Primary Melanoma Tumors Research Articles

Germline variants are associated with increased primary melanoma tumor thickness at diagnosis.

Germline genetic variants have been identified, which predispose individuals and families to develop melanoma. Tumor thickness is the strongest predictor of outcome for clinically localized primary melanoma patients. We sought to determine whether there is a heritable genetic contribution to variation in tumor thickness. If confirmed, this will justify the search for specific genetic variants influencing tumor thickness.To address this, we estimated the proportion of variation in tumor thickness attributable to genome-wide genetic variation (variant-based heritability) using unrelated patients with measured primary cutaneous melanoma thickness. As a secondary analysis, we conducted a genome-wide association study (GWAS) of tumor thickness.The analyses utilized 10 604 individuals with primary cutaneous melanoma drawn from nine GWAS datasets from eight cohorts recruited from the general population, primary care and melanoma treatment centers. Following quality control and filtering to unrelated individuals with study phenotypes, 8125 patients were used in the primary analysis to test whether tumor thickness is heritable. An expanded set of 8505 individuals (47.6% female) were analyzed for the secondary GWAS meta-analysis. Analyses were adjusted for participant age, sex, cohort and ancestry.We found that 26.6% (SE 11.9%, P = 0.0128) of variation in tumor thickness is attributable to genome-wide genetic variation. While requiring replication, a chromosome 11 locus was associated (P < 5 × 10−8) with tumor thickness. Our work indicates that sufficiently large datasets will enable the discovery of genetic variants associated with greater tumor thickness, and this will lead to the identification of host biological processes influencing melanoma growth and invasion.

CARDIAC TEMPONADE AS INITIAL PRESENTATION OF ADENOCARCINOMA OF LUNG

SESSION TITLE: Fellows Lung Cancer Posters SESSION TYPE: Fellow Case Report Posters PRESENTED ON: October 18-21, 2020 INTRODUCTION: Primary cardiac tumors are extremely uncommon and mostly cardiac tumors are associated with metastases from secondary tumors. Here we describe a case of a patient with primary lung cancer presenting with cardiac tamponade on initial presentation. CASE PRESENTATION: 70 years old male active smoker who presented to the emergency room for the complain of shortness of breath (SOB).He had Chest x-ray done which showed possible lower lobe infiltrates and was given antibiotics and sent home. He again presented to hospital four days later for worsening SOB. A CTA chest was done to rule out pulmonary embolism (PE) which was negative for PE but showed large pericardial effusion and left upper lobe small nodule. Patient had urgent ECHO done which showed very large effusion with tamponade physiology and also showed masses on free wall of left and right ventricles concerning for metastases. Cardiothoracic surgery was consulted and patient underwent pericardial window procedure. He also underwent CT guided biopsy of lung nodule which was consistent with moderately differentiate adenocarcinoma. His pericardial fluid cytology was also consistent with Metastatic adenocarcinoma. Post operatively patient did well and oncology service was consulted for cancer management. DISCUSSION: Metastatic tumors to heart are rare, although the lung cancer is the most common malignant tumor, review of literature showed only few cases of cardiac metastases associated with lung cancer. Various studies have shown the incidence of cardiac metastases ranging between 2.3% to 18.3% 1. Lung Cancer, melanoma and mediastinal primary tumors are the most common cancers associated with cardiac metastases. Bussani and etal reported adenocarcinoma as the most common lung cancer metastasizes to the heart in 26% of cases, followed by squamous cell carcinoma in 23.4% cases. One study showed Epicardium (88%) and pericardium (5-11%) are the most common site of cardiac metastasis and endocardium (< 3%) is the least involve site. 2 Metastases to heart occurs by direct extension, via blood stream, lymphatics and intracavitary diffusion through either the inferior vena cava or the pulmonary veins. Coronary arteries evaluation is recommended as they get compressed by metastatic tumor.3 Chemotherapy, radiotherapy, and surgery are the treatment options but usually prognosis is poor. CONCLUSIONS: Cardiac metastases are rare and high index of suspicion is required as variety of complications may occur due to metastasis including cardiac tamponade needing urgent pericardiocentesis. In patients with cardiac metastases a thorough evaluation should be performed to find secondary cancers. Reference #1: Bussani R, De-Giorgio F, Abbate A. Cardiac metastases. J Clin Pathol 60: 27-34, 2007. Reference #2: Abe S, Watanabe N, Ogura S. Myocardial metastasis from primary lung cancer: myocardial infarction-like ECG change and pathologic findings. Jpn J Med 30: 213-218, 1991. Reference #3: Butany J, Nair V, Naseemuddin A. Cardiac tumours: diagnosis and management. Lancet Oncol 6: 219-228, 2005. DISCLOSURES: No relevant relationships by Michael Caniglia, source=Web Response No relevant relationships by Adnan Khan, source=Web Response No relevant relationships by Sana Khan, source=Web Response No relevant relationships by Ramsha Zafar, source=Web Response

Abstract PR03: Nongenomic BAP1 aberrancy drives highly aggressive cutaneous melanoma phenotype

Abstract The purpose of this study was to determine the role of BAP1 levels in cutaneous melanoma (CM). BAP1 is a tumor suppressor in which loss of heterozygosity (LOH) from mutation and copy number alteration is well described in germline and somatic cancers. Although BAP1 genomic alterations in CM are extremely rare (2% of 665 samples from 5 datasets), marked variability in BAP1 expression is observed in CM. We show that low nuclear BAP1 levels portend a significantly worse clinical outcome in stage III CM (n=37, log rank p ≤0.01 for both overall survival and progression-free survival). Gene Set Enrichment Analysis (GSEA) revealed low BAP1 expression to be most highly ranked with an increased epithelial–mesenchymal transition (EMT) gene expression profile in CM tumors (n=379, FDR q = 1.34E-26) and cell lines (n=53, FDR q = 2.86E-116). We identify the expression of ZEB1, a master regulator of EMT, to be significantly associated with low BAP1 expression in CM tumors and cell lines (p= 1.5E-04 and 3.3E-05, respectively). Analysis of the BAP1 promoter indicates three canonical ZEB1 binding sites. Functional experiments show ZEB1 to bind to the BAP1 promoter, and luciferase activity assays indicate that ZEB1 acts as a transcriptional suppressor of BAP1 expression with differential utilization of the promoter binding sites. Targeted reduction of endogenous ZEB1 caused increased BAP1 levels, while targeted reduction of BAP1 did not modulate ZEB1 levels, consistent with ZEB1 having a suppressive effect on BAP1. Phenotypically, targeted reduction of BAP1 in CM cells resulted in a switch from a more differentiated, melanocytic state, to a less differentiated, more migratory and invasive phenotype. Extinguishing melanocyte-specific BAP1 in mice with a BRAF V600E mutant genetic background resulted in the emergence of primary melanoma tumors, with a marked EMT gene expression profile, and resultant metastases. Given the phenotypic changes associated with BAP1 levels in our mouse and human studies, we then tested the effect of modulating BAP1 on BRAF targeted therapy. Exogenous expression of BAP1 sensitized BRAF inhibitor (vemurafenib)-resistant melanoma cells, while the targeted reduction of BAP1 desensitized BRAF inhibitor-sensitive melanoma cells. BRAF mutant/BAP1 loss mice failed to exhibit a marked response to vemurafenib treatment compared to control mice. These data implicate regulation of BAP1 to be a major mechanism that characterizes a highly malignant and treatment-resistant subset of tumors. Our study indicates that nongenomic reduction in BAP1 through ZEB1 transcriptional modulation may be a key factor in aggressive CM. This abstract is also being presented as Poster A30. Citation Format: Haifeng Zhu, Junna Oba, Xiaoxing Yu, Caitlin A. Creasy, Marie-Andrée Forget, Fernando Carapeto, Cara L. Haymaker, Chang-Jiun Wu, Tatiana V. Karpinets, Wei-Lien Wang, Michael T. Tetzlaff, Alexander J. Lazar, Gordon B. Mills, Amanda R. Moore, Yu Chen, Jianhua Zhang, Jeffrey E. Gershenwald, Jennifer A. Wargo, Chantale Bernatchez, Patrick Hwu, P. Andrew Futreal, Scott E. Woodman. Nongenomic BAP1 aberrancy drives highly aggressive cutaneous melanoma phenotype [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR03.

Is repeated pulmonary metastasectomy justified?

Recurrence after pulmonary metastasectomy (PM) is frequent, but it is unclear to whom repeated pulmonary metastasectomy (RPM) offers highest benefits. Retrospective analysis of oncological and post-operative outcomes of consecutive patients who underwent PM from 2003 to 2018. Overall survival (OS) and disease-free interval (DFI) were calculated. Cox regression was used to identify variables influencing OS and DFI. In total, 264 patients (female/male: 114/150; median age: 62 years) underwent PM for colorectal cancer (32%), sarcoma (19%), melanoma (16%) and other primary tumors (33%). Pulmonary metastasectomy was approached by video-assisted thoracic surgery (VATS) in 73% and pulmonary resection was realized by non-anatomical resection in 76% of cases. The overall median follow-up time was 33 months (IQR 16–56 months) and overall 5-year survival rate was 62%. Local or distant recurrences were observed in 172 patients (65%) and RPM could be performed in 66 patients (25%) for a total of 116 procedures. RPM was realized by VATS in 49% and pulmonary resection by wedge in 77% of cases. In RPM patients, the 5-year survival rate after first PM was 79%. Post-operative cardio-pulmonary complication rate (13% vs. 12%; p = 0.8) and median length of stay (4 vs. 5 days; p = 0.2) were not statistically different between first PM and RPM. Colorectal cancer (HR 0.56), metachronous metastasis (HR 0.48) and RPM (HR 0.5) were associated with better survival. In conclusion, our results suggest that RPM offers favorable survival rates without increasing post-operative morbidity.

Does Distance to Treatment Affect Mortality Rate for Surgical Oncology Patients?

Using the National Cancer Database (NCDB), we seek to analyze the relationship of patient distance to hospital of treatment on mortality trends after surgery, since patients often travel large distances to referral centers. A retrospective cohort study of the NCDB from 2004 to 2013 was performed, and patients with gastrointestinal, melanoma, and head and neck primary site tumors who underwent surgery were included. We excluded cases with no recorded mortality status or distance from the hospital. A multivariable logistic regression was conducted with adjustments for population density, treating facility location, age, race, gender, education, income, insurance, comorbidities (Charlson-Deyo score), days from diagnosis to treatment, positive margin, tumor stage and grade, and lymph or vascular invasion. A total of 1 424 482 patients were included. Overall median distance to hospital was 9.7 miles (range 4.2-23.7 miles); 696 647 (48.91%) of the sample traveled a distance greater than 10 miles to the institution where the procedure was performed. The multivariable regression analysis demonstrated overall lower mortality for those patients travelling a longer distance to care for multiple tumor types, including: liver (OR .87, .77-.99, P = .032), pancreas (OR .82, .76-.89, P < .001), colon (OR .92, .89-.95, P < .001), rectum (OR .90, .83-.96, P = .003), melanoma (OR .83, .79-.88, P < .001), and tumors of the larynx (OR .80, .69-.94, P = .005). Increased distance traveled for surgical treatment has a significant correlation with decreased odds of mortality for multiple cancers, highlighting the importance of centralized referral patterns for oncology care.

Abstract 6509: Hypofractionated radiotherapy as a vaccine in combination with the TLR7/8 agonist resiquimod leads to significant abscopal tumor response and persistence antitumor immunity

Abstract High doses of ionizing radiation (IR) as delivered with hypofractionated radiotherapy (HFRT) can generate immunogenic cell death and elicit adaptive anti-tumor immunity through cross priming of tumor associated antigens. Resiquimod (R-848), a TLR7/8 agonist, generates a systemic antitumor immunity resulting in tumor regression and enhanced T-cell effector function in cutaneous T-cell lymphomas clinically. We hypothesized that combining HFRT with immunotherapy would boost the antitumorigenic effects of either monotherapies, and if so, could lead to a readily translated clinical modality. B16F10 (murine melanoma) and SCC7 (murine squamous cell carcinoma) cells were used as to grow flank subcutaneous syngeneic tumor models in C57BL/6J and C3H/HeJ mice respectively. Our studies indicate that intratumoral administration of resiquimod combined with HFRT to those tumors leads to a significantly stronger antitumor response than either single treatment in both tumor models. The combination therapy in the primary tumor led to complete tumor regression of the SCC7 tumors in 86% of the treated mice to just 14% rejection in the HFRT group alone. Tumor re-challenge of those mice led to tumor rejection in 83% in the combination group compared to 0% of the HFRT group indicating a potent systemic effect of the combined therapy. Furthermore, the combination therapy led to significant reduction of the index (treated) but moreover in the abscopal (untreated) tumors compared to either monotherapy confirming a systemic antitumor effect in this dual-tumor in vivo model. RT-qPCR analysis revealed an increase expression of Th-1 signature (IFNγ, IL-12, Prf1, GzmB) in both primary and abscopal tumors and flowcytometric analysis of the abscopal B16F10 tumors revealed an increase in CD8+ infiltration. A more thorough flowcytometric analysis following staining with the gp100 tetramer revealed an increased cross-priming against gp100 (PMEL) and enhanced gp100-specific T-cell infiltration within the tumor. Moreover, splenocytes that were re-stimulated in vitro with the gp100 peptide in an IFNγ ELISPOT assay, they increased in mice treated with the dual therapy. Finally, an in vitro study of B16F10 cells, which express TLR7/8 receptors, revealed a substantial increase in IFNβ and CCL5 expression with the dual treatment. Knocking down TLR7 and Tmem173 (STING) in those cells with siRNAs completely reversed the phenotypic IFN type I expression signature. In conclusion, the combination of HFRT with Resiquimod leads to very potent antitumor effects in both primary and abscopal melanoma and squamous cell carcinoma tumors driven by an enhanced systemic immunologic response. Our findings provide the rational for translating therapies combining radiotherapy and intratumoral resiquimod into early-phase clinical trials. Citation Format: Ilias V. Karagounis, Stefano Pierini, Noëlle François, Christoforos Thomas, Xiaowei Xu, Constantinos Koumenis, Andrea Facciabene, Amit Maity. Hypofractionated radiotherapy as a vaccine in combination with the TLR7/8 agonist resiquimod leads to significant abscopal tumor response and persistence antitumor immunity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6509.

39. CHARACTERIZING NOVEL INHIBITORS OF BRAIN METASTASIS-INITIATING CELLS

BACKGROUNDThe incidence of brain metastases (BM) is tenfold higher than primary brain tumors. BM commonly originate from primary lung, breast, and melanoma tumors with a 90% mortality rate within one year of diagnosis. Current standard of care for BM includes surgical resection with concurrent chemoradiation, but does not extend median survival past 16 months, posing a large unmet need to identify novel therapies against BM.METHODSFrom a large in-house biobank of patient-derived BM cell lines, the Singh Lab has generated murine orthotopic patient-derived xenograft (PDX) models of lung, breast, and melanoma BM that recapitulate the stages of BM progression as seen in human patients. Using these three PDX models, we identified a population of “pre-metastatic” brain metastasis-initiating cells (BMICs) that are newly arrived in the brain but have yet to form detectable tumors. Pre-metastatic BMICs are not detectable in human patients but are important therapeutic targets with the potential to prevent BM in at-risk patients.RESULTSRNA sequencing of pre-metastatic BMICs from all three PDX primary tumor models with subsequent Connectivity Map analysis identified novel compounds that have the potential of killing all three types of BMICs. In particular, we identified two compounds that have selective killing of BMICs in vitro from all three primary tumor cohorts while sparing non-cancerous cells. We further characterized their ability to inhibit the self-renewal and proliferative properties of BMICs. Ongoing in vivo work will investigate the compounds’ preclinical utilities in preventing BM.CONCLUSIONIdentification of novel small molecules that target BMICs could prevent the formation of BM completely and dramatically improve the prognosis of at-risk cancer patients.

Characterization of Uveal Melanoma Cell Lines and Primary Tumor Samples in 3D Culture.

PurposeUveal melanoma (UM) typically spreads to the liver, where it is incurable, as there are limited therapeutic interventions available. This study aimed to standardize laboratory methods for generating three-dimensional (3D) spheroids using UM cell lines and primary UM (PUM) samples for use in drug screening.MethodsSix UM cell lines and nine PUM, of differing genetic characteristics were cultured in two dimensions (2D) and three dimensions. 3D spheroid formation and growth were time monitored, and ImageJ software was used to calculate cross-sectional areas. PUM spheroids underwent immunohistochemistry for melanoma markers, nuclear BAP1, and cell proliferation. Chromosomal alterations in patient UM biopsies were compared with the corresponding 3D spheroid. In vitro drug assays testing doxorubicin and selumetinib assessed drug penetration and toxicity after 48 hours using imaging and the CellTiter-Glo 3D Cell Viability Assay.ResultsAll six UM cell lines formed spheroids of varying sizes and compactness; six of the nine PUM samples (67%) also formed spheroids, composed of MelanA+ proliferating melanocytes and admixed macrophages. PUM spheroids were genetically identical to the original sampled tumor. In vitro drug assays showed varying penetrations into UM cell line spheroids, with doxorubicin passing into the spheroid core and selumetinib having an effect largely on peripheral cells. Both drugs caused a dose-dependent reduction in viability of 3D spheroid cells.ConclusionsUM cell lines and PUM samples can successfully generate uniform 3D spheroids. PUM spheroids retain histological and genetic characteristics of the primary tumor. 3D spheroids are an important system for use in future high-throughput drug testing.Translational RelevanceThe use of 3D spheroids allows early-phase drug screening and is an important first step toward treatment personalization for UM patients.

Label-Free Infrared Spectral Histology of Skin Tissue Part II: Impact of a Lumican-Derived Peptide on Melanoma Growth.

Melanoma is the most aggressive type of cutaneous malignancies. In addition to its role as a regulator of extracellular matrix (ECM) integrity, lumican, a small leucine-rich proteoglycan, also exhibits anti-tumor properties in melanoma. This work focuses on the use of infrared spectral imaging (IRSI) and histopathology (IRSH) to study the effect of lumican-derived peptide (L9Mc) on B16F1 melanoma primary tumor growth. Female C57BL/6 mice were injected with B16F1 cells treated with L9Mc (n = 10) or its scrambled peptide (n = 8), and without peptide (control, n = 9). The melanoma primary tumors were subjected to histological and IR imaging analysis. In addition, immunohistochemical staining was performed using anti-Ki-67 and anti-cleaved caspase-3 antibodies. The IR images were analyzed by common K-means clustering to obtain high-contrast IRSH that allowed identifying different ECM tissue regions from the epidermis to the tumor area, which correlated well with H&E staining. Furthermore, IRSH showed good correlation with immunostaining data obtained with anti-Ki-67 and anti-cleaved caspase-3 antibodies, whereby the L9Mc peptide inhibited cell proliferation and increased strongly apoptosis of B16F1 cells in this mouse model of melanoma primary tumors.

The Potential Role of Selected miRNA in Uveal Melanoma Primary Tumors as Early Biomarkers of Disease Progression.

Uveal melanoma (UM) is the most common primary tumor of the eye diagnosed in adults, associated with a high risk of metastasis and thereby, poor prognosis. Among known risk factors for the development of metastatic disease is the loss of BAP1 expression and chromosome 3 monosomy in the primary tumor. However, the expression levels of specific micro RNAs (miRNA) in tumor tissue may also serve as a valuable marker for determining the risk of metastatic disease in patients with primary uveal melanoma. In our study, we analyzed the miRNA expression data of cases selected from The Cancer Genome Atlas study on uveal melanoma, and determined a panel of 15 miRNAs differentially expressed between patients with primary and metastatic disease. Next, 6 miRNAs were validated on a group of 46 tumor samples from primary and metastatic patients. We have shown, that expression of hsa-miR-592, hsa-miR-346, and hsa-miR-1247 was significantly increased, while hsa-miR-506 and hsa-miR-513c were decreased in the tumors of patients with metastatic disease. Hsa-miR-196b expression did not differ between the two subgroups, however, we showed significant correlation with BAP1 expression. Moreover, hsa-miR-592 also showed correlation with monosomy 3 tumors. Gene ontology analysis revealed involvement of those miRNAs with cellular processes mediating the metastatic process. Our results showed that miRNAs play an important role in the deregulation of several oncogenic pathways in UM and can, thereby, promote metastatic spread to distant organs. Moreover, differentially expressed miRNAs may be used as an interesting biomarker for the assessment of metastatic risk in uveal melanoma patients.

Enhanced Phenotype Definition for Precision Isolation of Precursor Exhausted Tumor-Infiltrating CD8 T Cells.

In the context of adoptive T cell transfer (ACT) for cancer treatment, it is crucial to generate in vitro large amounts of tumor-specific CD8 T cells with high potential to persist in vivo. PD-1, Tim3, and CD39 have been proposed as markers of tumor-specific tumor-infiltrating CD8 T lymphocytes (CD8 TILs). However, these molecules are highly expressed by terminally differentiated exhausted CD8 T cells (Tex) that lack proliferation potential. Therefore, optimized strategies to isolate tumor-specific TILs with high proliferative potential, such as Tcf1+ precursor exhausted T cells (Tpe) are needed to improve in vivo persistence of ACT. Here we aimed at defining cell surface markers that would unequivocally identify Types for precision cell sorting increasing the purity of tumor-specific PD-1+ Tcf1+ Tpe from total TILs. Transcriptomic analysis of Tpe vs. Tex CD8 TIL subsets from B16 tumors and primary human melanoma tumors revealed that Tpes are enriched in Slamf6 and lack Entpd1 and Havcr2 expression, which encode Slamf6, CD39, and Tim3 cell surface proteins, respectively. Indeed, we observed by flow cytometry that CD39– Tim3– Slamf6+ PD-1+ cells yielded maximum enrichment for tumor specific PD-1+ Tcf1+ OT1 TILs in B16.OVA tumors. Moreover, this population showed higher re-expansion capacity upon an acute infection recall response compared to the CD39+ counterparts or bulk PD-1+ TILs. Hence, we report an enhanced sorting strategy (CD39– Tim3– Slamf6+ PD-1+) of Tpes. In conclusion, we show that optimization of CD8 TIL cell sorting strategy is a viable approach to improve recall capacity and in vivo persistence of transferred cells in the context of ACT.

TERT, BRAF, and NRAS Mutational Heterogeneity between Paired Primary and Metastatic Melanoma Tumors

Mutational heterogeneity can contribute to therapeutic resistance in solid cancers. In melanoma, the frequencies of intertumoral and intratumoral heterogeneity are controversial. We examined mutational heterogeneity within individual patients with melanoma using multiplatform analysis of commonly mutated driver and nonpassenger genes. We analyzed paired primary and metastatic tumors from 60 patients and multiple metastatic tumors from 39 patients whose primary tumors were unavailable (n= 271 tumors). We used a combination of multiplex SNaPshot assays, Sanger sequencing, mutation-specific PCR, or droplet digital PCR to determine the presence of BRAFV600, NRASQ61, TERT-124C>T, and TERT-146C>T mutations. Mutations were detected in BRAF (39%), NRAS (21%), and/or TERT (78%). Thirteen patients had TERTmutant discordant tumors; seven of these had a single tumor with both TERT-124C>T and TERT-146C>T mutations present at different allele frequencies. Two patients had both BRAF and NRAS mutations; one had different tumors and the other had a single tumor with both mutations. One patient with a BRAFmutant primary lacked mutant BRAF in at least one of their metastases. Overall, we identified mutational heterogeneity in 18 of 99 patients (18%). These results suggest that some primary melanomas may be composed of subclones with differing mutational profiles. Such heterogeneity may be relevant to treatment responses and survival outcomes.

MX 2 is a novel regulator of cell cycle in melanoma cells.

MX2 protein is a dynamin-like GTPase2 that has recently been identified as an interferon-induced restriction factor of HIV-1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome-wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context-dependent.

Deep Learning Based on Standard H&E Images of Primary Melanoma Tumors Identifies Patients at Risk for Visceral Recurrence and Death.

Biomarkers for disease-specific survival (DSS) in early-stage melanoma are needed to select patients for adjuvant immunotherapy and accelerate clinical trial design. We present a pathology-based computational method using a deep neural network architecture for DSS prediction. The model was trained on 108 patients from four institutions and tested on 104 patients from Yale School of Medicine (YSM, New Haven, CT). A receiver operating characteristic (ROC) curve was generated on the basis of vote aggregation of individual image sequences, an optimized cutoff was selected, and the computational model was tested on a third independent population of 51 patients from Geisinger Health Systems (GHS). Area under the curve (AUC) in the YSM patients was 0.905 (P < 0.0001). AUC in the GHS patients was 0.880 (P < 0.0001). Using the cutoff selected in the YSM cohort, the computational model predicted DSS in the GHS cohort based on Kaplan-Meier (KM) analysis (P < 0.0001). The novel method presented is applicable to digital images, obviating the need for sample shipment and manipulation and representing a practical advance over current genetic and IHC-based methods.

Semaphorin-5A downregulation is associated with enhanced migration and invasion of BRAF-positive melanoma cells under vemurafenib treatment in melanomas with heterogeneous BRAF status.

Tumor heterogeneity affects the efficacy of anticancer treatment as tumor subclones with distinct molecular patterns may be present within one tumor, leading to differing sensitivities to chemotherapeutic agents. In the present study, six melanoma tissue fragments were obtained from different parts of tumor of four patients and then the effect of vemurafenib treatment on biological characteristics and molecular processes of cell cultures was estimated by using MTT-test, apoptosis, migration and invasion assays, PCR real time. There was different BRAF status determined between cells derived from the central and peripheral regions of primary melanoma tumors. BRAF-positive melanoma cells showed an increased apoptotic rate under vemurafenib treatment, as well as increased migration and invasion rates, whereas BRAF-negative melanoma cells did not exhibit such tendency. Furthermore, semaphorin-5A levels were diminished in BRAF-positive cells, but not in BRAF-negative ones, which could be related to increased migration and invasion. Melanoma cells derived from different regions of the same tumor may differ by mutations status, molecular processes and biological response to target therapy. The downregulation of semaphorin-5A may be involved in divergent effects of anticancer agents on tumor cell biology.

Abstract 3419: Sex, mutations, and mortality in early stage melanoma

Abstract Our goal is to investigate the relationship between sex, tumor mutation burden (TMB) and survival in untreated primary melanoma tumors, using data collected in an ongoing large P01 study on the Integration of Clinical and Molecular Biomarkers for Melanoma Survival (InterMEL). Here we present preliminary findings on sex, survival, and somatic TMB from 60 Stage II and Stage III primary melanoma tumors, none of which have received immunotherapy using the MSK IMPACT™ next generation sequencing assay. Our preliminary analysis includes 30 patients who died within 5 years (median survival of 31 months) and 30 patients who lived for more than 5 years since diagnosis (median follow-up of 86 months). We found the expected relationship between TMB and better survival in a univariate Cox regression analysis (HR=0.43, 95% CI=0.19 to 0.97, P=0.04), but no evidence of association between TMB and sex (Wilcoxon rank-sum test P=0.7) or any relationship between sex and survival (logrank test P=0.42). Our study is the first to investigate the relationship between sex, tumor mutational burden, and mortality in an early stage primary cohort that has not received immunotherapy. In our small sample, we observed the expected protective effect of TMB on survival, but no evidence of gender differences in TMB or survival. This is surprising given the robust, consistent, and well-documented female survival advantage (Roh et al., 2015; de Vries et al., 2008), and merits further investigation. Our results are an important first step to increasing our understanding of the relationship between mutational burden, survival, and biological sex. References Roh, M.R., Eliades, P., Gupta, S., Grant-Kels, J.M., and Tsao, H. (2015). Cutaneous melanoma in women. Int. J. Womens Dermatol. 1, 21–25. de Vries, E., Nijsten, T.E.C., Visser, O., Bastiaannet, E., van Hattem, S., Janssen-Heijnen, M.L., and Coebergh, J.-W.W. (2008). Superior survival of females among 10 538 Dutch melanoma patients is independent of Breslow thickness, histologic type and tumor site. Ann. Oncol. 19, 583–589. Citation Format: Matthew R. Schwartz, Li Luo, Marianne Berwick. Sex, mutations, and mortality in early stage melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3419.

Abstract 553: Melanoma subtypes that emerge during adaptive resistance to therapy are targets for bispecific T cell engager (BiTE®) antibody constructs directed to CDH19 and DLL3

Abstract The treatment landscape for metastatic melanoma has changed considerably over the last several years with the approval of targeted therapy, such as BRAF inhibitors, and immunotherapy, such as immune checkpoint inhibitors that block cytotoxic T lymphocyte associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). However, approximately 40% of patients do not respond to the current standard of care, and up to one-third of patients who initially respond to therapy can relapse. Novel therapies that act by a distinct mechanism of action may be needed to increase durable response rates in metastatic melanoma. Bispecific T cell engager (BiTE®) antibody constructs directly connect T cells to tumor cells and redirect T cell lysis to achieve tumor cell killing. BiTE®-mediated cytotoxicity is not dependent on a specific T cell receptor or peptide-MHC complex and has the potential to circumvent common immune resistance mechanisms. We have generated a BiTE® with half-life extension (HLE) that targets the atypical cadherin CDH19 on melanoma cells and CD3 on T cells (anti-CDH19/CD3 HLE BiTE®). We have also generated an HLE BiTE® that targets delta-like ligand 3 (DLL3) on tumor cells and CD3 on T cells, anti-DLL3/CD3 HLE BiTE®, or AMG 757. CDH19 and DLL3 are expressed on about half of primary and metastatic melanoma tumors prior to treatment. In this study, we investigated the expression of CDH19 and DLL3 and the activity of the corresponding BiTE® antibody constructs in a panel of 14 melanoma cell lines that were derived from patients that were resistant to either targeted therapy or immune checkpoint therapy and expanded the spectrum of recently defined multistage differentiation melanoma subtypes from melanocytic, transitional, neural crest-like and de-differentiated subtypes (Tsoi et al. Cancer Cell 2017). The cell surface expression of CDH19 and DLL3 was determined by flow cytometry. We then used T cell-dependent cytotoxicity (TDCC) assays to assess the activity of the anti-CDH19/CD3 HLE BiTE® and AMG 757 against the cell lines. 13/14 (93%) of the treatment-resistant melanoma cell lines expressed CDH19 on the cell surface, and 9/14 (64%) of the cell lines expressed cell surface DLL3. The anti-CDH19/CD3 HLE BiTE® and AMG 757 demonstrated potent cell killing of CDH19-positive or DLL3-positive melanoma cells, respectively, in TDCC assays, with EC50 values in the low picomolar range. These data show that melanoma cells that become resistant to targeted therapy and/or immune checkpoint blockade can be effectively targeted with BiTE® antibody constructs and suggest that the anti-CDH19/CD3 HLE BiTE® and AMG 757 may be potential novel therapies for treatment of relapsed and/or refractory metastatic melanoma. Citation Format: Julie M. Bailis, Fei Lee, Michael Giffin, Paul Hughes, Jennifer Tsoi, Lidia Robert, Thomas G. Graeber, Antoni Ribas, Angela Coxon. Melanoma subtypes that emerge during adaptive resistance to therapy are targets for bispecific T cell engager (BiTE®) antibody constructs directed to CDH19 and DLL3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 553.

Inhibition of Pannexin 1 Channels Reduces Tumorigenic Properties of Melanoma

Melanoma is the deadliest skin cancer and its incidence is still on the rise. Despite new advances with immunotherapy drugs, nearly 70% of patients still do not respond to treatments, underscoring the importance of finding new treatment targets and combination therapies. Pannexin 1 (PANX1 human; Panx1 mouse) is a channel‐forming glycoprotein expressed in many mammalian organs and tissues including the skin at early stages of development. Pannexin 1 channels allow the passage of ions and important signaling molecules up to 1 kDa, including ATP and other metabolites. We previously found that a knockdown of Panx1 resulted in reduced tumorigenic properties of mouse melanoma cell lines in vitro. In this study, we found that PANX1 is highly expressed in human melanoma tumors compared to normal skin. Using Western blot and immunohistochemistry, we showed that high PANX1 levels are present in melanoma tumors at all disease stages, as well as in patient‐derived cells and established melanoma cell lines. We demonstrated that inhibiting PANX1 function using shRNA or channel blockers, such as Carbenoxolone (CBX) and Probenecid (PBN), significantly reduced cell growth and cell migration, and substantially increased the production of melanin (used as a differentiation marker) in A375‐P and A375‐MA2 human melanoma cells. In addition, treatment with CBX or PBN in A375‐MA2 cells xenografted onto the chorioallantoic membrane of a chicken embryo model, significantly reduced primary melanoma tumor weight as well as tumor invasion. Blocking PANX1 channels using PBN reduced ATP release in A375‐P melanoma cells, suggesting a role for PANX1 in regulating the tumor microenvironment through purinergic signaling at the cell surface. In addition, our findings from cell‐surface biotinylation assays indicate that there is also a significant intracellular pool of PANX1 in melanoma cells that likely modulates signaling pathways such as Wnt/β‐catenin, since β‐catenin levels are significantly decreased upon shRNA knockdown of PANX1 in melanoma cells. Collectively, our findings suggest that PANX1 is a novel modulator of tumorigenic properties in human melanoma cells, contributing to signaling pathways that regulate melanoma progression and highlights PANX1 as a potential target for therapeutic intervention.Support or Funding InformationFunded by a Canadian Institutes of Health Research (CIHR) Project Grant to Silvia PenuelaThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Histopathologic discordance in melanoma can have substantial impacts on patient care

Recommended guidelines for sentinel lymph node biopsy, follow-up, and surveillance for cutaneous melanoma are based upon clinicopathologic staging. In effect, the accuracy of melanoma staging to estimate metastatic risk is critical to subsequent care, neither under-treating or over-treating the patient based on their tumor. Traditional staging continues to evolve based on additional data regarding clinicopathologic features and clinical outcomes. However, such features are subject to inter-observer variability, which puts a limit on their ability to improve prognostication. Reported discordance rates between initial and subsequent pathology review consistently impact both staging and disease management. Newer molecular techniques, such as gene expression profiling, can be used to help define the biology of the primary melanoma tumor and the best course of action after definitive surgical treatment.

Performance of a 31-gene expression profile test in cutaneous melanomas of the head and neck.

BackgroundWe report the performance of a gene expression profile test to classify the recurrence risk of cutaneous melanoma tumors of the head and neck as low‐risk Class 1 or high‐risk Class 2.MethodsOf note, 157 primary head and neck cutaneous melanoma tumors were identified. Survival analyses were performed using Kaplan‐Meier and Cox methods.ResultsGene expression profile class and node status stratified tumors into significantly different 5‐year survival groups by Kaplan‐Meier method (P < .0001 for all end points), and both were independent predictors of recurrence in multivariate analysis. Overall, 74% of distant metastases and 88% of melanoma‐specific deaths had Class 2 risk.ConclusionThe gene expression profile test identifies cases at increased risk for metastasis and death independent of a clinically or pathologically negative nodal status, suggesting that incorporation of this molecular tool could improve clinical management of patients with head and neck cutaneous melanoma, especially in those with a negative sentinel lymph node biopsy.