Table S3. Clinical outcomes by PTEN status in patients with BRAF V600 mutations

Table S2. Multivariate analysis of overall survival and time to CNS metastasis in all stage IIIB/C melanoma patients

Table S4. Clinical outcomes by PTEN status in patients without BRAF V600 or NRAS mutations

Table S1. Demographics, tumor characteristics, and clinical outcomes by tumor mutation status

Supplemental figure legends. Legends for supplemental figures

Supplementary Figure 1. Representative images of PTEN IHC results in FFPE melanoma tissue samples.

A 59-year-old male presented with 1 month of progressive dyspnea, 30-lb weight loss, and skin changes on the digits of the hands. In the 4 weeks prior to admission, he was admitted and treated twice for pneumonia at another hospital and received intravenous (IV) vancomycin, ceftriaxone, and azithromycin for a total of 10 days. After admission, he underwent computed tomography imaging of chest, which revealed findings suggestive of interstitial lung disease but given the fact that infection was not ruled out, empiric antibiotics were initiated. The skin lesions on the fingers were felt to be consistent with Gottron’s papules, and his overall constellation of findings were felt to be consistent with dermatomyositis (DM). Over the following 3 days, he developed diffuse, violaceous skin lesions, elevation of liver transaminases, and severe thrombocytopenia. The skin lesions progressed to epidermal necrosis. He developed erosions of the oral mucosa and scrotum. Before skin biopsy results were finalized, IV immunoglobulin and IV dexamethasone were started empirically for suspected DM and immune-mediated thrombocytopenia. His laboratory abnormalities normalized within a week. Biopsy results of the skin were consistent with Stevens-Johnson syndrome (SJS). Autoantibody test for anti-MDA5 were positive, confirming a diagnosis of anti-MDA5 associated DM. Subsequent development of SJS was likely due to antibiotic exposure in the preceding month. Simultaneous development of anti-MDA5 DM and SJS raises the question of a link between the 2 conditions. To our knowledge, this is the first reported association of these 2 conditions reported in the literature.

199 Background: KIT-mutated MM is a rare melanoma entity. Response rates of KIT-mutated MM to anti CTLA-4 and anti PD-1 have not previously been reported. Methods: A single-institution retrospective review identified patients (pts) with KIT-mutated MM treated with at least two doses of anti CTLA-4 or anti PD-1 between 2008-2017 with response determined by immune-related response criteria (irRC). Overall survival (OS) was from ICI start to death or last follow-up date, and progression-free-survival (PFS) was from ICI start to date of progression or death if pts deceased without disease progression. Results: Thirty-five pts treated with ipilimumab were identified. Median follow-up was 63.7 months. Median age at MM diagnosis was 65.4 years (range 26-81) and 54.3% were male. Subtypes were 51.4% mucosal, 22.9% acral-lentiginous, 22.9% cutaneous, and 2.9% unknown primary. KIT mutations were 57.1% exon 11, 25.7% exon 17, 11.4% exon 13, and 5.7% exon 2. Anatomical M stage at treatment initiation was 11.4% M1a, 34.3% M1b, and 54.3% M1c. Median OS was 11.8 months (8.4-35.6) and PFS was 3.0 months (2.8-5.8). Responses to ipilimumab were 8.6% complete response (CR), 11.4% partial response (PR), 28.6% stable disease (SD), and 51.4% progressive disease (PD) for a 48.6% clinical response rate (CRR). Twenty pts treated with anti PD-1 were identified. Median follow-up was 7.9 months. Median age at MM diagnosis was 66.7 years (range 42.6-86.8) and 70% were male. Subtypes were 40% mucosal, 30% cutaneous, 20% acral-lentiginous, and 10% unknown primary. KIT mutations were 45% exon 11, 35% exon 17, 10% exon 2, 5% exon 13 and and 5% exon 10. Anatomical M stage at treatment initiation was 10% M1a, 15% M1b, and 75% M1c. Median OS was 22.5 months (7.4-NA ) and PFS was 3.2 months (2.7-NA). Responses to anti PD-1 were 10.0% CR, 25.0% PR, 20.0% SD, and 45.0% PD for a 55.0% CRR. For both anti CTLA-4 and anti PD-1 treated cohorts, univariate analysis demonstrated no statistical significance between tumor response to ICI and clinical pt characteristics or KIT mutation exon. Conclusions: KIT-mutated MM demonstrated CRR of 48.6-55.0% to ICI. There was no correlation between pt characteristics or KIT exon mutation and response to therapy; however, analysis was limited by sample size.

Extramammary Paget disease (EMPD) is a locally aggressive cutaneous malignancy that usually arises in anogenital or axillary skin. Immune checkpoint inhibitors targeting programmed cell death receptor (PD-1) and/or its ligand (PD-L1) are approved for the treatment of several types of cancer, and response to these generally correlates with increased PD-L1 expression by tumor cells. The expression of PD-L1 and composition and density of the tumor-associated immune infiltrate in EMPD have been little studied. To determine whether EMPD might be amenable to immune checkpoint blockade, we analyzed the expression of PD-1 and PD-L1 and the composition and density of the tumor-associated immune infiltrate in EMPD and evaluated associations between biomarker expression and clinicopathologic parameters. Twenty-one EMPD tumors were evaluated for tumor cell PD-L1 expression and for relative expression and distribution of CD3, CD8, PD-1, and PD-L1 in the tumor-associated immune infiltrate by using a combination of visual and image analysis (Aperio ImageScope). In addition, PD-L1 expression was assessed in 10 cases of mammary Paget disease (MPD). In EMPD cases, PD-L1 was expressed by tumor cells (3/21; 14%) and the tumor-associated immune infiltrate (15/21; 71%), and PD-1 was expressed by the tumor-associated immune infiltrate in all cases analyzed (18/18). However, PD-L1 expression by EMPD tumor cells did not correlate with the density of CD3-, CD8-, or PD-1-positive cells in the tumor-associated immune infiltrate or other clinicopathologic parameters. Furthermore, the density of CD3, CD8, PD-1, and PD-L1 in the tumor-associated immune infiltrate did not correlate with any clinicopathologic parameters evaluated with the exception that CD3 positive values were significantly higher in patients who were still alive (median, 1310 cells/mm2; range, 543–2115;) than in those who died (median, 611 cells/mm2; range, 481–908; p = 0.049). In all MPD cases, PD-L1 was absent in tumor cells but present in the tumor-associated immune infiltrate, and PD-L1 expression in lymphocytes was lower in patients with HER2/neu-positive than in those with HER2/neu-negative disease (p = 0.07). Our findings raise the possibility of therapeutic targeting of the PD-1/PD-L1 axis in EMPD.

BRAFV600, NRAS, TP53, and BRAFNon‐V600 are among the most common mutations detected in non‐acral cutaneous melanoma patients. Although several studies have identified clinical and pathological features associated with BRAFV600 and NRAS mutations, limited data are available regarding the correlates and significance of TP53 and BRAFNon‐V600 mutations.

Numerous cutaneous manifestations have been associated with use of BRAF inhibitors, including two previously reported cases of granuloma annulare (GA) eruptions associated with vemurafenib therapy. Both of these patients were being treated for metastatic melanoma. In this report, we describe the case of a 71-year-old man who developed classic GA lesions while being treated with vemurafenib monotherapy for nonmelanoma cancer, specifically metastatic lung adenocarcinoma positive for BRAF V600 mutation. J Drugs Dermatol. 2017;16(10):1050-1052..

While clinical outcomes following immunotherapy have shown an association with tumor mutation load using whole exome sequencing (WES), its clinical applicability is currently limited by cost and bioinformatics requirements.

Numerous signaling pathways are involved in the molecular pathogenesis of melanoma. Two of the most prominent pathways include the RAS-RAF-MEK-ERK pathway and the PI3K-AKT pathway. While targeted inhibition of BRAF/MEK have resulted in improved survival in patients with BRAF V600-mutated melanoma, therapeutic resistance is often the end result. The PI3K-AKT pathway plays a significant role in BRAF-/MEK-inhibitor resistance in melanoma patients and may represent a crucial target for combination therapy. This chapter will describe the components of the PI3K-AKT pathway, highlight their role in cellular physiology and melanoma pathogenesis, and examine their potential for molecular-targeted therapy.

Somatic copy number alterations (SCNAs) affecting oncogenic drivers have a firmly established role in promoting cancer. However, no agreed-upon standard exists for calling locus-specific amplifications and deletions in each patient sample. Here, we report the correlative analysis of copy number amplitude and length with gene expression across 6,109 samples from The Cancer Genome Atlas (TCGA) dataset across 16 cancer types. Using specificity, sensitivity, and precision-based scores, we assigned optimized amplitude and length cutoffs for nine recurrent SCNAs affecting known oncogenic drivers, using mRNA expression as a functional readout. These cutoffs captured the majority of SCNA-driven, highly-expression-altered samples. The majority of oncogenes required only amplitude cutoffs, as high amplitude samples were almost invariably focal; however, CDKN2A and PTEN uniquely required both amplitude and length cutoffs as primary predictors. For PTEN, these extended to downstream AKT activation. In contrast, SCNA genes located peri-telomerically or in fragile sites showed poor expression-copy number correlations. Overall, our analyses identify optimized amplitude and length cutoffs as efficient predictors of gene expression changes for specific oncogenic SCNAs, yet warn against one-size-fits-all interpretations across all loci. Our results have implications for cancer data analyses and the clinic, where copy number and mutation data are increasingly used to personalize cancer therapy.

Capsular nevi (CN) are clusters of benign melanocytes situated in the capsule of lymph nodes and occur in up to 20% of lympadenectomy specimens. The molecular profile of CN in relation to prognostic parameters in patients with primary cutaneous melanoma (PCM) has not been previously investigated. We assessed BRAF V600E mutation by immunohistochemistry (IHC) in the CN of sentinel lymph nodes (SLN) in PCM patients and correlated the findings with demographic characteristics, PCM histopathologic and molecular features, and clinical outcome parameters. Seventy-eight cases of CN involving SLN of PCM patients were evaluated for BRAF V600E mutation by IHC. The results were correlated with patient demographics, PCM histopathologic and molecular features, and outcome measures. Thirty-six (46%) of 78 CN cases expressed BRAF V600E mutation by IHC. Nineteen (53%) of those BRAF-positive CN cases were from patients with at least American Joint Committee on Cancer stage II melanoma, whereas 62% of BRAF-negative CN cases (26/42) were from patients with stage I melanoma (P = .013). Twelve (33%) of the 36 BRAF-positive CN cases had metastatic melanoma involving lymph nodes, compared with 14% (6/42) of BRAF-negative CN cases (P = .061). CN mutation status was not associated with patient demographics, histopathologic or molecular features of the PCM, or survival outcomes. A high percentage of CN identified in the SLN of patients with PCM harbor BRAF V600E mutation. Positive mutation was associated with adverse clinicopathological parameters, specifically increased tumor stage and lymph node metastasis. These findings suggest that BRAF V600E mutation in CN of SLN may be useful as an adverse predictive biomarker in patients with melanoma.

9535Background: Our previous analysis of advanced CM pts and the TCGA demonstrated that BRAFV600 (V600), NRAS, TP53 and BRAFNon-V600 (NonV600) muts are common in CMs. As V600 and NRAS muts are both...

9057 Background: Cutaneous melanomas (CM) have a high rate of somatic mutations. We analyzed the prevalence and correlations of mutations in 201 cancer-related genes to identify clinically significant molecular events in a cohort of pts with advanced melanoma. Methods: Next generation sequencing (NGS) of all exons of 201 cancer-associated genes was performed on tumor and germline DNA from pts with a known primary non-acral CM enrolled in an IRB-approved research protocol. Demographics, primary tumor characteristics, date of diagnosis of stage IV disease, and overall survival (OS) were collected for all pts. The significance of associations between these characteristics and somatic mutations was analyzed by a variety of statistical methods. Results: NGS was performed on 106 advanced melanoma pts with a known non-acral primary CM. Genes with a mutation rate > 10% (n = 74) were assessed for clinical associations. NF1 (OR 12.9, p = 0.002) and PCLO (OR 3.1, p = 0.01) mutations were associated with male gender, while BRAF V600 mutations were more common in women (OR 0.38, p = 0.03). BRAF mutations were associated with younger age at diagnosis (p < 0.0001), and 12 genes were associated with older age. Mutations in 5 genes were associated with increased primary tumor Breslow thickness, most significantly MET (mean 8.3 vs 3.5 mm, p = 0.005). Among all pts with NGS data with distant metastases (n = 88), NRAS mutation was associated with significantly shorter OS from the diagnosis of stage IV (HR 3.2, p = 0.007), while mutations in BRAF (HR 0.35, p = 0.02) and 12 other genes were associated with significantly longer OS (HR 0.12 to 0.40). Among stage IV pts without BRAF V600 mutations (n = 52), NRAS mutation was again a significant predictor of shorter OS (HR 2.8, p = 0.04), and mutations in 12 other genes correlated with increased OS (HR 0.11 to 0.37). Conclusions: Somatic mutations in several genes in advanced non-acral CM pts were significantly associated with disease characteristics and clinical outcomes, and merit analysis in additional cohorts of pts. Analysis of copy number variations will be included in the final report for the cohort.

e20002 Background: The increasing availability of molecular profiling techniques amenable to FFPE tissue presents a key opportunity to study the molecular basis of melanoma. In order to optimize our methodological approaches for FFPE melanoma specimens for planned integrative analyses, we compared various DNA and RNA analyte extraction and melanin removal methods on analyte yield, quality, and molecular profiles. Methods: Primary (n = 2) and metastatic (n = 6) FFPE melanoma specimens with varying degrees of melanin pigmentation (3-none/minimal, 3-moderate, 2-high) were used for systematic testing of 3 RNA and 4 DNA standard isolation protocols (SIPs). The SIPs were used in combination with four potential methods of melanin removal (Zymo column [Zymo], CTAB, both, none). RNA expression was analyzed using a Nanostring 787 custom code set, and DNA was analyzed by next-generation sequencing (n = 202 targeted genes panel [T200]). Results: Significant differences in total RNA yield/mg tumor were observed between RNA SIPs and melanin removal methods. Although all (96/96) RNA analytes passed quality control (QC) regardless of RNA SIP, melanin removal method, or melanin content, Nanostring gene counts were significantly lower for RNA using 1 SIP. Comparison of analytes from the remaining 2 SIPs and 4 melanin removal methods revealed no significant differences in gene counts, hierarchical clustering results, or variability in gene expression above a conservative multiplicity-adjusted threshold (Bonferroni, 0.05/770). For DNA isolation, only 1 SIP consistently yielded sufficient DNA from highly pigmented specimens. DNA analytes that passed QC (44 of 64) were run on the T200 NGS panel. No differences between SIPs or melanin removal methods were observed for NGS mutation calls or coverage depth. Conclusions: In these studies, melanin did not affect Nanostring-based RNA profiling. The selected SIPs for DNA (QiAmp, Zymo) and RNA (HighPure, Zymo) yielded the best analyte quality and quantity from FFPE melanoma tumors for NGS and Nanostring-based profiling, and provide a robust framework for our ongoing and planned large scale integrated molecular profiling efforts in melanoma.