A Scoring System Based on [ 18 F]PFPN PET and Clinical Features for Predicting Prognosis in Patients With Mucosal Melanoma.

Background: Mucosal melanoma (MM) is a rare subtype of melanoma that responds poorly to immune checkpoint blockade (ICB), exhibiting low response rates and few durable responses. The biological mechanisms underlying ICB response and resistance in MM remain poorly understood, highlighting the need for novel therapeutic targets and biomarkers to improve patient outcomes. Hypothesis: While most MM patients respond poorly to ICB, a subset of durable responders (DRs) exists. We hypothesize that DRs have distinct biological and genomic features compared to rapid progressors. Methods: We performed multimodal analysis - including whole exome sequencing, bulk transcriptomics, H&E, and multiplexed Cyclic Immunofluorescence (CyCIF) imaging - on pre-treatment and post-progression tumor samples from 126 MM patients, with a focus on a subset of 80 patients treated with ICB to identify features associated with response. Results: Our genomic analysis of 80 MM patients revealed that DRs (PFS ≥ 1 year) had significantly fewer copy number alterations (CNAs) compared to those with progressive diseases (PFS ≤ 6 months). The most common MM driver mutations were identified including SF3B1, KIT, NF1, and NRAS. Notably, SF3B1 mutations were primarily found in anorectal primary tumors (10/17) and had fewer CNAs, a feature also observed in DRs, suggesting it may be associated with better ICB responses in MM. Bulk RNAseq analysis (n = 67) identified a tumor cluster enriched for epithelial-mesenchymal transition and hypoxia stress pathways, suggesting increased invasiveness and potential ICB resistance. These signatures were observed in 37% of our MM primary tumors, underscoring the aggressive nature of MM. CyCIF analysis of primary and metastatic MM lesions revealed increased CD8+ T cells and CD11C+ myeloid cells in metastatic sites. By defining the tumor/stromal border, we found fewer antitumorigenic CD68+ macrophages in the tumor center but more at the tumor border at the metastatic site compared to the primary sites, suggesting immune exclusion. In sinonasal primary tumors, non-metastatic patients had fewer protumorigenic CD163+ macrophages in both tumor center and border compared to those with distant metastases. Multimodal analysis of a longitudinally sampled MM patient treated with ICB showed distinct resistance clones associated with different microenvironments. Two lymph node metastases collected simultaneously after ICB showed clonal differences, with one (harboring a SOX17 mutation) exhibiting lower CD8+ T cell infiltration, suggesting the evolution of diverse resistance mechanisms. Conclusion: Our preliminary findings suggest that distinct genomic features, tumor states, and microenvironments differentiated ICB response and disease progression in MM, highlighting key tumor-intrinsic and microenvironmental factors associated with patient prognosis. Citation Format: Yingxiao Shi, Tuulia Vallius, Aikaterini Dedeilia, Roxanne Pelletier, Jia-Ren Lin, Mariana Lopez Leon, Christine Lian, Shishir Pant, Giuseppe Tarantino, Annette Wang, Jiajia Chen, Elizabeth I. Buchbinder, Peter K. Sorger, David Liu, Genevieve M. Boland. Integrative molecular and spatial analysis to reveal tumor intrinsic and extrinsic drivers of immunotherapy response in mucosal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5014.

Cutaneous melanoma (CM) has one of the highest response rates across cancers to immune checkpoint blockade (ICB) therapies, but mucosal melanoma (MM) responds poorly to ICB. MM patients have very few effective treatments options, making it critical to understand and improve anti-tumor immunity in these patients. MM is a rare subtype of melanoma, accounting for approximately 1% of all malignant melanoma diagnoses. MM occurs on mucosal surfaces such as nasal, vaginal, and anorectal, which provide a highly unique tumor microenvironment compared to CM arising in the skin. We compared the tumor immune microenvironment between CM and MM by multiplex immunofluorescence and discovered MM has decreased frequency of infiltrating immune cells. Using RNA sequencing, we investigated intra-tumoral differences in global gene expression between CM and MM that might explain the lack of immune cell infiltration and ICB response in MM. Compared to CM, MM had decreased expression of numerous innate immune genes in the RIG-I pathogen-sensing pathway. The RIG-I pathway produces an inflammatory response to destroy foreign pathogens, but is also important for ICB sensitivity and anti-tumor immunity in CM. Studies in CM demonstrate the RIG-I pathway enhances antigen presentation and promotes a favorable immune cell profile in the tumor microenvironment. Interestingly, the tumor microbiome, which is vastly different between CM and MM, has been shown to regulate the RIG-I pathway. We measured total fungal and bacterial load in MM and CM primary tumors and found increased levels of both bacteria and fungi in MM compared to CM. This raises the possibility that microbiome-mediated suppression of the RIG-I pathway underlies poor anti-tumor immunity and ICB response in MM, and that re-activation of this pathway may be a novel therapeutic strategy for overcoming ICB resistance in MM patients. Direct RIG-I agonists are still in clinical development, but FDA-approved hypomethylating agents, particularly 5’ aza-deoxycytidine (decitabine), can activate the RIG-I pathway and improve anti-tumor immunity by increasing the expression of RIG-I pathway activating genes, endogenous RNA retroviruses, tumor antigens, and natural killer (NK) cell ligands. We treated MM cell lines with decitabine and observed strong induction of RIG-I pathway genes, NK ligands, and tumor antigens. In conclusion, we have identified the loss of intra-tumoral RIG-I signaling as a potential microbiome-mediated mechanism underlying the poor tumor immune microenvironment and ICB response in MM. Combining decitabine, which is FDA-approved and now available orally, with ICB represents a novel treatment strategy for these difficult to treat MM patients that can be rapidly translated into the clinic. Citation Format: Morgan MacBeth, Richard Tobin, Robert Van Gulick, Martin D. McCarter, William A. Robinson, Kasey L. Couts. Loss of intra-tumoral RIG-I immune signaling is a potential microbiome-mediated mechanism underlying poor anti-tumor immunity and immunotherapy resistance in mucosal melanoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO048.

Background The objective was to assess the response rate and survival of patients with metastatic mucosal melanoma (MM) and uveal melanoma (UM) treated with anti-CTLA-4 or anti-PD-1 monoclonal antibodies (mAbs). Methods A multicenter retrospective study was performed in 25 dermatology departments in France. All patients with stage III-C to IV MM or UM who were treated with anti-CTLA-4 or anti-PD-1 mAbs between 2008 and 2016 were included and compared after adjustment for main prognostic factors with a second cohort of patients treated with chemotherapy. Tumor response was evaluated according to RECIST v. 1.1 criteria at Week 12. Results Four-hundred-and-thirty-nine patients were included, 229 MM (151 immunotherapy, 78 chemotherapy) and 210 UM (100 immunotherapy, 110 chemotherapy). Response rates of MM patients treated with immunotherapy were 18/151 (11.9%; 95% CI:7.2%-18.2%), versus 11/78 (14.1%, 95% CI:7.3%-23.8%) in patients treated with chemotherapy (p=0.87). No tumor response was observed in UM patients treated with immunotherapy, versus 4/110 responses (3.6%, 95% CI:1.0-9.0%) in patients treated with chemotherapy (p=0.15). The adjusted overall survival (OS) of MM patients treated with immunotherapy was longer than that of patients treated with chemotherapy HR=0.62 (95% CI: 0.43-0.91), p=0.014, with an unadjusted median OS of 15.97 months [interquartile range (IQR)=6.89-27.11] and 8.82 months [IQR=5.02-14.92], respectively. The adjusted OS of UM patients treated with immunotherapy was not significantly different from that of patients treated with chemotherapy (HR=0.98, 95% CI: 0.66–1.44) p=0.92, with an unadjusted median OS of 13.38 months [IQR=6.03-29.57] and 11.02 months [IQR=6.13-23.93], respectively. Conclusion Immunotherapy significantly improves OS for MM. The prognosis of metastatic UM remains poor.

10595 Background: Mucosal melanoma (MM) is a rare and aggressive subtype of melanoma. Outcomes for MM are significantly worse than for cutaneous melanoma (CM). While somatic mutations induced by UV radiation are known to cause CM, less is known about environmental and genetic alterations associated with MM. High penetrance alleles have been identified in familial cases of CM but the contribution of germline mutations to development of MM is not understood. Further, whilst population-based risk has been elaborated in CM, little is known for MM. The aim of this study is to characterize the germline genetic architecture of MM from high penetrance alleles to patient population genetics. Methods: A large retrospective cohort of MM patients (n=247) seen at MD Anderson Cancer Center were identified. Demographics, primary tumor sites, and presence of somatic mutations in BRAF, NRAS and KIT were extracted. Germline sequencing was performed on PBMC derived genomic DNA from the cohort using a gene panel of 322 major cancer genes. Frequency of identified germline alleles were compared to expected population control rate based on TOPMED using Fisher’s exact test. For genome wide association analysis (GWAS), genotyping will be performed on the MM cohort and the control group using the Infinium Global Screening Array. Cancer-free controls matched 3:1 based on MM cohort demographics have been assembled from existing case-control studies at MD Anderson. Following standard quality control procedures, we will impute variants from TOPMed using the Michigan Imputation Server. SNPtest v2.5.268 software will be utilized to perform association tests, using a multivariate logistic regression model incorporating gender and principal components. In addition to variants that may surpass a genome-wide significance score, we will inspect results for an enrichment of cancer genes. Results: Median age of MM cohort is 63, female predominant (60%), primarily White (84.6%), with gastrointestinal (36%), genitourinary (34%) and head & neck (30%) being the most common MM subtypes. Characteristics of the cohort were representative of the US MM population. Somatic mutations in KIT (26%) were most common. Germline mutations were found in 10.9% of the cohort, with most common detected variations in MITF (c.G952A:p.E318K) (2.4%) at OR of 13.9 (95%CI 5.0, 30.9), and CHEK2 (c.1100delC:p.T367fs) (1.6%) at OR of 16.4 (4.4, 42.9) compared to population control rate. Conclusions: Our study is the first to demonstrate the distribution of germline mutations in the largest cohort of MM patients. The MITF E318K allele is a known risk factor for CM, while CHEK2 1100delC allele has mixed evidence for conferring CM risk. GWAS data are currently being analyzed and these updated results will be presented in the context of the comprehensive analyses of germline risk alleles in MM patients.

Mucosal melanoma (MM) is epidemiologically, biologically, and molecularly distinct from cutaneous melanoma. Current treatment strategies have failed to significantly improve the prognosis for MM patients. This study aims to identify therapeutic targets and develop combination strategies by investigating the mechanisms underlying the tumorigenesis and progression of MM. We analyzed the copy number amplification of enhancer of zeste homolog 2 (EZH2) in 547 melanoma patients and investigated its correlation with clinical prognosis. Utilizing cell lines, organoids, and patient-derived xenograft models, we assessed the impact of EZH2 on cell proliferation and sensitivity to ferroptosis. Further, we explored the mechanisms of ferroptosis resistance associated with EZH2 by conducting RNA sequencing and chromatin immunoprecipitation sequencing. EZH2 copy number amplification was closely associated with malignant phenotype and poor prognosis in MM patients. EZH2 was essential for MM cell proliferation in vitro and in vivo. Moreover, genetic perturbation of EZH2 rendered MM cells sensitized to ferroptosis. Combination treatment of EZH2 inhibitor with ferroptosis inducer significantly inhibited the growth of MM. Mechanistically, EZH2 inhibited the expression of Krüpple-Like factor 14 (KLF14), which binds to the promoter of solute carrier family 7 member 11 (SLC7A11) to repress its transcription. Loss of EZH2 therefore reduced the expression of SLC7A11, leading to reduced intracellular SLC7A11-dependent glutathione synthesis to promote ferroptosis. Our findings not only establish EZH2 as a biomarker for MM prognosis but also highlight the EZH2-KLF14-SLC7A11 axis as a potential target for MM treatment.

To investigate the characteristics and prognosis of melanoma in a Chinese population. Total of162 advanced melanoma patients were analyzed retrospectively. Kaplan-Meier method and Log rank test were used for survival analysis. The median progression-free survival of mucosal and cutaneous melanoma patients was 13 versus 8 months (p=0.005), 14 versus 10 months in immunotherapy group (p =0.022), 6 versus 4 months in chemotherapy group (p =0.040). Age was an independent risk factor for mucosal melanoma patients. Staging and treatment regimen were independent risk factors for cutaneous melanoma patients. The lungs, liver and brain were most common metastasis locations. The prognosis of patients with advanced mucosal melanoma is better than cutaneous melanoma in China. There are significant differences between the two subtypes of melanoma.

9556 Background: Mucosal melanomas can be effectively treated with checkpoint inhibitors, although the response rates are lower than those observed for melanomas arising in cutaneous sites. The mechanistic basis for the lower efficacy of immunotherapies in mucosal melanoma has been suggested to be related to their lower mutational burden. However, there has been limited characterization of the genetics in this melanoma subtype. Methods: Tumor genotyping was performed on all mucosal melanoma patients seen within the Dana Farber Cancer Institute from 2011 until the present by Oncopanel analysis. Results: We identified a total of 57 mucosal melanoma patients whose tumors had been genotyped. Of these 42 received immunotherapy and had response data available. Within the cohort of mucosal melanoma patients, 37.3% had durable clinical benefit (DCB) to their first line of IO therapy. These patients had an average mutational burden/megabase of 6.41 (95% CI 3.53-11.01) but tumor mutational burden did not correlate with response in this cohort. The pattern of mutations in mucosal melanomas was distinct from cutaneous melanomas, as the most frequent mutations were in SF3B1, ATRX, KIT and NF1 genes. Patients with KIT aberrations had a higher DCB rate compared patients with wildtype KIT (73 vs. 33%). In addition, there were several genetic differences observed based upon the site of origin of the mucosal melanoma. A higher rate of SF3B1 mutations was observed in patients with melanoma of anal/rectal origin while patients with vulvar/vaginal melanoma had higher rates of ATRX mutations, which frequently correlated with p53 ( TP53) mutations. Conclusions: This analysis is one of the first to look at genetic patterns in a large cohort of a relatively rare type of melanoma and correlate with response. Our findings confirm the low mutational burden observed in mucosal melanoma despite the high response rate observed in these patients. In addition, this study uncovered a higher rate of response to immunotherapy in mucosal melanoma patients with a KIT mutation.

e21582 Background: Both chemotherapy and high-dose IFN-α2b (HDI) are effective treatment options in adjuvant setting for patients with resectable mucosal melanoma (MM). However, it is still unknown whether Ki67 level affects the selection of chemotherapy and HDI. Methods: Data from resected MM patients diagnosed as MM in Peking Cancer Hospital, were retrospectively collected and analyzed. Key inclusion criteria were: (1) diagnosed as resectable MM, with the date between Jan. 1, 2010, and Dec. 31, 2018; (2) Ki67 was identified by immunohistochemical staining; (3) received TMZ-based adjuvant chemotherapy or HDI. All patients were divided into two subgroups according to the Ki67 level proposed by previous publications: low (< 30%), high (> = 30%). Relapse-free survival (RFS) and melanoma-specific survival (MSS) were compared across different subgroups by log-rank tests. Multivariate Cox proportional hazards models were used to calculate hazard ratios (HRs), controlling for age, sex, primary site, lymphatic metastasis, LDH level, and gene mutational status (BRAF, c-KIT and NRAS). Results: In total, 1106 MM patients were screened and 175 met the inclusion criteria for analysis. 100 and 75 patients received temozolomide (TMZ)-based adjuvant chemotherapy and HDI therapy, respectively. Patients who received adjuvant chemotherapy had a superior RFS (21.0 vs. 9.6 months, HR = 0.47, P = 0.002) as compared to those with HDI, but no significant difference for MSS (45.9 vs. 37.6 months, HR = 0.63, P = 0.396). Longer RFS and MSS were observed in the ki67-low subgroup (HRs were 0.51, 95%CI 0.34-0.76 and 0.41, 95%CI 0.24-0.68 for RFS and MSS, respectively). For patients with low Ki67 (< 30%), two regimens showed no statistically different RFS (33.9 vs. 22.7 months, HR = 0.76, P = 0.329) and MSS (114.5 vs. 61.4 months, HR = 1.23, P = 0.967). However, for those with high Ki67, TMZ-based chemotherapy achieved an extended RFS compared with HDI (18.0 vs. 6.7 months, HR = 0.36, P < 0.001) and a trend toward improvement for MSS (41.4 vs. 25.1 months, HR = 0.47, P = 0.067). Conclusions: Ki67 level is an independent negative prognostic factor and impacts the selection of adjuvant treatment options for MM patients. Chemotherapy should be considered as the preference for patients with Ki67 > = 30%.

Introduction MM is a rare melanoma subtype with distinct biology, low immunogenicity and tumor mutational burden, and subsequently lower response rates to ICIs. The biology of MM is poorly understood. We sought to prospectively, uniformly, and comprehensively profile a cohort of MM patients to determine the interplay of molecular variation, germline predisposition, immunity, gut, mucosal and tumor microbiome and modifiable risk factors on ICI response and resistance in advanced MM. Methods Patients (pts) with MM treated with standard-of-care ICI as any treatment line presenting to MD Anderson are being prospectively enrolled for longitudinal collection for molecular, microbiome, and lifestyle factor profiling. Planned analyses include: whole genome sequencing of normal and tumor tissue, RNA sequencing, and T cell receptor sequencing from fresh tumor samples; germline sequencing and genotype analyses; NanoString Digital Spatial Profiling (DSP) from formalin-fixed paraffin embedded (FFPE) specimens; whole genome shotgun, microbiome profiling and 16S rRNA gene sequencing (16S) of fecal specimens and affected mucosal and tumor sites (swabs); participant lifestyle and patient reported outcomes (PROs) assessments. Fresh tissue is collected and used to develop PDX models and cell lines. Results 98 pts have been enrolled to date; 83 (85%) Caucasians, 8 (8%) Hispanic, 4 (4%) Asians. 67 (68%) females. Median age at MM diagnosis is 64 years (range 32-91 years). MM primary sites include 24 (24%) naso-oral, 29 (30%) urogenital, 26 (27%) anorectal, 7 (7%) conjunctival and 12 (12%) other. Clinical somatic mutation testing was performed in 76 (76%) pts and common mutations were KIT (n = 12, 16%), NRAS (n = 8, 11%) and BRAF V600 or non-V600 (n = 9, 12%). At data cut-off (November 2023), 18 (18%) pts deceased. Median follow-up from first diagnosis to last visit/death was 16 months (range 1-178 months). Sample collections at data cut-off include: blood samples (n = 95; 219 samples); fecal specimens (n = 50; 97 samples); mucosal swabs (tumor/tumor adjacent/oral cavity; n = 38; 101 samples); fresh frozen tumor/normal (n = 38; 55 samples), fresh tumor/normal (n = 28; 36 samples) and FFPE tumor (n = 21; 37 samples) tissue. PROs and dietary assessments have been collected from 47 pts. Fresh tissue for PDX model and cell line development has been collected from 11 pts. Conclusion This is an ongoing prospective study that is expected to drive insights into the tumor/microenvironment/host interactions and factors regulating immunogenicity to predict response and resistance to ICIs in a rare and understudied melanoma subtype. Interrogation of the role of the gut microbiome and its modifiable determinants will lead to the investigation of new therapeutic strategies to modulate the microbiome to improve treatment outcomes in MM. Data will be presented from initial cohort analyses. Citation Format: Florentia Dimitriou, Priyadharsini Nagarajan, Sabitha Prabhakaran, Neus Bota, Mark Knafl, Randy A. Chu, Ashish V. Damania, Pranoti V. Sahasrabhojane, Yasmine Hoballah, Jillian S. Losh, Nadim J. Ajami, Scott E. Woodman, Jennifer A. Wargo, Andrew Futreal, Jennifer L. McQuade. Delineating germline, tumor and extrinsic factors driving mucosal melanoma (MM) risk and response to immune checkpoint inhibitor (ICI) treatment [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 6699.

Background: Melanoma is a melanocytic malignancy that is classified into different subtypes, including cutaneous (CM), acral (AM), and mucosal melanoma (MM). Immune checkpoint inhibitors (ICI) targeting PD-1 and/or CTLA-4 have emerged as the standard of care for advanced metastatic melanoma. We investigated responses to ICIs, genomic profiles, and molecular differences among these different subtypes of melanoma. Methods: We performed a multi-center retrospective cohort study including patients with metastatic melanoma who received anti-PD1 +/- CTLA4 inhibitor ICI for metastatic disease. We employed the AACR GENIE (v13.1) cancer database, DepMap portal, and Enrichr web tool with MSigDB Hallmark 2020 database to analyze the incidence and distribution of significant alterations, differentially expressed genes, and pathway enrichment within distinct melanoma subtypes. Results: We identified 337 patients with advanced melanoma who received anti-PD1 +/- anti-CTLA4 for metastatic disease. CM was the most frequent melanoma subtype (81%), followed by MM (12%), and AM (8%). Patients with AM had the shortest OS and PFS CM (OS 3.4 years, PFS of 1.1 years), AM median (OS 1.4 years, PFS 3.8 months), and MM (OS 1.7 years, PFS 6 months; OS, P= 0.028; PFS P=0.029). Patients with CM or MM experienced longer OS with anti-PD1 +/- CTLA4 vs. anti-PD1 monotherapy, but no survival advantage was observed in patients with AM. In the GENIE dataset, we identified 2015/374/177 samples with CM/MM/AM, respectively. AM and MM patients were significantly more likely to be Female, Black or Asian than CM patients. BRAF V600 mutations were most frequent in CM (40%), followed by MM (7%) and AM (14%). However, MM and AM had an increase in alterations in cell cycle regulator genes. The incidence of CDK4 and CCND1 amplification, respectively was highest in AM (17%, 16%), followed by MM (6%, 6%) and CM (2%, 3%). We compared the pathways represented by differentially altered genes in AM vs. CM: G2M-Checkpoint (Q<0.05) and E2F targets (Q<0.05) pathways were enriched in AM. We also compared RNAseq data from n=4) AM and n=20 CM cell lines, and found the same pathways enriched amongst genes that were differentially expressed between AM and CM: G2M-Checkpoint (Q<0.01) and E2F targets (Q<0.01) Conclusions: Our findings highlight comparatively poor outcomes with ICIs in Acral Melanoma. AM has lower rates of actionable BRAF mutations; as such, very few patients with metastatic AM have access to effective 1st or 2nd line therapies. Thus, there is an urgent need to explore alternative therapeutic targets. Our data implicate cell cycle inhibitors as potentially important components of novel treatment strategies that could augment immunotherapy efficacy for patients with metastatic AM. Citation Format: Sadaf Solati, April A. N. Rose, Anna Spreafico, Adrian Sacher, Denis Yahiaoui, Wilson H. Miller. Comparing the clinical and genomic landscapes of acral, mucosal, and cutaneous metastatic melanomas treated with immune checkpoint inhibitors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A035.

Clinically node-negative head and neck mucosal melanoma: An analysis of current treatment guidelines & outcomes

Background: Mucosal melanoma (MM) is the rarest subtype of melanoma with markedly worse outcomes compared to cutaneous melanoma (CM). Unlike CM which is driven by somatic mutations due to UV exposure, risk factors for MM are less defined. While germline mutations in cancer susceptibility genes are well-studied in CM, they are less understood in MM. Herein, we aimed to identify the prevalence of germline mutations in cancer susceptibility genes in a large MM cohort. Methods: A cohort of 247 MM patients (pts) from MD Anderson Cancer Center with available blood for germline sequencing was identified. Pt characteristics were reflective of U.S. MM population with median age of 63, 60% females and 84.6% white. Clinicodemographic features of pts including tumor site, key somatic mutations (BRAF, NRAS, KIT) and personal history of other malignancies were abstracted, and their frequencies were compared in pts with and w/o germline mutation. Frequency of MITF and CHEK2 alleles were compared to expected population control rate based on TOPMED by Fisher’s exact test. Results: Among 247 pts, 10.9% had germline mutations, primarily in MITF (c.G952A:p.E318K) (2.4%) and CHEK2 (c.1100delC:p.T367fs) (1.6%) at OR of 13.9 (95%CI 5.0, 30.9) and 16.4 (4.4, 42.9) compared to population control rate (Table 1). MM sites of origin were gastrointestinal (36%), genitourinary (34%) and head & neck (30%). In terms of somatic mutations, KIT (26%) was most common, followed by NRAS (15%), non-V600E BRAF (6.5%) and BRAF V600E (2.4%). These variables did not differ between those with and w/o germline mutation. Pts with germline mutation had a higher incidence of non-melanoma malignancies (48% vs 29%; p=0.044). Table 1. Summary of identified germline mutations in the mucosal melanoma cohort. Germline mutation Protein MITF (N= 6) p.E318K CHEK2 (N=4) p.T367fs MUYTH p.G368D ATM splice ATM p.L263fs ATM p.R1875X ATM p.L1327X ATM p.A2067D ATM p.R2034X BRCA2 p.A1689fs CHEK2 p.S400T ERCC3 p.R109X MITF p.C29Y NF1 p.L1183R PALB2 p.V221X POLE p.D287E TP53 p.R306X TP53 p.V73Wfs*50 TSC1 p.Q844X Conclusions: This study reports a high prevalence of germline cancer susceptibility mutations in the largest cohort of MM to date, including mutations which are actionable. There is overlap with known MITF risk alleles for CM. We are currently performing a GWAS to comprehensively understand the genetic architecture of risk in MM. Citation Format: Afsaneh Amouzegar, Xiaogang Wu, James Long, Sabitha Prabhakaran, Latasha Little, Curtis Gumbs, Jared Malke, Julie Simon, Jianhua Zhang, Jennifer Wargo, Paul Scheet, Justin W. Wong, Priyadharsini Nagarajan, Jennifer L. McQuade, Andrew Futreal. Germline mutations in cancer susceptibility genes in patients with mucosal 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 7334.

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease.

Comparative investigation of cell cycle and immunomodulatory genes in mucosal and cutaneous melanomas: Preliminary data suggest a potential promising clinical role for p16 and the PD-1/PD-L1 axis

9561 Background: MM is a rare melanoma subtype with distinct biology and low response rates to ICIs. We seek to prospectively profile a cohort of MM patients to determine the interplay of gut, mucosal and tumor microbiome and modifiable risk factors on ICI response and resistance. Methods: Patients (pts) with MM presenting at MD Anderson are being prospectively enrolled for longitudinal collection for molecular, microbiome, and lifestyle factor profiling. Planned analyses will evaluate (1) fecal specimens [whole metagenome shotgun (WMS) sequencing, microbiome profiling and 16S rRNA gene sequencing (16S)]; (2) mucosal surface swabs (WMS sequencing); (3) formalin-fixed paraffin embedded (FFPE) tumor specimens [Bruker Digital Spatial Profiling (DSP)]; and (4) fresh tumor samples [whole exome (WES) and whole genome (WGS) of normal and tumor tissue, RNA sequencing, and T cell receptor sequencing]. Results: 82 pts with sequenced fecal specimens and mucosal surface swabs were enrolled as of 12/2024. MM primary sites included 25 (30%) naso-oral (including sinonasal), 14 (17%) urogenital, 30 (37%) anorectal, 10 (12%) conjunctival and 3 (4%) other. Disease stages were IVM1a/M1b in 9 (9%) pts, and IVM1c/M1d in 27 (33%) pts. Initial analysis of gut and mucosal surface swabs’ microbiome by MM primary site displayed a wide range of intrasample heterogeneity and microbial signatures that correlate with the MM primary site. WES and WGS data analysis indicate low tumor mutational burden (TMB) of 1.34mut/Mb (median, range 0.52 – 14.72). Common mutations included SF3B1 (23%), KIT (15%) and NRAS (8%). Anorectal and urogenital tumors contributed to mutational signatures associated with DNA mismatch repair and microsatellite instability (COSMIC v3.211). Conjunctival and naso-oral tumors showed an association with UV exposure. Results in the gut microbiome analysis from 78 pts treated with ICIs showed compositional differences in the presence and proportion of bacterial taxa in responders (R), compared to non-responders (NR). Distinct bacteria such as Streptococcus, Collinsella , and Blautia were identified in R, and Butyricicoccus in NR. DSP analysis by treatment response showed higher expression of CD56 and CD20 in the immune and tumor compartments in R. Conclusions: This is an ongoing prospective study that is expected to drive insights into the tumor/microenvironment/host interactions and factors regulating immunogenicity to predict response and resistance to ICIs in a rare and understudied melanoma subtype. Interrogation of the role of the gut microbiome and its modifiable determinants will lead to the investigation of new therapeutic strategies to modulate the microbiome to improve treatment outcomes in MM.