Subset Of Melanomas Research Articles

Challenges in Diagnosis of Melanoma from the Perspective of a Pathologist

Introduction: Melanoma is an aggressive melanocytic neoplasm with a continuously increasing incidence worldwide. Vast majority of melanoma arises from skin whereas a small subset of melanoma can arise as a primary malignancy of mucosal membranes. Gastrointestinal (GI) melanoma is usually metastatic in origin whereas primary GI melanomas are extremely rare. Anorectal Melanoma comprises of 1/% of all malignant neoplasms of this area. About 70% of these lesions are pigmented whereas 30% of them are amelanotic. Upto 25% of the patients with melanoma develops recurrent disease locally or in regional lymph nodes. Materials And Methods: This is a prospective study conducted in a tertiary care hospital comprising of six cases of Melanoma. We studied the cases according to their age, sex, clinical presentation, site, radiological findings, histopathological findings, immunohistochemical findings and treatment provided. Results: This study comprises of six cases of Melanoma among which five cases are primary one case is recurrent. Among the five primary cases, four cases have GI origin (sigmoid colon, rectum, anal canal) one case originated from skin involving left foot. There is a single case of recurrent MM involving right inguinal lymph nodes with a history of primary MM in the right lower limb. The patients belong to the age group of 6th to 8th decade with male preponderance. Histopathological examination of formalin fixed paraffin tissue sections was done followed by Hematoxylin & Eosin and Immunohistochemical staining which confirmed the diagnosis of Melanoma. Conclusion: Melanoma is an aggressive melanocytic neoplasm that carries a poor prognosis. It commonly involves skin. Its occurrence in GI tract is rare in which primary lesions are exceedingly unusual. Diagnosis and Treatment require multidisciplinary team approach with surgery remaining as a cornerstone of treatment.

A Multimodal Drug-Diet-Immunotherapy Combination Restrains Melanoma Progression and Metastasis.

The genetic landscape of cancer cells can lead to specific metabolic dependencies for tumor growth. Dietary interventions represent an attractive strategy to restrict the availability of key nutrients to tumors. In this study, we identified that growth of a subset of melanoma was severely restricted by a rationally designed combination therapy of a stearoyl-CoA desaturase (SCD) inhibitor with an isocaloric low-oleic acid diet. Despite its importance in oncogenesis, SCD underwent monoallelic codeletion along with PTEN on chromosome 10q in approximately 47.5% of melanoma, and the other SCD allele was methylated, resulting in very low-SCD expression. Although this SCD-deficient subset was refractory to SCD inhibitors, the subset of PTEN wild-type melanoma that retained SCD was sensitive. As dietary oleic acid could potentially blunt the effect of SCD inhibitors, a low oleic acid custom diet was combined with an SCD inhibitor. The combination reduced monounsaturated fatty acids and increased saturated fatty acids, inducing robust apoptosis and growth suppression and inhibiting lung metastasis with minimal toxicity in preclinical mouse models of PTEN wild-type melanoma. When combined with anti-PD1 immunotherapy, the SCD inhibitor improved T-cell functionality and further constrained melanoma growth in mice. Collectively, these results suggest that optimizing SCD inhibitors with diets low in oleic acid may offer a viable and efficacious therapeutic approach for improving melanoma treatment. Significance: Blockade of endogenous production of fatty acids essential for melanoma combined with restriction of dietary intake blocks tumor growth and enhances response to immunotherapy, providing a rational drug-diet treatment regimen for melanoma.

Isomer-sourced structure iteration methods for in silico development of inhibitors: Inducing GTP-bound NRAS-Q61 oncogenic mutations to an “off-like” state

The NRAS-mutant subset of melanoma represent some of the most aggressive and deadliest types associated with poor overall survival. Unfortunately, for more than 40 years, no therapeutic agent directly targeting NRAS mutations has been clinically approved. In this work, based on microsecond scale molecular dynamics simulations, the effect of Q61 mutations on NRAS conformational characteristics is revealed at the atomic level. The GTP-bound NRAS-Q61R and Q61K mutations show a specific targetable pocket between Switch-II and α-helix 3 whereas the NRAS-Q61L non-polar mutation category shows a different targetable pocket. Moreover, a new isomer-sourced structure iteration method has been developed for the in silico design of potential inhibitor prototypes for oncogenes. We show the possibility of a designed prototype HM-387 to target activated NRAS-Q61R and that it can gradually induce the transition from the activated NRAS-Q61R to an “off-like” state.

A Phase II Study of ERK Inhibition by Ulixertinib (BVD-523) in Metastatic Uveal Melanoma.

Uveal melanoma is a rare and aggressive subset of melanoma that is minimally responsive to traditional therapies. Greater than 80% of uveal melanomas have a mutation in GNAQ or GNA11 which lead to downstream signaling through the MAPK pathway. Ulixertinib (BVD-523) is a potent and reversible small-molecule ATP-competitive inhibitor of both ERK1 and ERK2 protein kinases. We performed a phase II study to determine the efficacy and safety of BVD-523 in patients with metastatic uveal melanoma. This was conducted as a Simon two-stage design with a sample size of 25 patients and an initial evaluation of efficacy after 13 patients. From April 2018 to April 2019, 13 patients were enrolled. Patients were predominantly female (69%) with a median age of 64 years (34-76). Sites of metastases included liver (84.6%) and lung (30.8%). Grade 3 and 4 toxicities associated with therapy were consistent with ERK inhibitors and included liver function test (LFT) elevation, hyponatremia, pruritis, amylase elevation, anemia, and rash. The best response, per RECIST 1.1, was stable disease in 4 patients, and disease progression in 7 patients. Two patients were unevaluable for response due to withdrawal from study. Median time to progression was 2.0 months. There were eight deaths due to disease progression with a median overall survival of 6.9 months. ERK inhibition with ulixertinib (BVD-523) did not demonstrate activity in patients with metastatic uveal melanoma. The toxicities observed were consistent with what would be expected with MAPK pathway inhibition. Uveal melanoma is a difficult to treat disease with minimal therapy options. The majority of uveal melanomas have mutations in GNAQ or GNA11 leading to activation of the MAPK pathway. Efforts to target MEK in uveal melanoma has had mixed results. This phase II trial of ERK inhibition with BVD-523 examined the potential role of this agent in uveal melanoma therapy.

Misdiagnosis and Clinical Insights into Acral Amelanotic Melanoma-A Systematic Review.

Acral amelanotic melanomas (AAMs), a rare subset of melanomas located on acral sites such as the palms, soles, and subungual areas, are diagnostically challenging due to their lack of typical pigmentation and often benign clinical appearance. Misdiagnosis is common, leading to delays in treatment and potentially worse outcomes. This systematic review aims to synthesise evidence on cases of AAM initially misdiagnosed as other conditions, to better understand their clinical and epidemiological characteristics, diagnostic pitfalls, and management strategies. A comprehensive search of the MEDLINE/PubMed, EMBASE, and SCOPUS databases was conducted up to March 2024. Case reports and small case series of AAMs initially misdiagnosed as other conditions were included. Data on patient demographics, clinical presentation, and diagnostic methods were collected and analyzed. Of the 152 records identified, 26 cases from 23 articles met the inclusion criteria. A demographic analysis revealed that the gender distribution appears to be perfectly balanced, with an age range of 38 to 91 years. Misdiagnoses included non-healing ulcers or traumatic lesions (37.5%), benign proliferative lesions (29.2%) and infectious lesions (20.8%). The foot was the most affected site (53.8%). Notably, a histological evaluation was performed in 50% of cases involving the upper extremities, in contrast to only 7.1% of cases involving the foot and 0% of cases of the heel. This discrepancy suggests a reluctance to perform biopsies in the lower extremities, which may contribute to a higher misdiagnosis rate in these areas. The underutilization of biopsy in the diagnosis of lower extremity lesions contributes significantly to the misdiagnosis and delay in treatment of AAMs. Especially when the clinical assessment and dermoscopy are inconclusive, biopsies of suspicious lesions are essential. Immunohistochemistry and markers such as PRAME are critical in differentiating melanoma from other malignancies such as clear cell sarcoma. This review highlights the need for increased vigilance and a proactive diagnostic approach to increase early detection rates and improve prognostic outcomes.

PRAME Immunohistochemistry for Distinguishing Vulvar and Vaginal Melanoma From Benign Melanocytic Nevi.

Vulvovaginal melanoma (VVM) is a rare but deadly disease, accounting for 5% of all vulvar malignancies, with a 5-yr survival rate of only 47% for all stages of the disease. VVM is a distinct subset of melanoma, with a unique genomic profile and underlying pathogenesis unassociated with sun exposure. Distinguishing these rare malignancies from very common pigmented lesions of the vulva and vagina is challenging as histologic features often overlap between entities. PReferentially expressed Antigen in MElanoma (PRAME) is a melanoma-associated protein, and immunohistochemistry (IHC) for PRAME distinguishes cutaneous, oral mucosal, and retinal melanoma from atypical nevi. Given the biological differences between VVM and cutaneous melanoma, the utility of PRAME IHC for the diagnosis of VVM is unknown. We accrued a cohort of 20 VVM and 21 benign vulvar melanocytic nevi. We found that nuclear PRAME IHC staining with 4+ intensity was present in 85% of the VVM and 0% of the nevi. With the assistance of PRAME IHC, we found evidence of close or positive margin involvement in 3 of 10 cases where margins were originally diagnosed as negative for melanoma in situ. Our study is the first to assess PRAME IHC in a cohort of VVM cases and provides confidence for using PRAME IHC to assist with diagnosis and margin assessment in this rare disease.

Abstract 2: Vertical MAPK pathway targeting in novel genetically engineered mouse and cell line models of NF1-altered melanoma: the mSK-Mel murine cohort

Abstract Large scale clinical genomic sequencing efforts have revealed inactivating mutations in the RAS-GTPase Neurofibromin 1 (NF1) in a significant subset of melanomas. To date, immunotherapy and MAPK pathway-directed targeted therapies have been largely inactive in this molecularly defined cohort and immunogenic models that reflect the distinct co-mutation patterns found in NF1-mutant melanoma patients are lacking. Leveraging a population-scale tumor genomic profiling initiative, we identified TP53 as a gene significantly co-altered with NF1 in melanoma. We thus generated and molecularly characterized a cohort of genetically engineered mice with targeted deletion of NF1 in melanocytes. Melanocyte-specific, homozygous knockout of NF1 induced hyperpigmentation yet was insufficient for tumorigenesis. Addition of TP53 knockout and/or conditional activating mutation in BRAF (BRAFVE), resulted in melanoma formation with variable and high penetrance, respectively, along with histologic features consistent with human melanomas. Tumor latency and overall survival in NF1/TP53 double knockout mice was similar to NF1/BRAFVE double mutants. NF1 knockout did not shorten the latency to tumor formation in the setting of BRAFVE/TP53 mutation but did intensify melanocytic hyperpigmentation in all genetic backgrounds tested. To facilitate preclinical and functional studies, we derived 22 congenic cell lines from harvested mouse tumors from NF1 knockout mice, with and without BRAFVE mutation, and tested their sensitivity to targeted agents. As expected, loss of NF1 conditioned the response to BRAF inhibition, while NF1-mutant cells retained sensitivity to MEK inhibition. To abrogate the effects of adaptive RAS reactivation after MEK inhibitor therapy, combined MEK/SHP inhibition in NF1/TP53 knockout cells and BRAF/SHP inhibition in NF1/TP53/BRAFVE mutant cells strongly blunted ERK phosphorylation and cell proliferation better than single agent therapy. However, this response to the addition of SHP inhibition was transient and ERK rebound was driven by continued MEK activation and dependance. In syngeneic xenograft models of NF1/TP53/BRAFVE mutation, MEK inhibition alone, or in combination with RAF and/or SHP inhibition, induced tumor regression and delayed the onset of resistance and progression as compared to doublet RAF/SHP inhibitor therapy. Overall, we demonstrated the efficacy and feasibility of vertical MAPK pathway targeting in a novel cohort of genetically relevant mouse and cell line models of NF1-mutant melanoma and provide justification for future studies of vertical MAPK pathway targeting to achieve maximal ERK pathway inhibition in this molecularly defined patient cohort. Citation Format: Alexis M. Jones, Aphrothiti J. Hanrahan, Moriah H. Nissan, Sebastien Monette, Ziyu Chen, Wenhuo Hu, Sandra Misale, Isabell Schulze, Naresh Vasani, Cailian Liu, Xia Yang, Mohsen Abu-Akeel, Elisa de Stanchina, Nikolaus Schultz, Michael F. Berger, Neal Rosen, Taha Merghoub, David B. Solit. Vertical MAPK pathway targeting in novel genetically engineered mouse and cell line models of NF1-altered melanoma: the mSK-Mel murine cohort [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 2.

Tertiary lymphoid structures in desmoplastic melanoma have increased lymphocyte density, lymphocyte proliferation, and immune cross talk with tumor when compared to non-desmoplastic melanomas

ABSTRACT Tertiary lymphoid structures (TLS) are ectopic lymphoid structures that can arise in human cancers and are associated with improved overall survival (OS) and response to immune checkpoint blockade (ICB) in several cancers, including non-desmoplastic metastatic melanoma (NDMM). Desmoplastic melanoma (DM) has one of the highest response rates to ICB, and we previously identified that primary DM (PDM) contains TLS. Despite the association of TLS with survival and ICB response, it is unknown whether TLS or associated markers of immune activity can differ between PDM and NDMM. We hypothesized that PDM would contain higher frequencies of TLS than NDMM, that T and B-cell densities and proliferation would be greater in TLS of PDM than TLS of NDMM, and that proliferation rates of T and B-cells in PDM TLS would be concordant with those of intratumoral lymphocytes. We found that four features of TLS in PDM distinguish them from TLS in NDMM. TLS were peritumoral in NDMM but intratumoral in PDM. CD8+ T-cell and CD20+ B-cell densities and proliferative fractions were higher in PDM TLS than NDMM TLS. Additionally, the proliferative fractions of T- and B-cells were concordant between the TLS and tumor site in PDM and discordant in NDMM. Collectively, these data suggest that TLS and associated immune markers can differ across melanoma subsets and suggest that PDM TLS may be more immunologically active and have enhanced immune cell trafficking between tumor and TLS compared to NDMM.

SRC-RAC1 signaling drives drug resistance to BRAF inhibition in de-differentiated cutaneous melanomas

Rare gain-of-function mutations in RAC1 drive drug resistance to targeted BRAF inhibition in cutaneous melanoma. Here, we show that wildtype RAC1 is a critical driver of growth and drug resistance, but only in a subset of melanomas with elevated markers of de-differentiation. Similarly, SRC inhibition also selectively sensitized de-differentiated melanomas to BRAF inhibition. One possible mechanism may be the suppression of the de-differentiated state, as SRC and RAC1 maintained markers of de-differentiation in human melanoma cells. The functional differences between melanoma subtypes suggest that the clinical management of cutaneous melanoma can be enhanced by the knowledge of differentiation status. To simplify the task of classification, we developed a binary classification strategy based on a small set of ten genes. Using this gene set, we reliably determined the differentiation status previously defined by hundreds of genes. Overall, our study informs strategies that enhance the precision of BRAFi by discovering unique vulnerabilities of the de-differentiated cutaneous melanoma subtype and creating a practical method to resolve differentiation status.

Identification of Melanoma Subsets Based on DNA Methylation Sites and Construction of a Prognosis Evaluation Model.

Background Melanoma is a lethal skin malignant tumor, and its formation or development is regulated by various genetic and epigenetic molecules. Although there are traditional methods provided for the doctors to evaluate the patients' prognosis or make the diagnosis, the novel method based on epigenetic markers is still needed to make the early diagnosis. Results We identified 256 melanoma-independent prognosis-related methylation sites (P < 0.0001) and divided patients into seven methylation subgroups. Methylation levels and survival time in the C2 subgroup were lower than that of other clusters (P < 0.05). We established the predicted model of prognosis risk for melanoma using the significantly changed methylation sites in C2. The model efficiently divided patients into high- and low-risk groups (area under the receiver operating characteristic curve, 0.833). Risk scores and patient survival time were negatively correlated (rs = −0.325, P < 0.0001). Genes corresponding to the independent prognosis-associated methylation sites were enriched in cancer- and immunology-related pathways. We identified 35 hub genes. DOK2, GBP4, PSMB9, and NLRC5 were significantly changed according to methylation subgroups, survival, tumor stages, and T categories and were positively correlated, which was validated in the testing group (P < 0.05). The levels of DOK2, GBP4, PSMB9, and NLRC5 had an opposite trend to their methylation sites in patients with poor prognosis. Conclusions We identified seven DNA methylation subtypes and constructed a highly effective prognosis risk assessment model. The transcript levels of key genes corresponding to the independent prognosis-related methylation sites were significantly changed in patients according to prognosis and positively correlated with each other, indicating they may collaboratively promote melanoma formation. These findings further our understanding of the mechanism of melanoma and provide new targets for diagnosis and treatment.

Investigating molecular profiles of subtypes of mucosal melanomas: Is vulvovaginal melanoma a distinct entity? (263)

<b>Objectives:</b> Vulvovaginal melanomas (VVM) are considered to be a subtype of mucosal melanoma (MM) that are less immunogenic and less responsive to immuno-oncology therapy (IO) than cutaneous melanoma (CM). Due to the rarity of MM, there is a paucity of data regarding the molecular and immune profile differences between MM subtypes. Herein we compared VVM with other subtypes of mucosal melanoma and explored the significance of IO agents on survival. <b>Methods:</b> Tumor samples were analyzed using next-generation sequencing (TruSeq, 45 genes; NextSeq, 592 genes and NovaSeq, WES), IHC, and WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). PD-L1 expression was tested by IHC using 28-8 (Agilent) and SP-142 (Spring Biosciences) (positive cut-off ≥1%). MSI was tested by FA, IHC, and NGS. TMB was measured by totaling somatic mutations per tumor (TMB-high cut-off ≥ 10 mutations per MB). Immune cell fraction was calculated by Q uantiSeq. (Finotello 2019, Genome Medicine). Survival was extracted from insurance claims data and calculated from the time of IO treatment to the last contact using Kaplan-Meier survival curves for molecularly defined cohorts. Statistical significance was determined using Chi-square and Wilcoxon rank-sum test and adjusted for multiple comparisons (q<0.05). <b>Results:</b> A total of 183 VVM, 84 anorectal melanomas (ARM), and 95 head and neck mucosal melanomas (HNM) tumors were evaluated. The median ages of each cohort were 66, 67, and 71, respectively. The <i>SF3B1</i> RNA splicing gene was more frequently mutated among VVM and ARM compared with HNM (26.7%, 36.2%, and 1.8%, respectively, q<0.01). There was also a trend toward higher rates of <i>TP53</i> and <i>KIT</i> mutations in VVM and ARM compared with HNM. <i>BRCA1</i> and <i>APC</i> mutations were found exclusively in ARM tumors. <i>KIT</i> was the most common biomarker amplification (14.5% VVM, 10.5% ARM, and HNM). Otherwise, rates of biomarker amplification were low overall. When evaluating IO therapy-related biomarkers, VVM had the highest rate of PD-L1 positivity (34.6%) compared to ARM (18.5%) and HNM (33.3%). High TMB was present in 4.2% of ARM, 4.8% of HNM, and 0% of VVM tumors. MSI-H/dMMR tumors were absent in all three cohorts. Immune cell infiltrates were similar among all MM tumors, except for an increase in regulatory T cell infiltration in VVM tumors (q<0.01). Treatment and survival data were available for 119 VVM, 61 ARM, 42 HNM, and 4839 CM patients. Of these, 17% VVM (<i>n</i>=20), 31% ARM (<i>n</i>=19), 14% HNM (<i>n</i>=6) and 22% CM (<i>n</i>=1056) received IO therapy. Median survival was 17.5 months (mo) for VVM, 16.8 mo for ARM, and 33 mo for HNM, none of which were significantly different. There was a trend toward better survival for CM patients on IO therapy compared to MM patients (36.6 mo vs 18.8 mo, HR: 0.67, 95% CI: 0.45-1.06, p=0.087). <b>Conclusions:</b> MM represents a unique subset of melanoma, with worse survival and response to IO therapy than CM. VVM and ARM appear to be more similar on a molecular level than HNM.

Iris and Ciliary Body Melanocytomas Are Defined by Solitary GNAQ Mutation Without Additional Oncogenic Alterations

To analyze the genetic features of melanocytomas and melanomas of the anterior uvea and assess the value of molecular testing for diagnosis and prognostication. Retrospective case-control study. Patients with melanocytoma (n= 16) and melanoma (n= 19) of the anterior uvea. Targeted next-generation sequencing was performed on formalin-fixed, paraffin-embedded tumor tissue from anterior uveal melanocytic tumors and correlated with clinicopathologic features. Presence or absence of accompanying oncogenic alterations beyond GNAQ/GNA11 and their association with histologic features and local recurrence. Hotspot missense mutations in GNAQ/GNA11 were identified in 91% (32/35) of all cases. None of the melanocytomas with or without atypia demonstrated chromosomal imbalances or additional oncogenic variants beyond GNAQ mutation, and none recurred over a median follow-up of 36 months. Additional alterations identified in a subset of melanomas include mutations in BAP1 (n= 3), EIF1AX (n= 4), SRSF2 (n= 1), PTEN (n=1), and EP300 (n= 1); monosomy 3p (n= 6); trisomy 6p (n= 3); trisomy 8q (n= 2); and an ultraviolet mutational signature (n= 5). Local recurrences were limited to melanomas, all of which demonstrated oncogenic alterations in addition to GNAQ/GNA11 (n= 5). A single melanoma harboring GNAQ and BAP1 mutations and monosomy 3 was the only tumor that metastasized. In this study, anterior segment uveal melanocytomas did not display oncogenic alterations beyond GNAQ/GNA11. Therefore, they are genetically similar to uveal nevi rather than uveal melanoma based on their molecular features known from the literature. Molecular testing can be performed on borderline cases to aidrisk stratification and clinical management decisions.

A Phase Ib Study of Sotrastaurin, a PKC Inhibitor, and Alpelisib, a PI3Kα Inhibitor, in Patients with Metastatic Uveal Melanoma.

Simple SummaryUveal melanoma is a rare subset of melanoma characterized by the presence of early initiating GNAQ/11 mutations, with downstream activation of several pathways which are thought to contribute to cell growth. Based on clinical and preclinical data supporting targeting of protein kinase C (PKC) and the phosphatidylinositol 3-kinase (PI3K) pathway, we conducted a phase Ib study to assess the safety of combined sotrastaurin, a PKC inhibitor, and alpelisib, a PI3K inhibitor. We found that sotrastaurin and alpelisib can be safely administered, however there was no evidence of clinical efficacy.Uveal melanoma (UM) is a rare subset of melanoma characterized by the presence of early initiating GNAQ/11 mutations, with downstream activation of the PKC, MAPK, and PI3Kα pathways. Activity has been observed with the PKC inhibitors sotrastaurin (AEB071) and darovasertib (IDE196) in patients with UM. Inhibition of the PI3K pathway enhances PKC inhibition in in vivo models. We therefore conducted a phase Ib study of sotrastaurin in combination with the PI3Kα inhibitor alpelisib to identify a tolerable regimen that may enhance the activity of PKC inhibition alone. Patients with metastatic uveal melanoma (n = 24) or GNAQ/11 mutant cutaneous melanoma (n = 1) were enrolled on escalating dose levels of sotrastaurin (100–400 mg BID) and alpelisib (200–350 mg QD). The primary objective was to identify the maximum tolerated dose (MTD) of these agents when administered in combination. Treatment-related adverse events (AE) occurred in 86% (any grade) and 29% (Grade 3). No Grade 4–5-related AEs occurred. Dose Level 4 (sotrastaurin 200 mg BID and alpelisib 350 mg QD) was identified as the maximum tolerated dose. Pharmacokinetic analysis demonstrated increasing concentration levels with increasing doses of sotrastaurin and alpelisib, without evidence of interaction between agents. Pharmacodynamic assessment of pMARCKS and pAKT protein expression with drug exposure suggested modest target inhibition that did not correlate with clinical response. No objective responses were observed, and median progression-free survival was 8 weeks (range, 3–51 weeks). Although a tolerable dose of sotrastaurin and alpelisib was identified with pharmacodynamic evidence of target inhibition and without evidence of a corresponding immunosuppressive effect, limited clinical activity was observed.

Identification of MicroRNA-mRNA Networks in Melanoma and Their Association with PD-1 Checkpoint Blockade Outcomes.

Simple SummaryWe conducted a network analysis of microRNA–mRNA associations in melanoma tissue and cell lines to identify the microRNAs central to melanoma biology and their associated gene expression profiles. Further, we evaluated expression of these microRNAs in melanoma patient biopsies and found that increased expression of miR-100-5p and miR-125b-5p were associated with improved outcomes with anti-PD-1 immunotherapy. Further investigation of these microRNAs as biomarkers and potential targets to improve immunotherapy response in melanoma is warranted.Metastatic melanoma is a deadly malignancy with poor outcomes historically. Immuno-oncology (IO) agents, targeting immune checkpoint molecules such as cytotoxic T-lymphocyte associated protein-4 (CTLA-4) and programmed cell death-1 (PD-1), have revolutionized melanoma treatment and outcomes, achieving significant response rates and remarkable long-term survival. Despite these vast improvements, roughly half of melanoma patients do not achieve long-term clinical benefit from IO therapies and there is an urgent need to understand and mitigate mechanisms of resistance. MicroRNAs are key post-transcriptional regulators of gene expression that regulate many aspects of cancer biology, including immune evasion. We used network analysis to define two core microRNA–mRNA networks in melanoma tissues and cell lines corresponding to ‘MITF-low’ and ‘Keratin’ transcriptomic subsets of melanoma. We then evaluated expression of these core microRNAs in pre-PD-1-inhibitor-treated melanoma patients and observed that higher expression of miR-100-5p and miR-125b-5p were associated with significantly improved overall survival. These findings suggest that miR-100-5p and 125b-5p are potential markers of response to PD-1 inhibitors, and further evaluation of these microRNA–mRNA interactions may yield further insight into melanoma resistance to PD-1 inhibitors.

Enhancing adoptive CD8 T cell therapy by systemic delivery of tumor associated antigens

Adoptive T-cell transfer (ACT) offers a curative therapeutic option for subsets of melanoma and hematological cancer patients. To increase response rates and broaden the applicability of ACT, it is necessary to improve the post-infusion performance of the transferred T cells. The design of improved treatment strategies includes transfer of cells with a less differentiated phenotype. Such T cell subsets have high proliferative potential but require stimulatory signals in vivo to differentiate into tumor-reactive effector T cells. Thus, combination strategies are needed to support the therapeutic implementation of less differentiated T cells. Here we show that systemic delivery of tumor-associated antigens (TAAs) facilitates in vivo priming and expansion of previously non-activated T cells and enhance the cytotoxicity of activated T cells. To achieve this in vivo priming, we use flexible delivery vehicles of TAAs and a TLR7/8 agonist. Contrasting subcutaneous delivery systems, these vehicles accumulate TAAs in the spleen, thereby achieving close proximity to both cross-presenting dendritic cells and transferred T cells, resulting in robust T-cell expansion and anti-tumor reactivity. This TAA delivery platform offers a strategy to safely potentiate the post-infusion performance of T cells using low doses of antigen and TLR7/8 agonist, and thereby enhance the effect of ACT.

PARP Inhibitors in Melanoma-An Expanding Therapeutic Option?

Simple SummaryMelanomas with homologous recombination DNA damage repair pathways represent a subset of melanoma that may benefit from PARP inhibitors and immunotherapy. PARP inhibitors have an established role in treating cancers with underlying BRCA mutation through synthetic lethality; however, there is increasing evidence that it can be applied to a larger population including other types of homologous recombination defects. These gene mutations can be found in 20–40% of cutaneous melanoma. To date, PARP inhibitors and immunotherapy have been overlooked in the management of melanoma. This review explores the reasons for combining PARP inhibitors and immunotherapy. There is evidence to suggest that PARP inhibitors can improve the therapeutic effect of immune checkpoint inhibitors. Therefore, this combination approach has the potential to impact future treatment of patients with melanoma, particularly those with homologous recombination DNA damage repair defects. Immunotherapy has transformed the treatment landscape of melanoma; however, despite improvements in patient outcomes, monotherapy can often lead to resistance and tumour escape. Therefore, there is a need for new therapies, combination strategies and biomarker-guided decision making to increase the subset of patients most likely to benefit from treatment. Poly (ADP-ribose) polymerase (PARP) inhibitors act by synthetic lethality to target tumour cells with homologous recombination deficiencies such as BRCA mutations. However, the application of PARP inhibitors could be extended to a broad range of BRCA-negative cancers with high rates of DNA damage repair pathway mutations, such as melanoma. Additionally, PARP inhibition has the potential to augment the therapeutic effect of immunotherapy through multi-faceted immune-priming capabilities. In this review, we detail the immunological role of PARP and rationale for combining PARP and immune checkpoint inhibitors, with a particular focus on a subset of melanoma with homologous recombination defects that may benefit most from this targeted approach. We summarise the biology supporting this combined regimen and discuss preclinical results as well as ongoing clinical trials in melanoma which may impact future treatment.

Metastatic acral melanoma treatment outcomes: a systematic review and meta-analysis.

Acral melanomas are a unique subset of melanomas occurring on the palms, soles, and nails. There is poor prognosis with surgery alone and no specific guidelines for the treatment of metastatic acral melanoma. This meta-analysis explored the systemic therapy outcomes for metastatic acral melanoma. Medline, Pubmed, EMBASE, and the grey literature were searched from 2010 to August 2020 for studies specifying the treatment outcome of metastatic acral melanoma. Studies were assessed by two investigators. A random-effects meta-analysis was performed and pooled Kaplan-Meier curves for progression-free survival and overall survival were created. Critical appraisal was performed using the Newcastle-Ottawa Scale. Nineteen nonrandomized studies were included, comprising 646 patients with acral melanomas and 1609 patients with nonacral melanomas treated with systemic therapy including chemotherapy, KIT-targeted drugs, as well as anti-CTLA-4 and anti-PD-1 checkpoint inhibitor therapy. Thirteen studies included Kaplan-Meier curves for progression-free survival or overall survival and 11 studies reported treatment responses. Patients with acral melanomas had worse prognosis than nonacral cutaneous melanoma (acral overall survival: median 15 months, 95% CI, 13.7-16.3 months; nonacral cutaneous: median 24 months, 95% CI, 22.6-25.4 months, P < 0.001). Acral melanoma patients treated with anti-PD-1 monotherapy had higher overall survival at 12 months (53%) compared with anti-CTLA-4 monotherapy (34%; P < 0.001). This study provides estimates of treatment response for metastatic acral melanoma, demonstrating low activity across a breadth of approved drug therapies, including anti-PD-1, the most active therapy in melanoma to date. Further research into treatments for metastatic acral melanoma is needed.

Abstract 1218: Targeting B-RAF and checkpoint inhibitor resistant melanoma with RSK inhibitor PMD-026 in pre-clinical models

Abstract Cutaneous melanoma is a highly invasive skin cancer. Mortality for melanoma is low if caught before metastasis occurs, but is poor if metastasis has occurred at diagnosis. Melanomas are among the most mutated cancer genomes and have a high neoantigen load, which is related to better response to immunotherapies. A subset of melanomas contains mutations in BRAF(V600E) and respond initially to BRAF inhibitors, though they become resistant through escape pathways. Focused development of BRAF and immunotherapies has resulted in the clinical use of targeted therapies against the oncogenic BRAF/MEK/ERK pathway and immune checkpoint inhibitors for the treatment of metastatic melanoma. Although some patients with metastatic melanoma benefit from these therapies, others either do not respond or become resistant. Because of this, new targets and drugs are needed for patients who are either intrinsically resistant or have acquired resistance to targeted therapies and immunotherapies. RSKs are serine/threonine kinases downstream of BRAF/ERK that activate transcription, proliferation and cell survival. Thus RSKs are potential new targets and the availability of a RSK inhibitor called PMD-026, now in Phase 1b clinical trials for breast cancer, provides a tool to explore this possibility in pre-clinical models of melanoma. PMD-026 is an ATP-competitive inhibitor of the N-terminal kinase domain. This inhibitor is highly stable and effective in cell-based assays with an average IC50 of 2nM and 200-400 nM in soft agar and inhibits tumor growth in multiple mouse models of breast cancer given 70-100 mg/kg given orally. It is currently the only RSK inhibitor that can be given to mice orally with demonstrated in vivo activity. To explore the opportunity for RSK inhibitors in melanoma, we screened 1,959 PDx models for RSK1-4 expression, which was evaluated based on RNAseq. In melanoma, RSK1 and RSK2 were highly expressed, unlike RSK3 and RSK4. In a study of 21 cases of melanoma, RSK2 was activated in 52% of the cases based on nuclear localization using our companion diagnostic. Using FRET based assays, PMD-026 inhibited RSK2 binding with an IC50=14 nM compared to BID1870 with IC50=94 nM demonstrating competitive in vitro potency. Importantly, PMD-026 inhibits viability of melanoma cells including those that are resistant to the BRAF inhibitor. Surprisingly, we also found that it significantly reduces PD-L1 expression at the cell surface of several melanoma lines. This is through down-regulation of PD-L1 transcription and subsequent reduced PD-L1 cell surface expression. As expected, in our melanoma models, PMD-026 inhibited RSK signaling through pYB-1, a transcription factor known to bind to the PD-L1 promoter. Testing in pre-clinical mouse models is in progress. We propose that PMD-026 may therefore potentiate melanoma immunotherapy approaches and also improve response of patients to BRAFV600E targeted therapies. Citation Format: Joe William Ramos, Stephanie Si, Takeo Fujii, Brien Haun, Won Seok Yang, Aarthi Jayanthan, Sandra E. Dunn. Targeting B-RAF and checkpoint inhibitor resistant melanoma with RSK inhibitor PMD-026 in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1218.

Immunohistochemical expression of PAX8, PAX2, and cytokeratin in melanomas.

Deviations from the classic melanocytic immunophenotype in melanoma can present a diagnostic challenge. PAX8 and PAX2 are common markers for renal or Müllerian differentiation. While most PAX8+ or PAX2+ carcinomas are seldom confused with melanoma, some cases may show a more ambiguous immunophenotype, especially when MiTF family altered renal cell carcinoma (MiTF-RCC) is in the differential diagnosis. Neither PAX8 nor PAX2 expression has been reported in melanoma to date. We aimed to better characterize PAX8, PAX2, and cytokeratin immunoreactivity in a large series of melanomas. Tissue microarrays consisting of 263 melanomas were immunostained for PAX8, PAX2, and cytokeratin and graded by an h-score. PAX8 expression was seen in 7.9% of melanomas and was significantly associated with spindle cytomorphology. PAX2 was positive in one (0.4%) melanoma. Cytokeratin positivity was seen in three (1.2%) cases and was associated with metastases. PAX8 is expressed in a subset of melanomas and may be strong/extensive. As PAX8 positivity does not exclude a diagnosis of melanoma, it should be used in conjunction with other immunohistochemical markers, such as cytokeratin and PAX2, when melanoma, MiTF-RCC, and other PAX8+ tumors are in the differential diagnosis.

Targeting non-canonical activation of GLI1 by the SOX2-BRD4 transcriptional complex improves the efficacy of HEDGEHOG pathway inhibition in melanoma

Despite the development of new targeted and immune therapies, the prognosis of metastatic melanoma remains bleak. Therefore, it is critical to better understand the mechanisms controlling advanced melanoma to develop more effective treatment regimens. Hedgehog/GLI (HH/GLI) signaling inhibitors targeting the central pathway transducer Smoothened (SMO) have shown to be clinical efficacious in skin cancer; however, several mechanisms of non-canonical HH/GLI pathway activation limit their efficacy. Here, we identify a novel SOX2-BRD4 transcriptional complex driving the expression of GLI1, the final effector of the HH/GLI pathway, providing a novel mechanism of non-canonical SMO-independent activation of HH/GLI signaling in melanoma. Consistently, we find a positive correlation between the expression of GLI1 and SOX2 in human melanoma samples and cell lines. Further, we show that combined targeting of canonical HH/GLI pathway with the SMO inhibitor MRT-92 and of the SOX2-BRD4 complex using a potent Proteolysis Targeted Chimeras (PROTACs)-derived BRD4 degrader (MZ1), yields a synergistic anti-proliferative effect in melanoma cells independently of their BRAF, NRAS, and NF1 mutational status, with complete abrogation of GLI1 expression. Combination of MRT-92 and MZ1 strongly potentiates the antitumor effect of either drug as single agents in an orthotopic melanoma model. Together, our data provide evidence of a novel mechanism of non-canonical activation of GLI1 by the SOX2-BRD4 transcriptional complex, and describe the efficacy of a new combinatorial treatment for a subset of melanomas with an active SOX2-BRD4-GLI1 axis.