We assume that VHL or PTEN mutations may contribute to the development of human renal cancer. However, the overall mutation rate was low in all specimens investigated, and the development and prognosis of the disease were not exclusively associated with these types of genetic alterations.

DP08 Next-generation sequencing: the future for malignant melanoma diagnostic precision? A case of a BAP1 mutation

Abstract A 48-year-old man presented to the hand surgeons with a 5-year history of a subcutaneous swelling on his right wrist, associated with paraesthesia and pain, initially thought to be a benign ganglion. Excision biopsy of the lesion on histology revealed an encapsulated pleomorphic tumour attached to a nerve, thought to represent a sarcoma. Immunohistochemistry showed a positive reaction to S100, SOX10, calretinin and synaptophysin. Ki67 showed a 20% proliferation index. Multiple pathology opinions were sought, and further testing was performed at a specialist genomic laboratory. Detailed questioning regarding family history of malignancy revealed that the patient’s mother had died from metastatic uveal melanoma, a maternal aunt had died of a brain tumour and his maternal grandmother had lung cancer. Pan-cancer gene panel DNA next-generation sequencing showed a BAP1 mutation with a very high variant allele fraction, suggestive of germline inheritance. Given the clinical history, morphology, immunoprofile and DNA result, a favoured diagnosis of metastatic malignant melanoma with a BAP1 mutation was made. The patient was referred to the specialist skin cancer multidisciplinary team meeting and reviewed by the dermatology team. Full skin check examination revealed two long-standing atypical naevi (> 10 years), one on the left forearm and another on his back, and these were excised urgently. Histology of the back lesion showed a polypoid melanocytic tumour comprising large epithelioid melanocytes with mild-to-moderate nuclear pleomorphism, multinucleated cells and intranuclear inclusions. This was in keeping with a pT4a nodular malignant melanoma with 7-mm Breslow thickness, associated with BAP1-inactivated syndrome. Histology of the left forearm lesion was consistent with a combined BAP1-inactivated melanocytoma, otherwise known as a ‘BAPoma’. An ophthalmology review did not identify a primary uveal melanoma. Staging scans of his chest, abdomen and pelvis and magnetic resonance imaging of his head showed no evidence of metastatic disease. The patient was referred for genetics counselling, wide local excision of the melanoma on his back, for consideration of adjuvant therapy with oncology and long-term follow-up with dermatology. BAP1 tumour predisposition syndrome is a newly recognized cancer syndrome that predisposes the patient to uveal melanoma, malignant mesothelioma, renal cell carcinoma, cutaneous melanomas and a range of other cancers (Masoomian B, Shields JA, Shields CL. Overview of BAP1 cancer predisposition syndrome and the relationship to uveal melanoma. J Curr Ophthalmol 2018; 30:102–9). BAP1-related cancers tend to occur earlier in life and are more aggressive. Our case highlights the need for early identification of germline mutations through sequencing to improve outcomes and treatment-related morbidity by early diagnosis in these rare cancer syndromes.

The differences in immune features and genomic profiling between squamous cell carcinoma and adenocarcinoma – A multi-center study in Chinese patients with uterine cervical cancer

BackgroundSquamous cell carcinoma (SCC) and adenocarcinoma (AC) of the uterine cervix have distinct biological behaviors and different treatment responses. Studies on immune features and genomic profiling of these two pathologic types were limited and mainly focused on small patient cohorts. MethodsFrom 2014 to 2021, 336 (254 SCC vs. 82 AC) cervical cancer patients who were diagnosed/treated in 7 medical centers in China were enrolled in the study. Next-generation sequencing of 425 cancer-relevant genes was performed on tumor tissues and liquid biopsies. Somatic alterations and immune response-related biomarkers were analyzed. Patient prognosis and immune infiltration were analyzed using data from The Cancer Genome Atlas (TCGA). ResultsAC tended to have more immunotherapy resistance-related STK11 alterations (P = 0.039), a higher proportion of microsatellite instability (P = 0.21), and more actionable mutations (P = 0.161). In contrast, higher tumor mutational burdens (TMB; P = 0.01), a higher proportion of TMB-high patients (P = 0.016), and more PD-L1-high patients (P = 0.0013) were observed in SCC. Multiple genetic alterations and aberrant signaling pathways were specifically enriched in AC (e.g., TP53, KRAS, ERBB2, and ARID1A alterations) or SCC (e.g., PIK3CA, FBXW7, EP300, and BAP1 mutations). Notably, AC-enriched genetic changes were significantly associated with decreased infiltrations of various B cells, T cells, and dendritic cells, whereas SCC-associated molecular features tended to be associated with increased CD4+ T cell infiltrations. ConclusionsThis was the first multi-center study revealing the immunologic and genomic features between SCC and AC in Chinese patients with cervical cancer. Our findings have illustrated the difference in genetic profiles of those two cervical cancer subtypes, which may suggest the possibility of differential treatment regimens, with better immunotherapy efficacy in SCC and targeted therapy options more favorable in AC.

Genomic profiles of renal cell carcinoma in a small Chinese cohort.

Our aim was to describe the molecular characteristics of Renal Cell Carcinoma (RCC) and develop a small panel of RCC-associated genes from a large panel of cancer-related genes. Clinical data of 55 patients with RCC diagnosed in four hospitals from September 2021 to August 2022 were collected. Among the 55 patients, 38 were diagnosed with clear cell RCC (ccRCC), and the other 17 were diagnosed with non-clear cell RCC (nccRCC), including 10 cases of papillary renal cell carcinoma, 2 cases of hereditary leiomyomatosis and RCC syndrome (HLRCC), 1 eosinophilic papillary RCC, 1 tubular cystic carcinoma, 1 TFE3 gene fusion RCC, and 2 RCC with sarcomatoid differentiation. For each patient, 1123 cancer-related genes and 79 RCC-associated genes were analyzed. The most frequent mutations in a large panel of 1123 cancer-related genes in the overall population of RCC patients were VHL (51%), PBRM1 (35%), BAP1 (16%), KMT2D (15%), PTPRD (15%), and SETD2 (15%). For ccRCC patients, mutations in VHL, PBRM1, BAP1, and SERD2 can reach 74%, 50%, 24%, and 18%, respectively, while for nccRCC patients, the most frequent mutation was FH (29%), MLH3 (24%), ARID1A (18%), KMT2D (18%), and CREBBP (18%). The germline mutation rate in all 55 patients reached 12.7% (five with FH, one with ATM, and one with RAD50). The small panel containing only 79 RCC-associated genes demonstrated that mutations of VHL, PBRM1, BAP1, and SETD2 in ccRCC patients were 74%, 50%, 24%, and 18% respectively, while for the nccRCC cohort, the most frequent mutations were FH (29%), ARID1A (18%), ATM (12%), MSH6 (12%), BRAF (12%), and KRAS (12%). For ccRCC patients, the spectrum of mutations by large and small panels was almost the same, while for nccRCC patients, the mutation spectrum showed some differences. Even though the most frequent mutations (FH and ARID1A) in nccRCC were both demonstrated by large panels and small panels, other less frequent mutations such as MLH3, KMT2D, and CREBBP were not shown by the small panel. Our study revealed that nccRCC is more heterogeneous than ccRCC. For nccRCC patients, the small panel shows a more clear profile of genetic characteristics by replacing MLH3, KMT2D, and CREBBP with ATM, MSH6, BRAF, and KRAS, which may help predict prognosis and make clinical decisions.

Genomic determinants of progression-free survival after second line of therapy (PFS-2) for patients on immunotherapy-based combination therapies for advanced clear cell renal cell carcinoma.

4545 Background: The role of somatic genomic mutations in predicting outcomes in clear cell renal cell carcinoma (ccRCC) is poorly defined. Both ipilimumab/nivolumab (IO/IO) and several combinations of VEGFR-targeted tyrosine kinase inhibitor (TKI/IO) are preferred first-line treatment options for metastatic ccRCC. The impact of somatic mutations on PFS-2 for IO combination therapies was evaluated in this retrospective analysis. Methods: One-hundred forty-one patients with ccRCC had sequencing with MSK-IMPACT and initiated 1st line combination IO at Memorial Sloan Kettering (MSK) Cancer Center between 1/1/2014 and 12/30/2020. Treatment groups are defined as IO/IO (n=76) and TKI/IO (n=65). PFS-2 is defined as time from start of 1st line to progression on next therapy, or death, and is estimated using Kaplan-Meier method. PFS-2 association with each mutation was assessed via log-rank test and a model-based approach. Restricted mean survival time (RMST) up to 48 months was calculated for PFS-2 and modeled using a generalized linear model adjusted for IMDC risk. To test for heterogeneity of effect among treatment groups, an interaction test between somatic mutation and treatment group is performed in the model. Results: VHL, PBRM1, SETD2, mTOR pathway, BAP1, and TP53 were the most common somatic mutations. Only BAP1 was significantly associated with PFS-2: median PFS-2 was 44 months for patients without BAP1 mutation and 17 months for patients with BAP1 mutation (log-rank p=0.006). After adjusting for IMDC risk group, 48 month RMST for PFS-2 was 13 months lower in patients with BAP1 mutation. An interaction test performed in the RMST model showed no significant variation in the treatment effect of IO/IO or TKI/IO based on the presence or absence of BAP1 mutation (p=0.97), although sample size limits power to detect differences. Results for other somatic mutations are shown. Conclusions: PFS-2 was significantly inferior for ccRCC patients with somatic BAP1 mutation treated with IO-based therapy. Interaction test did not suggest a significant advantage for IO/IO or TKI/IO for mutation-present or -absent groups. Clinical stratification remains key for selecting TKI/IO or IO/IO for patients. [Table: see text]

Impact of BAP1 mutational status on immunotherapy outcomes in advanced malignant pleural mesothelioma: A single institution experience.

e20537 Background: Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor of mesothelial cells usually diagnosed at advanced stages with dismal prognosis. Most patients progress after 1st line systemic chemotherapy. In the last decade, single or dual agent immune checkpoint inhibitor (ICI) therapy has emerged as a therapeutic option. BAP1 is a commonly mutated tumor suppressor gene in MPM that has been shown to correlate negatively with survival in other solid tumors. We sought to determine the impact of BAP1 mutation on outcomes with ICIs when used as second line systemic therapy. Methods: We retrospectively reviewed a cohort of MPM patients who received ICIs at the Cleveland Clinic foundation between 2017-2022. Patients must have progressed after 1st line systemic chemotherapy prior to ICI. We then compared oncologic outcomes of ICI therapy based on BAP1 mutational status on Next-Generation Sequencing (NGS). End points of interest were overall response rate (ORR) and progression free survival (PFS) while on ICIs. Results: There were a total of 43 MPM patients who received ICIs following progression on pemetrexed/carboplatin (Median age 74 years, IQR 66-81, range 57-92) with a male predominance (M:F ratio 39:4). Histologies included- 58% epithelioid, 28% sarcomatoid and 14% mixed/biphasic histology. Single agent ICI therapy with nivolumab or pembrolizumab was used in 65%. Median follow up was 3 months. BAP1 mutation on NGS was detected in 16%. Other mutated genes included CDKN2A- 7%, BRAF- 5%, MTAP 2% and PTEN 2%. We compared baseline characteristics and outcomes between BAP1 wild type (WT) and BAP1 mutated (MT) groups. Median age at mesothelioma diagnosis were similar 74 vs 70 years (0.3347) with no significant differences in either asbestos or smoking exposure (Table 1). An analysis of outcomes revealed an ORR to ICI of 33% (36% vs 29% respectively [p = 0.9]). PFS was similar between both groups - 4 months [95% CI, 3-NA] for BAP1 WT vs 4 months [95% CI, 2-18] for BAP1 MT group. Conclusions: BAP1 mutation had no prognostic significance on either response rates or disease progression while on ICI therapy in our cohort of advanced MPM patients. The identification of predictive biomarkers for immunotherapy of MPM is an area of unmet clinical need. [Table: see text]

Association of somatic mutations and histologic subtype/grade on prognosis and PD-L1 expression in mesothelioma.

8507 Background: Mesothelioma includes epithelioid, sarcomatoid, and biphasic histologic subtypes, and the epithelioid subtype is histologically graded based on mitotic count and nuclear atypia. Correlation between the most common somatic mutations ( BAP1, NF2, TP53, CDKN2A), and overall survival is poorly understood. High PD-L1 expression is a poor prognostic factor for survival and may be a predictor of response to immunotherapy. Few studies have evaluated the relationships between these factors. Methods: We performed a retrospective study in 217 patients with pleural, peritoneal and bicavitary mesothelioma as part of an IRB-approved biobanking protocol. Histologic subtype, grade of epithelioid mesothelioma, tumor stage, and overall survival time were determined. Next-generation DNA sequencing (NGS) was used to identify the prevalence of somatic mutations. PD-L1 immunohistochemistry was obtained on 184 samples and quantified as percentage of tumor cells. Histologic subtype, nuclear grade, clinical stage, PD-L1 expression, survival time, and the prevalence of each mutation were compared using chi-squared, Kruskal-Wallis, or Cox proportional hazard tests. All p-values were determined in R version 4.2.0 using a two-sided alpha error of 0.05 to mark statistical significance. Results: The most frequent somatic mutations were found in BAP1 (99/217, 45.6%), CDKN2A (47/217, 21.7%), TP53 (37/217, 17.1%), and NF2 (31/217, 14.3%). Patients with CDKN2A-mutated mesothelioma experienced shorter survival (p=0.032). TP53 mutations were associated with higher nuclear grade (p=0.015), higher mitotic count (p=0.013), and greater nuclear atypia (p=0.015). TP53 mutations were more prevalent in pleural than peritoneal mesothelioma (p=0.016) and in tumors without lymph node metastasis (p=0.05). As expected, patients with epithelioid mesothelioma had superior overall survival (p=0.02) and their tumors had lower PD-L1 expression (p=0.007) than either sarcomatoid or biphasic mesotheliomas. Neither PD-L1 expression nor mutations in BAP1, NF2, CDKN2A, or TERT were associated with nuclear grade, atypia, or mitotic count. No association was found between BAP1 or NF2 mutations and tumor stage, lymph node metastasis, or PD-L1 expression. Conclusions: To our knowledge, this is the first study of this scale to correlate PD-L1 expression, somatic mutation data, histologic features, and survival in mesothelioma patients. The most significant findings were that patients with CDKN2A mutations had shorter survival and TP53 mutations, which were more prevalent in pleural mesotheliomas, were associated with higher nuclear grade, higher mitotic count, and greater nuclear atypia. An ongoing analysis evaluates the contribution of germline mutations, which occurred in a significant proportion (15.7%) of this mesothelioma patient cohort.

Survival outcomes associated with liver-directed therapies in patients with metastatic uveal melanoma.

9590 Background: Metastatic uveal melanoma (mUM) is characterized by a hepatotropic pattern of spread and poor outcomes. Tebentafusp, the only FDA approved therapy, is restricted to patients (pts) with the HLA-A*02:01 genotype. As a result, a variety of liver-directed therapies (LDT) are commonly used for management of hepatic metastases. Methods: Pts with mUM with hepatic metastases who underwent LDT, including radiofrequency ablation, microwave ablation, hepatic metastatectomy, Yttrium-90 radioembolization (SIRT), transarterial chemoembolization (TACE), bland embolization, and immunoembolization (IE), were identified from an institutional database. Data collected included: age, gender, tumor location and size, baseline liver function tests (LFT) and lactate dehydrogenase (LDH), molecular tumor characteristics (where available), type(s) of LDT received, systemic therapies received, and vital status. Time from onset of hepatic metastasis until death (Liver-OS), as well as time from initial diagnosis of primary uveal melanoma until death (Ocular-OS) were calculated. Results: A total of 119 pts were identified. The median age at diagnosis was 53.8 years (range 26-83), 47% were female. At the time of LDT initiation, 33% had bilobar disease and 16% had extrahepatic metastasis. 18% pts had elevated LFTs and 55% had elevated LDH. 72% of pts had M1a disease, 24% had M1b and 4% had M1c disease. 45% of pts received more than one form of LDT. SIRT (31%) was the commonly administered initial LDT, followed closely by IE (26%) and TACE (9%). 86% of pts received systemic therapy including 42% who received immune checkpoint blockade overlapping with LDT. Molecular profiling in 26 pts revealed recurrent mutations in BAP1 (n = 18) and SF3B1 (n = 8). The median ocular-OS across all pts was 71.0 months (95% confidence intervals (CI), 62.8-99.4). The median liver-OS across all pts was 31.8 months (95% CI 25.2-35.3). In pts who received TACE, IE, or SIRT exclusively (n = 44), the median Liver-OS was 15.2 months (95% CI 12.8-NA) for TACE (n = 8), 23.3 months (95% CI 15.8-NA) for SIRT (n = 28), and 28.2 months (95% CI 22.6-NA) for IE (n = 8). Conclusions: Our results provide additional support for the utilization of LDT in pts with mUM, as demonstrated by improved OS compared to historical survival data.

EZH2/EZH1 inhibitor tulmimetostat (CPI-0209) in patients with advanced solid tumors or hematologic malignancies: Preliminary phase II results.

3094 Background: Many preclinical tumor models with mutations in ARID1A and BAP1 are highly sensitive to EZH2 inhibition. Tulmimetostat (CPI-0209) is an investigational oral, next-generation, dual EZH2/EZH1 inhibitor with durable target coverage. Phase I dose escalation established a recommended Phase II dose of 350 mg (Lakhani et al. ASCO 2021). We report preliminary Phase II findings from the ongoing Phase I/II study (NCT04104776). Methods: Phase II is evaluating tulmimetostat 350 mg once daily in 28-day cycles in 6 disease-based cohorts (Table). The Simon 2-stage study design requires 1 response (complete [CR] or partial [PR]) in stage 1 (n = 10 patients [pts]) for expansion to stage 2 (plus n = 19) (except in M4, see table). Primary endpoint is objective response rate (ORR) based on disease-specific criteria; secondary objectives include safety. Results: As of Nov 08, 2022, 62 pts received ≥1 dose (safety evaluable); 48 had ≥1 postbaseline tumor assessment (efficacy evaluable). Pts had median 3.2 years (0.6–19.6) since diagnosis; 77.4% had ≥2 prior lines of therapy. Cohorts M2, M5 and M6 had completed stage 1 enrollment. At cut-off, best responses among evaluable patients (Table) show 4 cohorts have at least 1 confirmed response, including 2 CR and 1 PR in 7 pts with peripheral T-cell lymphoma (PTCL). 47 pts (75.8%) experienced TEAEs leading to dose modifications, 20 (32.3%) had serious TEAEs, and 5 (8.1%) discontinued treatment due to TEAEs. The most frequent TEAEs (≥15% of pts) (any Grade/Grade ≥3) considered at least possibly related to tulmimetostat were thrombocytopenia (51.6%/27.4%), diarrhea (45.2%/12.9%), nausea (37.1%/0%), anemia (30.6%/16.1%), fatigue (29.0%/0%), alopecia (25.8%/1.6%), dysgeusia (24.2%/0%), vomiting (21.0%/0%), and neutropenia (17.7%/14.5%). Conclusions: Based on response criteria of ORR ≥1/10 pts, the ovarian (M2), endometrial (M3), and mesothelioma (M5) cohorts achieved eligibility for stage 2 expansion (not relevant in M4). The safety profile of tulmimetostat is consistent with EZH2 inhibition and previous data. These preliminary findings in heavily pretreated pts with multiple tumor types, including tumors with ARID1A alterations or BAP1 loss, support ongoing investigation of tulmimetostat. Clinical trial information: NCT04104776 . [Table: see text]

Novel genetically engineered mouse models for clear cell renal cell carcinoma

Genetically engineered mouse models (GEMMs) are important immunocompetent models for research into the roles of individual genes in cancer and the development of novel therapies. Here we use inducible CRISPR-Cas9 systems to develop two GEMMs which aim to model the extensive chromosome p3 deletion frequently observed in clear cell renal cell carcinoma (ccRCC). We cloned paired guide RNAs targeting early exons of Bap1, Pbrm1, and Setd2 in a construct containing a Cas9D10A (nickase, hSpCsn1n) driven by tetracycline (tet)-responsive elements (TRE3G) to develop our first GEMM. The founder mouse was crossed with two previously established transgenic lines, one carrying the tet-transactivator (tTA, Tet-Off) and one with a triple-mutant stabilized HIF1A-M3 (TRAnsgenic Cancer of the Kidney, TRACK), both driven by a truncated, proximal tubule-specific γ-glutamyltransferase 1 (ggt or γGT) promoter, to create triple-transgenic animals. Our results indicate that this model (BPS-TA) induces low numbers of somatic mutations in Bap1 and Pbrm1 (but not in Setd2), known tumor suppressor genes in human ccRCC. These mutations, largely restricted to kidneys and testis, induced no detectable tissue transformation in a cohort of 13 month old mice (N = 10). To gain insights into the low frequencies of insertions and deletions (indels) in BPS-TA mice we analyzed wild type (WT, N = 7) and BPS-TA (N = 4) kidneys by RNAseq. This showed activation of both DNA damage and immune response, suggesting activation of tumor suppressive mechanisms in response to genome editing. We then modified our approach by generating a second model in which a ggt-driven, cre-regulated Cas9WT(hSpCsn1) was employed to introduce Bap1, Pbrm1, and Setd2 genome edits in the TRACK line (BPS-Cre). The BPS-TA and BPS-Cre lines are both tightly controlled in a spatiotemporal manner with doxycycline (dox) and tamoxifen (tam), respectively. In addition, whereas the BPS-TA line relies on paired guide RNAs (gRNAs), the BPS-Cre line requires only single gRNAs for gene perturbation. In the BPS-Cre we identified increased Pbrm1 gene-editing frequencies compared to the BPS-TA model. Whereas we did not detect Setd2 edits in the BPS-TA kidneys, we found extensive editing of Setd2 in the BPS-Cre model. Bap1 editing efficiencies were comparable between the two models. Although no gross malignancies were observed in our study, this is the first reported GEMM which models the extensive chromosome 3p deletion frequently observed in kidney cancer patients. Further studies are required (1) to model more extensive 3p deletions, e.g. impacting additional genes, and (2) to increase the cellular resolution, e.g. by employing single-cell RNAseq to ascertain the effects of specific combinatorial gene inactivation.

Organoid models of fibrolamellar carcinoma mutations reveal hepatocyte transdifferentiation through cooperative BAP1 and PRKAR2A loss

Fibrolamellar carcinoma (FLC) is a lethal primary liver cancer, affecting young patients in absence of chronic liver disease. Molecular understanding of FLC tumorigenesis is limited, partly due to the scarcity of experimental models. Here, we CRISPR-engineer human hepatocyte organoids to recreate different FLC backgrounds, including the predominant genetic alteration, the DNAJB1-PRKACA fusion, as well as a recently reported background of FLC-like tumors, encompassing inactivating mutations of BAP1 and PRKAR2A. Phenotypic characterizations and comparisons with primary FLC tumor samples revealed mutant organoid-tumor similarities. All FLC mutations caused hepatocyte dedifferentiation, yet only combined loss of BAP1 and PRKAR2A resulted in hepatocyte transdifferentiation into liver ductal/progenitor-like cells that could exclusively grow in a ductal cell environment. BAP1-mutant hepatocytes represent primed cells attempting to proliferate in this cAMP-stimulating environment, but require concomitant PRKAR2A loss to overcome cell cycle arrest. In all analyses, DNAJB1-PRKACAfus organoids presented with milder phenotypes, suggesting differences between FLC genetic backgrounds, or for example the need for additional mutations, interactions with niche cells, or a different cell-of-origin. These engineered human organoid models facilitate the study of FLC.

Spatial immunophenotypes predict clinical outcome in intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (iCCA) is a severe malignant tumour that shows only modest responses to immunotherapy. We aimed to identify the spatial immunophenotypes of iCCA and delineate potential immune escape mechanisms. Multiplex immunohistochemistry (mIHC) was performed to quantitatively evaluate the distribution of 16 immune cell subsets in intratumour, invasive margin and peritumour areas in a cohort of 192 treatment-naïve patients with iCCA. Multiregion unsupervised clustering was used to determine three spatial immunophenotypes, and multiomics analyses were carried out to explore functional differences.Results: iCCA displayed a region-specific distribution of immune cell subsets with abundant CD15+ neutrophil infiltration in intratumour areas. Three spatial immunophenotypes encompassing inflamed (35%), excluded (35%) and ignored (30%) phenotypes were identified. The inflamed phenotype showed characteristics of abundant immune cell infiltration in intratumour areas, increased PD-L1 expression and relatively favourable overall survival. The excluded phenotype with a moderate prognosis was characterized by immune cell infiltration restricted to the invasive margin or peritumour areas and upregulation of activated hepatic stellate cells, extracellular matrix and Notch signalling pathways. The ignored phenotype, with scarce immune cell infiltration across all subregions, was associated with MAPK signalling pathway elevation and a poor prognosis. The excluded and ignored phenotypes, constituting non-inflamed phenotypes, shared features of an increased angiogenesis score, TGF-β and Wnt-β catenin pathway upregulation and were enriched for BAP1 mutations and FGFR2 fusions. We identified three spatial immunophenotypes with different overall prognoses in iCCA. Tailored therapies based on the distinct immune evasion mechanisms of the spatial immunophenotypes are needed. The contribution of immune cell infiltration in the invasive margin and peritumour areas has been proved. We explored the multiregional immune contexture of 192 patients to identify three spatial immunophenotypes in intrahepatic cholangiocarcinoma (iCCA). By integrating genomic and transcriptomic data, phenotype-specific biological behaviours and potential immune escape mechanisms were analysed. Our findings provide a rationale to develop personalized therapies for iCCA.

BAP1-related signature predicts benefits from immunotherapy over VEGFR/mTOR inhibitors in ccRCC: a retrospective analysis of JAVELIN Renal 101 and checkmate-009/010/025 trials.

In patients with advanced clear cell renal cell carcinoma, despite the undoubted benefits from immune checkpoint inhibitor (ICI)-based therapies over monotherapies of angiogenic/mTOR inhibitors in the intention-to-treat population, approximately a quarter of the patients can scarcely gain advantage from ICIs, prompting the search for predictive biomarkers for patient selection. Clinical and multi-omic data of 2428 ccRCC patients were obtained from The Cancer Genome Atlas (TCGA, n = 537), JAVELIN Renal 101 (avelumab plus axitinib vs. sunitinib, n = 885), and CheckMate-009/010/025 (nivolumab vs. everolimus, n = 1006). BAP1 mutations were associated with large progression-free survival (PFS) benefits from ICI-based immunotherapies over sunitinib/everolimus (pooled estimate of interaction HR = 0.71, 95% CI 0.51-0.99, P = 0.045). Using the top 20 BAP1 mutation-associated differentially expressed genes (DEGs) generated from the TCGA cohort, we developed the BAP1-score, negatively correlated with angiogenesis and positively correlated with multiple immune-related signatures concerning immune cell infiltration, antigen presentation, B/T cell receptor, interleukin, programmed death-1, and interferon. A high BAP1-score indicated remarkable PFS benefits from ICI-based immunotherapies over angiogenic/mTOR inhibitors (avelumab plus axitinib vs. sunitinib: HR = 0.55, 95% CI 0.43-0.70, P < 0.001; nivolumab vs. everolimus: HR = 0.72, 95% CI 0.52-1.00, P = 0.045), while these benefits were negligible in the low BAP1-score subgroup (HR = 1.16 and 1.02, respectively). In advanced ccRCCs, the BAP1-score is a biologically and clinically significant predictor of immune microenvironment and the clinical benefits from ICI-based immunotherapies over angiogenic/mTOR inhibitors, demonstrating its potential utility in optimizing the personalized therapeutic strategies in patients with advanced ccRCC.

Obesity paradox in uveal melanoma: high body mass index is associated with low metastatic risk

BackgroundMetabolic factors and obesity may influence the development and progression of cancer. In this study, we examine their association with the risk of developing metastases of uveal melanoma.MethodsData on metabolic...

Abstract 1379: Studying the autophagic flux in response to hypoxia in BAP1-mutated pleural mesothelioma cells linked to asbestos exposure

Abstract Introduction. Malignant mesothelioma is a rare cancer linked to asbestos exposure. It comes from the oncogenic transformation of mesothelioma cells, a protective membrane that covers several internal cavities including the pleura, peritoneum and pericardium. Malignant pleural mesothelioma (MPM) accounts for more than 90% of cases. The average survival of these patients is approximately 8 to 12 months, due to the resistance of tumors towards treatments. A well-known hallmark in cancer is tumor suppression gene inactivation. BAP1 is the most mutated gene in pleural mesothelioma (25-60%). BAP1 has a deubiquitinase activity involved in chromatin modifications, homologous recombination, DNA damage and programmed cell death. Moreover, hypoxia is an important microenvironment component within tumors, and it is demonstrated in patients with MPM. The bio-persistent localization of asbestos in mesothelioma cells has been shown to induce chronic inflammation that activates autophagic flux to survive. Autophagy is induced by several types of cellular stresses, including hypoxia. This suggests that MPM cells are dependent on autophagic flux when subjected to stress to ensure their continuity and proliferation. Hypothesis/Objectives. The hypothesis of this project is that MPM has vulnerabilities in the autophagy/lysosome process, which can thus be targeted by chemical inhibitors to improve treatment response and efficacy. The specific objectives are to i) examine autophagy flux in MPM cells with BAP1 mutations, ii) demonstrate autophagy modulation in MPM cells towards the hypoxic response, and iii) assess the potential for autophagy and lysosome inhibitors in MP cells with mutations on BAP1 or towards the hypoxic response. Methods/Results. To achieve this, molecular biology approaches are used to reintroduce (Gateway system), or repress (CRISRP/Cas9) the expression of BAP1 in Mero-25 and Mero-41 cells, respectively. Models were confirmed by western blot. The hypoxic chamber is used to manipulate cells in 1% O2, in which we confirmed the activation of autophagy in response to hypoxia in a kinetic timeframe of 24, 48 and 72hr. Subsequently, the autophagic flux was studied in presence of Bafilomycin A1 indicating an activation of this cellular process. The potential and cytotoxicity of autophagy or lysosome inhibitors will be evaluated by XTT and clonogenic assays. Conclusion. Malignant pleural mesothelioma has an unmet need in novel therapeutic strategies. Our studies could contribute to improve anticancer treatments using combinatory approaches that modulate autophagy. Citation Format: Chloe Michaud, Sandra Turcotte. Studying the autophagic flux in response to hypoxia in BAP1-mutated pleural mesothelioma cells linked to asbestos exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1379.

Epigenetic and transcriptomic characterization reveals progression markers and essential pathways in clear cell renal cell carcinoma

Identifying tumor-cell-specific markers and elucidating their epigenetic regulation and spatial heterogeneity provides mechanistic insights into cancer etiology. Here, we perform snRNA-seq and snATAC-seq in 34 and 28 human clear cell renal cell carcinoma (ccRCC) specimens, respectively, with matched bulk proteogenomics data. By identifying 20 tumor-specific markers through a multi-omics tiered approach, we reveal an association between higher ceruloplasmin (CP) expression and reduced survival. CP knockdown, combined with spatial transcriptomics, suggests a role for CP in regulating hyalinized stroma and tumor-stroma interactions in ccRCC. Intratumoral heterogeneity analysis portrays tumor cell-intrinsic inflammation and epithelial-mesenchymal transition (EMT) as two distinguishing features of tumor subpopulations. Finally, BAP1 mutations are associated with widespread reduction of chromatin accessibility, while PBRM1 mutations generally increase accessibility, with the former affecting five times more accessible peaks than the latter. These integrated analyses reveal the cellular architecture of ccRCC, providing insights into key markers and pathways in ccRCC tumorigenesis.

Exploration of Morphological Features of Clear Cell Renal Cell Carcinoma With PBRM1, SETD2, BAP1, or KDM5C Mutations.

The last decade has seen great advances in genomic profiling and prognosis-associated factors of clear cell renal cell carcinoma (RCC), the most common entity in kidney cancer. Following VHL, PBRM1, SETD2, BAP1, and KDM5C have been validated as the most common co-occurring gene mutations in clear cell RCC by multicenter studies. However, the morphological features of clear cell RCC with co-occurring gene mutations remain unclear. In this study, we presented 20 clear cell RCCs that underwent next-generation sequencing, of which 1 tumor was reclassified as ELOC-mutated RCC. PBRM1, SETD2, BAP1, and KDM5C were the most common mutations, following VHL. Morphologically, clear cell RCC with PBRM1 or KDM5C mutation usually displayed a low-grade pattern. Cystic changes and hyalinized stroma were often observed. The Ki67 index was <10%. These observations indicated good prognosis. However, mutated SETD2 may increase the malignancy of clear cell RCC with PBRM1 mutation. Two clear cell RCCs with mutated PBRM1 and SETD2 developed local or distant metastases. Clear cell RCC with BAP1 mutations always had high-grade patterns, and rhabdoid differentiation was also observed, indicating that BAP1 mutation was associated with poor outcomes. Papillary architecture was often a feature of BAP1 mutation, which is uncommon in clear cell RCC. PDL1 was positive in only one tumor with BAP1 mutation, and the positivity rate was limited to 5%. B7H3 was negative in all tumors. Morphologic findings in this small cohort may suggest why PBRM1 mutation does not correlate with decreased survival, whereas BAP1 mutation usually predicts poor outcomes.

Mesotheliomas and Benign Mesothelial Tumors: Update on Pathologic and Imaging Findings

A diverse spectrum of benign entities and malignant neoplasms originate from the monotonous mesothelium that lines the serosal membranes of the pleural, pericardial, and peritoneal cavities. The mesothelium of myriad sites shows a common origin from the lateral plate mesoderm; primary mesothelial tumors thus demonstrate similar pathogenesis, imaging findings, and treatment options. Significant changes have been made in the 2021 World Health Organization (WHO) classification schemata of the pleural and pericardial tumors on the basis of recent advances in pathology and genetics. While malignant mesotheliomas are biologically aggressive malignancies that occur primarily in patients exposed to asbestos with attendant poor survival rates, well-differentiated papillary mesothelial tumors and adenomatoid tumors charter a benign clinical course with an excellent prognosis. Mesothelioma in situ is a newly characterized entity represented by recurrent unexplained pleural effusions without any identifiable mass at imaging or thoracoscopy. Immunohistochemical markers based on BAP1, MTAP, CDKN2A, and TRAF7 gene mutations help differentiate diffuse mesotheliomas from benign mesothelial proliferations and localized mesotheliomas. Cross-sectional imaging modalities, including US, CT, MRI, and fluorine 18-fluorodeoxyglucose (FDG) PET/CT, permit diagnosis and play a major role in staging and assessing surgical resectability. Imaging studies are invaluable in providing noninvasive and quantitative assessment of tumor response in patients with unresectable disease. Owing to significant overlap in patient characteristics and pathomorphology, accurate diagnosis based on advanced histopathology techniques and genetic abnormalities is imperative for optimal management and prognostication. While patients with nonepithelioid pleural mesotheliomas benefit from immunotherapy, novel targeted therapies for CDKN2A-, NF2-, and BAP1-altered mesotheliomas are under consideration. © RSNA, 2023 Quiz questions for this article are available through the Online Learning Center.

Predictive biomarkers for the oncolytic virus, JX-594, through the HIF-dependent interferon beta pathway in metastatic renal cell carcinoma.

710 Background: Although combination approaches with an immunotherapy are established as the first-line setting in metastatic renal cell carcinoma (mRCC), immunotherapy are only efficacious for a subset of patients. Our group has been working on improving the benefit of immunotherapy using JX-594 (pexastimogene devacirepvec, Pexa-vec) in mRCC. The purpose of this study is to develop predictive biomarker for JX-594 in metastatic clear cell RCC (ccRCC). Methods: Using ccRCC cell lines representative of each four mutations (786-O (VHL mutation), Caki-2 (VHL and PBRM1 mutation), A-498 (VHL and SETD2 mutation), and 769-P (VHL and BAP1 mutation)), four cell line-derived xenograft (CDX) mouse models have been developed. 2-10x106 tumor cells were subcutaneously injected into the right flank of wild-type BALB/c nude mice. When tumors reached &gt;50 mm3, mice were treated with either PBS or 1x107 plaque-forming units of JX-594 by intratumoral injection in day 0, 3, and 6. Interferon beta (IFN-β) expression were measured with qRT-PCR after the JX-594 treatment in 786-O CDX model. Results: All CDX models demonstrated significant decrease in tumor size by the JX-594 compared to control. Most rapid tumor growth was observed in BAP1-deficient xenografts while PBRM1-deficient xenografts demonstrated the slowest growth among four xenografts. The decrease of tumor volume by the JX-594 was most pronounced in BAP1-deficient tumors compared to VHL, PBRM1, and SETD2 mutation tumors. Even after adjusting the relative differences of tumor growth rate in each model, xenografts with BAP1 loss were still associated with significantly better response to JX-594. IFN-β expression significantly decreased in BAP1-deficient tumors compared to other tumors, while the expression level was recovered dramatically after JX-594. Conclusions: BAP1-deficient tumors demonstrated rapid tumor progression while they were most significantly reduced after JX-594. BAP1-deificient tumors had decreased levels of IFN-β expression while it was most dramatically recovered after JX-594, suggesting the mechanism of JX-594 through the HIF-dependent interferon beta induction. This is the first study to elucidate the potential of BAP1 as predictive biomarker for JX-594 and its underlying mechanisms.

Genomic alterations in metastatic renal cell carcinoma (mRCC): Impact on survival and clinical outcomes.

700 Background: Genomic alterations in RCC can serve as biomarkers for response to therapy. Wild type VHL has shown inferior response to VEGF inhibitors, while patients (pts) with mutated VHL have improved outcomes with immunotherapy (IO)/tyrosine kinase inhibitor (TKI) therapy. We describe the impact of genomic alterations on outcomes and response to front-line therapy. Methods: Pts with mRCC who underwent genomic sequencing between 2015-2022 at Indiana University and were treated with front-line therapy were included. Kaplan-Meier method was used to analyze progression free survival (mPFS) and overall survival (OS) using the log rank test to compare groups. Results: 141 pts were included. Median age was 58yrs (range, 24-81). Tumor histology was 68% clear cell, 10% papillary, 9% unclassified, 1% chromophobe, 1% poorly differentiated, and 9% other. 18% of pts had sarcomatoid features; 13% had rhabdoid. 47% pts had metastasis at diagnosis and 53% at relapse. Metastasis sites were lungs 60%, regional lymph nodes (LNs) 41%, bone 36%, distant LNs 21%, liver 17%, and brain 8%. IMDC risk was 18% good, 48% intermediate, 17% poor, and 17% unknown. Front-line therapy was IO/IO in 21%, IO/TKI in 18%, single-agent TKI in 29%, single-agent IO in 6%, and other in 26%. Genetic alterations included VHL in 50%, PBRM1 in 29%, SETD2 in 23%, BAP1 in 16%, and TP53 in 15%. Median follow-up was 2.74yrs (range 0.1-18.6). Overall mPFS with first line therapy was 1.2yrs (95%CI 0.9-1.5). 2-yr OS was 80% (95%CI: 71-86). For pts with a VHL mutation, mPFS was 1.4yrs (95% CI; 0.9-1.8) compared to 0.9yrs (0.5-1.4) for pts without (p=0.38). 2-yr OS for pts with a VHL mutation was 93% (95% CI: 82-97) vs. 68% (95%CI: 54-78) for pts without (p=0.01). No other mutations impacted overall mPFS or 2-yr OS. mPFS for pts treated with IO/IO was 0.6yrs (95%CI 0.3-0.9) vs. 0.9yrs (95%CI 0.5-1.4) for IO/TKI (p=0.09). 2-yr OS for pts treated with IO/IO was 70% (95% CI 44-85) compared to 94% (95% CI 65-99) for IO/TKI (p=0.29). For pts with SETD2 mutations treated with IO/IO, mPFS was 0.7yrs (95%CI 0.3-1.2) vs. 1.4yrs (95%CI 0.8-1.9) for IO/TKI (p=0.01). For pts with BAP1 mutations treated with IO/IO, mPFS was 0.4yrs (95%CI 0.2-1.2) vs. NE (95%CI 0.2-NE) for IO/TKI (p=0.04). The table outlines outcomes by genomic mutation and therapy. Conclusions: VHL mutation was associated with improved 2-yr OS. Pts with SETD2 and BAP1 mutations treated with IO/TKI had improved mPFS, though no differences in OS. [Table: see text]