Abstract Desmoplastic melanomas (DMs) comprise 4% of the overall melanoma burden and have a 5-year survival rate of 85%. DMs are dermal tumors characterized by spindled melanocytes situated within abundant desomplastic stroma. These unusual histological features commonly lead to misdiagnosis. Currently, there are no known genetic drivers. A better understanding of the underlying biology of desmoplastic melanoma would provide biomarkers and therapeutic opportunities. Towards this goal, we performed low-coverage genome and high-coverage exome sequencing of 20 DMs in a discovery cohort, followed by targeted sequencing of 293 candidate genes on a validation cohort of 42 cases. Additionally, high-resolution aCGH was performed on samples from both cohorts. A high mutation burden (median 62 mutations/Mb) ranked desmoplastic melanoma among the most highly mutated cancers sequenced to date. Mutation patterns strongly indicate that UV-radiation is the dominant mutagen and implicate a superficially located cell of origin despite their predominantly intradermal presentation. Novel alterations included recurrent promoter mutations and amplification of NF-kappa B inhibitor epsilon, NFKBIE (IkBϵ) in 14.5% of samples. The promoter mutations typically affect both alleles and occur over a highly conserved DNA region. The mutations are predicted to disrupt a canonical Ets Like Factor 1 (ELF1) binding site. In total, these data imply aberrant NF-kappa B signaling as a pathogenic feature of desmoplastic melanoma. Commonly mutated oncogenes in melanomas, in particular BRAF V600E and NRAS Q61K/R, were absent. Instead, other genetic alterations known to activate the MAPK and PI3K signaling cascades were identified in 73% of samples, affecting NF1, CBL, ERBB2, MAP2K1, MAP3K1, BRAF, EGFR, PTPN11, MET, RAC1, SOS2, NRAS, and PIK3CA. Rb and p53 pathway alterations occurred respectively in 71% and 66% of tumors, affecting RB1, FBXW7, CDK4, PPP6C, CCND1, CDKN2A, TP53, and MDM2. Finally, TERT promoter mutations or amplifications occurred in 90% of tumors. The consequences of the mutations on protein expression levels was confirmed by immunostaining for NF1, EGFR, Rb, CDK4, CCND1, p16, p53, and Mdm2. Collectively, many of these oncogenic mutations are potentially druggable. In conclusion, desmoplastic melanomas harbor distinct genetic alterations that explain their unique biology, and this study illuminates genetic biomarkers and nominates targets for therapeutic intervention. Citation Format: Alan H. Shain, Maria Garrido, Thomas Botton, Eric Talevich, Iweh Yeh, Zack Sanborn, Jongsuk Chung, Nicholas Wang, Hojabr Kakavand, Graham Mann, John Thompson, Thomas Wiesner, Ritu Roy, Adam Olshen, Alexander Gagnon, Joe Gray, Nam Huh, Joe Hur, Klaus Busam, Richard Scolyer, Raymond Cho, Rajmohan Murali, Boris Bastian. Exome sequencing of desmoplastic melanoma reveals recurrent NFKBIE promoter mutations and diverse MAPK/PI3K pathway activating mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2968. doi:10.1158/1538-7445.AM2015-2968

Repeat tumor biopsies to study genomic changes during therapy are difficult, invasive and data are confounded by tumoral heterogeneity. The analysis of circulating tumor DNA (ctDNA) can provide a non-invasive approach to assess prognosis and the genetic evolution of tumors in response to therapy. Mutation-specific droplet digital PCR was used to measure plasma concentrations of oncogenic BRAF and NRAS variants in 48 patients with advanced metastatic melanoma prior to treatment with targeted therapies (vemurafenib, dabrafenib or dabrafenib/trametinib combination) or immunotherapies (ipilimumab, nivolumab or pembrolizumab). Baseline ctDNA levels were evaluated relative to treatment response and progression-free survival (PFS). Tumor-associated ctDNA was detected in the plasma of 35/48 (73%) patients prior to treatment and lower ctDNA levels at this time point were significantly associated with response to treatment and prolonged PFS, irrespective of therapy type. Levels of ctDNA decreased significantly in patients treated with MAPK inhibitors (p < 0.001) in accordance with response to therapy, but this was not apparent in patients receiving immunotherapies. We show that circulating NRAS mutations, known to confer resistance to BRAF inhibitors, were detected in 3 of 7 (43%) patients progressing on kinase inhibitor therapy. Significantly, ctDNA rebound and circulating mutant NRAS preceded radiological detection of progressive disease. Our data demonstrate that ctDNA is a useful biomarker of response to kinase inhibitor therapy and can be used to monitor tumor evolution and detect the early appearance of resistance effectors.

BRAF kinase inhibitors have dramatically affected treatment of BRAF(V600E) (/) (K)-driven metastatic melanoma. Early responses assessed using [(18)F]fluorodeoxyglucose uptake-positron emission tomography (FDG-PET) have shown dramatic reduction of radiotracer signal within 2 weeks of treatment. Despite high response rates, relapse occurs in nearly all cases, frequently at sites of treated metastatic disease. It remains unclear whether initial loss of (18)FDG uptake is due to tumor cell death or other reasons. Here, we provide evidence of melanoma cell volume reduction in a patient cohort treated with BRAF inhibitors. We present data demonstrating that BRAF inhibition reduces melanoma glucose uptake per cell, but that this change is no longer significant following normalization for cell volume changes. We also demonstrate that volume normalization greatly reduces differences in transmembrane glucose transport and hexokinase-mediated phosphorylation. Mechanistic studies suggest that this loss of cell volume is due in large part to decreases in new protein translation as a consequence of vemurafenib treatment. Ultimately, our findings suggest that cell volume regulation constitutes an important physiologic parameter that may significantly contribute to radiographic changes observed in clinic.

TPS9600 Background: The outcome of high risk stage IIIb and IIIc melanoma patients is poor, with a 5 year overall survival (OS) rate of &lt; 50%. Adjuvant high dose IPI significantly improves 5 year progression free survival (PFS) and OS. In stage IV patients the combination of IPI and NIVO improves response rates (RR) and PFS compared to monotherapy, but at cost of higher toxicity. Neo-adjuvant treatment may be a favorable approach as immune checkpoint inhibition (ICI) is of greatest value at the moment of TCR triggering and therefore dependent on the amount of antigen present. The phase Ib OpACIN study comparing neo-adjuvant and adjuvant IPI and NIVO showed that neo-adjuvant treatment is feasible as all patients underwent surgery on time. The neo-adjuvant pathological RR was 80%, although 18/20 patients (90%) stopped early due to ≥1 grade 3 or 4 immune-related adverse events (irAEs). To date (median follow-up 45 wks), none of the 8 responders in the neo-adjuvant arm have relapsed. This raises the question whether the neo-adjuvant IPI and NIVO schedule can be adjusted to reduce toxicity but preserve efficacy. Methods: The aim of the multi-center phase 2 OpACIN-neo trial is to identify an optimal neo-adjuvant combination scheme of IPI and NIVO. 90 patients with resectable stage III melanoma will be randomized 1:1:1 between three combination schemes of IPI and NIVO. Patients in arm A will receive 2 courses standard regimen IPI 3mg/kg + NIVO 1mg/kg q3wks, in arm B IPI 1mg/kg + NIVO 3mg/kg q3wks, and in arm C 2 courses of IPI 3mg/kg q3wks directly followed (2-24hr) by 2 courses NIVO 3mg/kg q2wks. All patients will undergo surgery at week 6. The primary endpoints are rate of grade 3/4 irAEs, pathological RR, and radiologic RECIST 1.1. An interim analysis is planned after 13 patients have been accrued to each arm (according to the Simon 2-stage design). Major inclusion criteria are: ≥1 measurable lymph node metastases (according to RECIST 1.1) that can be biopsied, no history of in-transit metastases in the last 6 months, and naïve for ICI. Baseline biopsies and blood samples (week 0, 6, 12) will be taken for translational research. The first center started inclusion in December 2016, until now 2 patients have been enrolled. Clinical trial information: NCT02977052.

Aims Triple‐negative breast cancer ( TNBC ) patients generally have a poor outcome; there is a pressing need to identify more effective therapeutic strategies. Clinical trials targeting programmed death 1/programmed death ligand 1 ( PD 1/ PDL 1) in melanoma and non‐small‐cell lung cancer have reported high response rates, and tumoral PDL 1 expression has been suggested as a potential biomarker to enrich for patient response to these treatments. There are only very limited data to date reporting the expression of PDL 1 in TNBC . Methods and results PDL 1 immunohistochemistry was performed on 161 primary TNBC s and assessed in the tumour as well as immune cells in the stromal compartment. PDL 1 expression was very common in TNBC , expressed in the tumour cell membrane (64%), cytoplasm (80%) and stromal (93%) cellular compartments. Cytoplasmic tumoral expression of PDL 1 was associated with a lower risk of breast cancer‐specific death [hazard ratio ( HR ) 0.45, P = 0.035] while stromal PDL 1 expression was associated with a lower rate of deaths from all causes ( HR 0.305, P = 0.0042). Membranous expression of PDL 1 was not associated with outcome. While both PDL 1 expression and tumour‐infiltrating lymphocytes were associated with a better outcome, only lymphovascular invasion and high tumour‐infiltrating lymphocytes were independently prognostic for breast cancer‐specific death. Conclusion While PDL 1 expression is frequent in TNBC , it was not independently prognostic. There were differences in outcome depending on the cellular compartment of PDL 1 expression. These data provide further impetus for investigating the utility of immune checkpoint therapies in TNBC , given the clinical significance of tumour‐infiltrating lymphocytes ( TIL s) and PDL 1 expression in this cohort.

Immune checkpoint inhibitors have revolutionized the treatment of patients with advanced-stage metastatic melanoma, as well as patients with many other solid cancers, yielding long-lasting responses and improved survival. However, a subset of patients who initially respond to immunotherapy, later relapse and develop therapy resistance (termed "acquired resistance"), whereas others do not respond at all (termed "primary resistance"). Primary and acquired resistance are key clinical barriers to further improving outcomes of patients with metastatic melanoma, and the known mechanisms underlying each involves various components of the cancer immune cycle, and interactions between multiple signaling molecules and pathways. Due to this complexity, current knowledge on resistance mechanisms is still incomplete. Overcoming therapy resistance requires a thorough understanding of the mechanisms underlying immune evasion by tumors. In this review, we explore the mechanisms of primary and acquired resistance to immunotherapy in melanoma and detail potential therapeutic strategies to prevent and overcome them. <i>Clin Cancer Res; 24(6); 1260-70. ©2017 AACR</i>.

Reflectance confocal microscopy can assist in the differential diagnosis of lip MEL and MAC. An RCM Lip Score that we developed based on study results is proposed and needs to be validated on an independent data set.