Background Melanoma is the leading cause of skin cancer-related deaths worldwide. While there have been significant improvements in the treatment of advanced melanoma in the past decade, biomarker development lagged behind. Objectives The majority of liquid biopsy biomarkers rely on the analyses of oncogenic mutations; however, about 20% of melanoma patients are wild type. Therefore, validation of universal predictive and prognostic biomarkers is urgently needed. Methods We analysed plasma samples in a discovery cohort (n = 20) and expansion cohort (n = 166) of metastatic melanoma patients and healthy donors (n = 116). Total plasma circulating cell-free DNA (cfDNA) concentrations were measured on the Qubit(R)platform using assays for single-(ss) and double (ds)-stranded DNA, DNA spectrophotometry and RNase P qPCR. We explored the diagnostic, predictive and prognostic potential of cfDNA concentration by bio-statistical methods and established a cfDNA threshold for risk stratification. Results Our selected best method was Qubit(R)dsDNA assay which quantified higher plasma cfDNA concentrations in melanoma patients than in healthy controls (AUC 72%). Measurement of baseline cfDNA concentration revealed that high cfDNA was associated with presence of metastases and higher AJCC stage (P < 0.05). Furthermore, high baseline cfDNA was an indicator of shorter overall survival in patients with oncogenic mutations (HR 2.12,P = 0.0008), and in wild-type patients (HR 5.55,P < 0.0001). Conclusions We provide evidence that total cfDNA can be used as a biomarker for melanoma irrespective of the tumour genotype and can provide information on tumour load, risk of progression and risk of death.

BACKGROUND:GNAQ and GNA11 mutant nonuveal melanoma represent a poorly characterized rare subgroup of melanoma with a gene mutation profile similar to uveal melanoma.OBJECTIVES:To characterize these tumours in terms of clinical behaviour and genetic characteristics.METHODS:Patients with nonuveal GNAQ/11 mutated melanoma were identified from the prospective multicentre tumour tissue registry ADOREG, Tissue Registry in Melanoma (TRIM) and additional cooperating skin cancer centres. Extensive data on patient, tumour and treatment characteristics were collected retrospectively. Targeted sequencing was used to determine tumour mutational burden. Immunohistochemistry staining was performed for programmed death-ligand 1 and BRCA1-associated protein (BAP)1. Existing whole-exome cutaneous and uveal melanoma data were analysed for mutation type and burden.RESULTS:We identified 18 patients with metastatic GNAQ/11 mutant nonuveal melanoma. Tumours had a lower tumour mutational burden and fewer ultraviolet signature mutations than cutaneous melanomas. In addition to GNAQ and GNA11 mutations (nine each), six splicing factor 3b subunit 1 (SF3B1), three eukaryotic translation initiation factor 1A X-linked (EIF1AX) and four BAP1 mutations were detected. In contrast to uveal melanoma, GNAQ/11 mutant nonuveal melanomas frequently metastasized lymphatically and concurrent EIF1AX, SF3B1 and BAP1 mutations showed no apparent association with patient prognosis. Objective response to immunotherapy was poor with only one partial response observed in 10 treated patients (10%).CONCLUSIONS:Our findings suggest that GNAQ/11 mutant nonuveal melanomas are a subtype of melanoma that is both clinically and genetically distinct from cutaneous and uveal melanoma. As they respond poorly to available treatment regimens, novel effective therapeutic approaches for affected patients are urgently needed. What is already known about this topic? The rare occurrence of GNAQ/11 mutations in nonuveal melanoma has been documented. GNAQ/11 mutant nonuveal melanomas also harbour genetic alterations in EIF1AX, SF3B1 and BAP1 that are of prognostic relevance in uveal melanoma. What does this study add? GNAQ/11 mutant nonuveal melanomas show metastatic spread reminiscent of cutaneous melanoma, but not uveal melanoma. GNAQ/11 mutant nonuveal melanomas have a low tumour mutational burden that is higher than uveal melanoma, but lower than cutaneous melanoma. What is the translational message? Primary GNAQ/11 mutant nonuveal melanomas are a subtype of melanoma that is clinically and genetically distinct from both cutaneous and uveal melanoma. As metastatic GNAQ/11 mutant nonuveal melanomas respond poorly to available systemic therapies, including immune checkpoint inhibition, novel therapeutic approaches for these tumours are urgently needed. Linked Comment: Rafei-Shamsabadi. Br J Dermatol 2020; 183:806-807.

Background Intratumoral injection of immunomodulating agents is a promising approach to prime antitumor immune responses and to control tumor growth while avoiding major systemic toxicities. Intratumoral CV8102, a TLR7/8/RIG-1 agonist based on noncoding single stranded RNA showed dose dependent single agent activity and synergism with PD-1 blockade in preclinical models. Trial design CV-8102-008 (NCT03291002) is a phase I, open-label, dose escalation and expansion study of intratumoral CV8102 in patients with advanced, inoperable cutaneous melanoma (cMEL), squamous cell carcinoma of the skin (cSCC) or head and neck (hnSCC) or adenoid cystic carcinoma (ACC). Eight intratumoral injections of CV8102 are provided over a 12 weeks period, unless disease progression requiring initiation of next-line therapy or unacceptable toxicity occurs. Patients showing evidence of clinical benefit on single agent CV8102 are eligible for further treatment. Currently the first part of the trial, with escalating doses of single agent CV8102 and CV8102 in combination with anti-PD-1 antibodies to determine the maximum tolerated/ recommended dose (MTD/RCD) guided by a Bayesian logistic regression model with overdose control. and flexible cohort sizes is ongoing. Safety, biological and tumor response according to RECIST 1.1/ir RECIST of CV8102 alone/in combination with anti-PD-1 antibodies will be further characterized in expansion cohorts including anti-PD-1 naive and refractory patients. Clinical trial identification NCT03291002. Legal entity responsible for the study CureVac AG. Funding CureVac AG. Disclosure J. Krauss: Advisory / Consultancy: Vaccibody; Advisory / Consultancy: Zelluna; Research grant / Funding (self): Heidelberg ImmunoTherapeutics. T. Eigentler: Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: Novartis; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pierre Fabre; Advisory / Consultancy, Speaker Bureau / Expert testimony: Bristol-Myers Squibb. C. Weishaupt: Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self): BMS; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: CureVac; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: La Roche Posay; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Leo Pharma; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Novartis; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Pierre Fabre; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Sanofi; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony: Takeda. P. Terheyden: Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: BMS; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Pierre-Fabre; Honoraria (self), Advisory / Consultancy: Roche; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Merck Serono; Honoraria (self): CureVac. L. Heinzerling: Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Amgen; Advisory / Consultancy: MSD; Advisory / Consultancy: BMS; Advisory / Consultancy: Roche; Advisory / Consultancy: CureVac; Advisory / Consultancy: Pierre-Fabre; Advisory / Consultancy: Sanofi. P. Mohr: Advisory / Consultancy, Shareholder / Stockholder / Stock options: Pierre Fabre; Advisory / Consultancy, Research grant / Funding (institution), Shareholder / Stockholder / Stock options: Merck; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Roche; Advisory / Consultancy, Shareholder / Stockholder / Stock options: GSK; Advisory / Consultancy, Research grant / Funding (institution), Shareholder / Stockholder / Stock options: BMS; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Novartis; Advisory / Consultancy: LEO; Advisory / Consultancy, Shareholder / Stockholder / Stock options: Amgen. B. Weide: Advisory / Consultancy: CureVac GmbH; Advisory / Consultancy, Research grant / Funding (self): BMS; Advisory / Consultancy, Research grant / Funding (self): MSD. S. Ochsenreither: Honoraria (self), Advisory / Consultancy: MSD; Honoraria (self), Advisory / Consultancy: BMS; Honoraria (self), Advisory / Consultancy: Merck; Honoraria (self), Advisory / Consultancy: CureVac; Honoraria (self), Advisory / Consultancy: Glenmark; Honoraria (self), Advisory / Consultancy: Incyte. R. Gutzmer: Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): JohnsonJ Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Novartis; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Amgen; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Merck-Serono; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Roche Pharma; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Bristol-MyersSquibb; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Almirall-Hermal; Honoraria (self), Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: SUN; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Pierre-Fabre; Honoraria (self), Advisory / Consultancy: LEO; Honoraria (self), Advisory / Consultancy: 4SC; Honoraria (self), Advisory / Consultancy: Incyte; Honoraria (self), Advisory / Consultancy: Takeda. J.C. Becker: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Merck Serono; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer; Honoraria (self), Advisory / Consultancy: CureVac; Honoraria (self), Advisory / Consultancy: eTheRNA; Honoraria (self), Advisory / Consultancy: Lytix; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy: Rigontec; Honoraria (self), Advisory / Consultancy: Takeda; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: 4SC; Research grant / Funding (institution): Alcedis; Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Bristol-Myers Squibb; Travel / Accommodation / Expenses: Incyt. F. Funkner: Full / Part-time employment: CureVac AG. R. Heidenreich: Full / Part-time employment: CureVac AG. S. Kays: Full / Part-time employment: CureVac AG. U. Klinkhardt: Full / Part-time employment, Former: CureVac; Full / Part-time employment: Boehringer Ingelheim. U.S. Gnad-Vogt: Full / Part-time employment, Officer / Board of Directors: CureVac AG. B. Scheel: Full / Part-time employment: CureVac AG. O. Schonborn-Kellenberger: Full / Part-time employment, consultant to CureVac AG: Cogitars GmbH. T. Seibel: Full / Part-time employment: CureVac AG. All other authors have declared no conflicts of interest.

Despite multidisciplinary local and systemic therapeutic approaches, the prognosis for most patients with brain metastases is still dismal. The role of adaptive and innate anti-tumor response including the Human Leukocyte Antigen (HLA) machinery of antigen presentation is still unclear. We present data on the HLA class II-chaperone molecule CD74 in brain metastases and its impact on the HLA peptidome complexity. We analyzed CD74 and HLA class II expression on tumor cells in a subset of 236 human brain metastases, primary tumors and peripheral metastases of different entities in association with clinical data including overall survival. Additionally, we assessed whole DNA methylome profiles including CD74 promoter methylation and differential methylation in 21 brain metastases. We analyzed the effects of a siRNA mediated CD74 knockdown on HLA-expression and HLA peptidome composition in a brain metastatic melanoma cell line. We observed that CD74 expression on tumor cells is a strong positive prognostic marker in brain metastasis patients and positively associated with tumor-infiltrating T-lymphocytes (TILs). Whole DNA methylome analysis suggested that CD74 tumor cell expression might be regulated epigenetically via CD74 promoter methylation. CD74(high) and TILhigh tumors displayed a differential DNA methylation pattern with highest enrichment scores for antigen processing and presentation. Furthermore, CD74 knockdown in vitro lead to a reduction of HLA class II peptidome complexity, while HLA class I peptidome remained unaffected. In summary, our results demonstrate that a functional HLA class II processing machinery in brain metastatic tumor cells, reflected by a high expression of CD74 and a complex tumor cell HLA peptidome, seems to be crucial for better patient prognosis.

Background: Checkpoint inhibition is an effective treatment in patients with metastatic melanoma (MM). T cell activation can induce tumor rejection but also possibly severe autoimmune side effects (irAE). Autoantibody biomarkers from serum have potential to predict irAEs such as an ipilimumab-induced colitis. Methods: We use a cancer immunotherapy array consisting of 850 human protein antigens from 4 classes: 1. Tumor-associated antigens (TAA), 2. cancer pathway proteins, 3. autoimmune antigens, 4. cytokines/interleukins. Protein antigens were covalently coupled to magnetic beads and serum AABs were analyzed by Luminex FlexMap 3D. First, we screened pre-immunotherapy sera from 142 patients with MM (Heidelberg Cohort: 82 Ipilimumab (Ipi) treated; 11 Ipi/Nivolumab (Nivo); 40 Pembrolizumab (Pembro), 119 healthy controls (HC)). In this cohort, 41.5% (n = 59) experienced irAEs of any grade and 7% (n = 10) had colitis of grade 3 or 4. In a second study, 200 MM patients from 5 European sites (53 Ipi/Nivo; 111 Pembro, 100 HC) were analyzed. 25.6% (n = 42) had grade 3 or 4 irAEs, 12.8% (n = 21) had diarrhea or colitis of any grade and 9.8% (n = 16) had grade 3 or 4 colitis. Results: 40 different AABs were significantly more prevalent in MM compared to HC including NY-ESO1, NY-ESO 2 and other TAAs, cytokines, and nuclear proteins. Significant correlations of AABs were seen in Ipi-treated patients who experienced irAEs, both in mono- but also in combination therapy, allowing to dichotomize MM in risk groups. Also different sets of AABs were seen in Pembro-treated patients with irAEs. The protein antigens represent a variety of biological processes: they are involved in melanoma progression including transcription factors or components of the E3 ubiquitin ligase complex, cytokeratins, and proteins involved in cell adhesion. Conclusions: In MM, screening of AABs prior to start of Checkpoint inhibition holds potential to predict risk for irAEs such as colitis. As irAEs are especially frequent in Ipi-based treatment regimes, AABs presented here may serve as useful biomarkers for a risk-based treatment decision. Legal entity responsible for the study: Jessica C. Hassel and Protagen AG. Funding: Protagen AG. Disclosure: J.C. Hassel: Consulting role: Merck, Amgen; Honoraria: Bristol-Myers Squibb, Merck, Novartis, Roche and Pfizer; Science projects support: BMS. J. Mangana: Temporary advisory relationship and receives travel support: MSD, Merck. C. Pföhler: Consulting role: Merck Serono, Novartis, Roche, Amgen, BMS; Honoraria: Merck Serono, Novartis, Roche, Amgen, BMS. B. Weide: Consulting role: Curevac, Philogen, BMS; Honoraria: MSD, BMS, Roche, Amgen, Philogen; Science projects support: BMS, Philogen. L. Hakim-Meibodi: Travel grants: BMS. F. Meier: Honoraria: Roche, BMS, GSK, Novartis, MSD; Travel support: Roche, BMS; Research funding: Wyeth/Pfizer, Merck-Serono, Novartis. H.-D. Zucht, P. Budde; M. Tuschen: Employee: Protagen AG. P. Schulz-Knappe: Board member and chair holder: Protagen AG. All other authors have declared no conflicts of interest.

BACKGROUND:Early detection is a key factor in improving survival from melanoma. Today, the clinical diagnosis of cutaneous melanoma is based mostly on visual inspection and dermoscopy. Preclinical studies in freshly excised or paraffin-embedded tissue have shown that the melanin fluorescence spectra after stepwise two-photon excitation, a process termed dermatofluoroscopy, differ between cutaneous melanoma and melanocytic naevi. However, confirmation from a larger prospective clinical study is lacking.OBJECTIVES:The primary end point of this study was to determine the diagnostic accuracy of dermatofluoroscopy in melanoma detection. Secondary end points included the collection of data for improving the computer algorithm that classifies skin lesions based on melanin fluorescence and the assessment of safety aspects.METHODS:This was a prospective, blinded, multicentre clinical study in patients with pigmented skin lesions (PSLs) indicated for excision either to rule out or to confirm cutaneous melanoma. All included lesions underwent dermoscopy and dermatofluoroscopy in vivo before lesions were excised and subjected to histopathological examination.RESULTS:In total, 369 patients and 476 PSLs were included in the final analysis. In 101 of 476 lesions (21·2%) histopathology revealed melanoma. The observed sensitivity of dermatofluoroscopy was 89·1% (90 of 101 melanomas identified), with an observed specificity of 44·8%. The positive and negative predictive values were 30·3% and 93·9%, respectively. No adverse events occurred.CONCLUSIONS:Dermatofluoroscopy is a safe and accurate diagnostic method to aid physicians in diagnosing cutaneous melanoma. Limitations arise from largely amelanotic or regressing lesions lacking sufficient melanin fluorescence.

Immune checkpoint blockers (ICB) have become pivotal therapies in the clinical armamentarium against metastatic melanoma (MMel). Given the frequency of immune related adverse events and increasing use of ICB, predictors of response to CTLA-4 and/or PD-1 blockade represent unmet clinical needs. Using a systems biology-based approach to an assessment of 779 paired blood and tumor markers in 37 stage III MMel patients, we analyzed association between blood immune parameters and the functional immune reactivity of tumor-infiltrating cells after ex vivo exposure to ICB. Based on this assay, we retrospectively observed, in eight cohorts enrolling 190 MMel patients treated with ipilimumab, that PD-L1 expression on peripheral T cells was prognostic on overall and progression-free survival. Moreover, detectable CD137 on circulating CD8 + T cells was associated with the disease-free status of resected stage III MMel patients after adjuvant ipilimumab + nivolumab (but not nivolumab alone). These biomarkers should be validated in prospective trials in MMel.

INTRODUCTION:PD-L1 is established as a predictive marker for therapy of non-small cell lung cancer with pembrolizumab. Furthermore, PD-L1 positive melanoma has shown more favorable outcomes when treated with anti-PD1 antibodies and dacarbazine compared to PD-L1 negative melanoma. However, the role of PD-L1 expression with regard to response to checkpoint inhibition with anti-CTLA-4 is not clear, yet. In addition, the lack of standardization in the immunohistochemical assessment of PD-L1 makes the comparison of results difficult. In this study, we investigated the PD-L1 gene expression with a new fully automated technique via RT-PCR and correlated the findings with the response to the anti-CTLA-4 antibody ipilimumab. MATERIALS AND METHODS:Within a retrospective multi-center trial, PD-L1 gene expression was evaluated in 78 melanoma patients in a total of 111 pre-treatment tumor samples from 6 skin cancer centers and analyzed with regard to response to ipilimumab. For meaningful statistical analysis, the cohort was enriched for responders with 30 responders and 48 non-responders. Gene expression was assessed by quantitative RT-PCR after extracting mRNA from formalin-fixed paraffin embedded tumor tissue and correlated with results from immunohistochemical (IHC) stainings. RESULTS AND DISCUSSION:The evaluation of PD-L1 expression based on mRNA level is feasible. Correlation between PD-L1 expression as assessed by IHC and RT-PCR showed varying levels of concordance depending on the antibody employed. RT-PCR should be further investigated to measure PD-L1 expression, since it is a semi-quantitative method with observer-independent evaluation. With this approach, there was no statistical significant difference in the PD-L1 expression between responders and non-responders to the therapy with ipilimumab. The evaluation of PD-L1 expression based on mRNA level is feasible. Correlation between PD-L1 expression as assessed by IHC and RT-PCR showed varying levels of concordance depending on the antibody employed. RT-PCR should be further investigated to measure PD-L1 expression, since it is a semi-quantitative method with observer-independent evaluation. With this approach, there was no statistical significant difference in the PD-L1 expression between responders and non-responders to the therapy with ipilimumab.

Despite recent therapeutic advances, a diagnosis of metastatic melanoma still foreshadows a grim prognosis for the majority of patients. One of the factors believed to be associated with the failure of current conventional cancer therapies are the so-called Cancer Stem Cells (CSCs). CSCs share common features with corresponding tissue stem cells, such as self-renewal capacity and the ability to give rise to progeny with the potential to proliferate and differentiate. Therapeutically targeting CSCs may thus eliminate the root cause of the cancer, however this would require the accurate identification of markers distinguishing CSCs from normal cells. The expression of candidate CSC markers is associated with a poor prognosis in a number of cancer types, but their clinical significance remains unclear and to the best of knowledge there have been no clinical studies in melanoma. Here, we aimed to determine the clinical significance of four molecules identifying putative CSCs, namely CD133, ABCG2, ALDH1A1 and CD44v7/8. This article is protected by copyright. All rights reserved.