Elevated serum lactate dehydrogenase (sLDH) is associated with poor clinical outcomes in patients with stage IV metastatic melanoma (MM). It is currently unknown if sLDH elevation correlates with distinct molecular, metabolic, or immune features of melanoma metastases. The identification of such features may identify rational therapeutic strategies for patients with elevated sLDH. Thus, we obtained sLDH levels for melanoma patients with metastases who had undergone molecular and/or immune profiling. Our analysis of multi-omics data from independent cohorts of melanoma metastases showed that elevated sLDH was not significantly associated with differences in immune cell infiltrate, point mutations, DNA copy number variations, promoter methylation, RNA expression, or protein expression in melanoma metastases. The only significant association observed for elevated sLDH was with the number of metastatic sites of disease. Our data support that sLDH correlates with disease burden, but not specific molecular or immunological phenotypes, in metastatic melanoma.

TPS10087 Background: Previous OpACIN and OpACIN-neo studies, investigating neoadjuvant ipilimumab (IPI) plus nivolumab (NIVO), demonstrated high pathologic response rates (74-78%) and favorable long-term outcomes in patients (pts) achieving pathologic response; at 36 and 18 months follow-up, respectively, only 1/71 (1.4%) pts with response has relapsed. In contrast, pts without pathologic response (pNR) have a poor prognosis; 15/23 (65.2%) have relapsed so far. This emphasizes the need for baseline biomarkers predictive of non-response and new neoadjuvant treatment combinations for these pts. In our previous studies, baseline interferon-gamma (IFN-γ) signature low pts were less likely to respond to neoadjuvant IPI plus NIVO. The DONIMI study tests the combination of NIVO +/- IPI with domatinostat (DOM), a class 1 histone deacetylase inhibitor, according to the IFN-γ signature in the tumor. Based on the signature previously described by Ayers et al. we have developed a neoadjuvant IFN-γ signature algorithm that will be used for the first time to classify pts in this prospective trial. Methods: The aim of this two-center investigator-initiated phase 1b study is to assess the safety and feasibility of neoadjuvant NIVO +/- DOM +/- IPI in 45 stage III melanoma pts with RECIST 1.1 measurable de-novo or recurrent disease. IFN-γ signature high pts (n = 20) will be randomized (stratified by center) to Arm A (2 cycles NIVO 240mg q3wk) or Arm B (2 cycles NIVO 240mg q3wk + DOM 200mg twice daily (BID), d1-14, q3wk). IFN-γ signature low pts (n = 25) will be randomized to Arm C (2 cycles NIVO 240mg q3wk + DOM 200mg BID, d1-14, q3wk) or Arm D (2 cycles NIVO 240mg q3wk + IPI 80mg q3wk + DOM 200mg once daily (OD), d1-14, q3wk). Based on safety data of the first 5 pts in arm D, the remaining pts will be treated with either a higher dosing scheme (200mg BID, d1-14, q3wks), a lower dosing scheme (100mg OD, d1-14, q3wks) or the same dosing scheme (200mg OD, d1-14, q3wks). The primary endpoint is safety and feasibility. A treatment arm will be declared as not feasible if 2/5 or 3/10 pts cannot adhere to the preplanned time of surgery (week 6 +/- 1week) due to treatment-related adverse events. Biopsies (week 0, 3), blood samples (week 0, 3, 6, 12) and feces (week 0, 3, 6) will be collected for translational research. The first patient was enrolled on January 23 th , 2020. Clinical trial information: NCT04133948.

Most international clinical guidelines recommend 5-10 mm clinical margins for excision of melanoma in situ (MIS). While the evidence supporting this is weak, these guidelines are generally consistent. However, as a result of the high incidence of subclinical extension of MIS, especially of the lentigo maligna (LM) subtype, wider margins will often be needed to achieve complete histologic clearance. In this review, we assessed all available contemporary evidence on clearance margins for MIS. No randomized trials were identified and the 31 non-randomized studies were largely retrospective reviews of single-surgeon or single-institution experiences using Mohs micrographic surgery (MMS) for LM or staged excision (SE) for treatment of MIS on the head/neck and/or LM specifically. The available data challenge the adequacy of current international guidelines as they consistently demonstrate the need for clinical margins > 5 mm and often > 10 mm. For LM, any MIS on the head/neck, and/or ≥ 3 cm in diameter, all may require wider clinical margins because of the higher likelihood of subclinical spread. Histologic clearance should be confirmed prior to undertaking complex reconstruction. However, it is not clear whether wider margins are necessary for all MIS subtypes. Indeed, it seems that this is unlikely to be the case. Until optimal surgical margins can be better defined in a randomized trial setting, ideally controlling for MIS subtype and including correlation with histologic excision margins, techniques such as preliminary border mapping of large, ill-defined lesions and, most importantly, sound clinical judgement will be needed when planning surgical clearance margins for the treatment of MIS.

Interventions to improve histopathology agreement may reduce melanoma 'over-calling' and 'under-calling'.

© 2018 UICC Despite recent advances in targeted and immune-based therapies, advanced stage melanoma remains a clinical challenge with a poor prognosis. Understanding the genes and cellular processes that drive progression and metastasis is critical for identifying new therapeutic strategies. Here, we found that the GTPase RAB27A was overexpressed in a subset of melanomas, which correlated with poor patient survival. Loss of RAB27A expression in melanoma cell lines inhibited 3D spheroid invasion and cell motility in vitro, and spontaneous metastasis in vivo. The reduced invasion phenotype was rescued by RAB27A-replete exosomes, but not RAB27A-knockdown exosomes, indicating that RAB27A is responsible for the generation of pro-invasive exosomes. Furthermore, while RAB27A loss did not alter the number of exosomes secreted, it did change exosome size and altered the composition and abundance of exosomal proteins, some of which are known to regulate cancer cell movement. Our data suggest that RAB27A promotes the biogenesis of a distinct pro-invasive exosome population. These findings support RAB27A as a key cancer regulator, as well as a potential prognostic marker and therapeutic target in melanoma.

Among the histogenic subtypes of melanoma, nodular melanoma (NM) is the major contributor for thicker and fatal melanomas and it has been associated with melanoma-specific death in thin tumours, highlighting an important subgroup of 'aggressive thin' melanomas. This review provides a synthesis of the distinct characteristics of NM, with respect to epidemiology and risk factors, clinical presentation, histopathology, molecular and dermoscopic aspects, and screening practices. The real challenges are to find better biomarkers of aggressiveness and to know whether the control of such aggressive melanomas can be influenced by targeted interventions such as early detection, drug interventions and preventive strategies.

10015 Background: Early results of the OpACIN-neo study testing 3 different dosing schedules of neoadjuvant IPI + NIVO demonstrated that 2 cycles IPI 1mg/kg + NIVO 3mg/kg (IPI1NIVO3, arm B) was the most favorable schedule with 20% grade 3-4 immunotherapy-related adverse events (irAEs) and a pathologic response rate (pRR) of 77%. After a median follow-up (FU) of 8.3 months, none of the 64 patients (pts) with a pathologic (path) response ( < 50% viable tumor cells) versus 9/21 (43%) without a path response had relapsed. Here, we present the updated 2-year RFS, EFS and long-term toxicity data. Methods: In the phase 2 multi-center OpACIN-neo trial, 86 stage III melanoma pts with resectable and RECIST 1.1 measurable lymph node metastasis were randomized between 3 different dosing schedules of neoadjuvant IPI + NIVO: arm A: 2x IPI3+NIVO1 Q3W (n = 30), arm B: 2x IPI1+NIVO3 Q3W (n = 30), and arm C: 2x IPI3 Q3W followed by 2x NIVO3 Q2W (n = 26). Lymph node dissection was scheduled at week 6. Primary endpoints were toxicity, radiologic RR and pRR; RFS and EFS were secondary endpoints. Results: After a median FU of 24.6 months, the median RFS and EFS was not reached in any of the 3 arms. In total, 2 pts progressed before surgery, 12 pts relapsed (11 pts without path response and 1 pt with pCR) and 5 pts died (4 due to melanoma and one pt due to toxicity). Estimated 24-months RFS was 84% (95% CI 76-92%) for the total population, 97% (95% CI 93-100%) for pts with a path response and 36% (95% CI 17-74%) for pts without a path response. Estimated 24-months EFS for the total population was 82% (95% CI 74-91%). RFS and EFS did not differ between the arms. Of the 81 pts alive, 55 (68%) have ongoing irAEs; only 2 (3%) pts have ≥ grade 3 irAEs. Most frequent ongoing irAEs were vitiligo (35%), fatigue (14%), sicca syndrome (11%), rash (10%), arthralgia (7%) and endocrine toxicities (20%). 17 pts need hormone replacement therapy: 11 (14%) thyroid hormone and 7 (9%) hydrocortisone. No difference between treatment arms was observed. Ongoing surgery-related AEs were observed in 31 (38%) pts of which lymphedema was seen most frequently (17 pts; 21%). Conclusions: Extended follow-up data shows that 2 cycles of neoadjuvant IPI + NIVO without adjuvant therapy induces durable RFS. While almost no ongoing high-grade irAEs were observed, the majority of pts have low-grade ongoing toxicities. These outcomes strongly support the need to test 2 cycles of neoadjuvant IPI1+NIVO3 versus adjuvant anti-PD-1 in a randomized phase 3 trial. Clinical trial information: NCT02977052.

Melanomas were divided into those etiologically related to sun exposure and those that are not, as determined by their mutational signatures, anatomic site, and epidemiology. Melanomas on the sun-exposed skin were further divided by the histopathologic degree of cumulative solar damage (CSD) of the surrounding skin, into low and high CSD, on the basis of degree of associated solar elastosis. Low-CSD melanomas include superficial spreading melanomas and high-CSD melanomas incorporate lentigo maligna and desmoplastic melanomas. The "nonsolar" category includes acral melanomas, some melanomas in congenital nevi, melanomas in blue nevi, Spitz melanomas, mucosal melanomas, and uveal melanomas. The general term <i>melanocytoma</i> is proposed to encompass "intermediate" tumors that have an increased (though still low) probability of disease progression to melanoma.

Abstract Metastatic melanoma is a lethal disease with a dismal 5-year survival rate. Thus, intense efforts to boost novel therapeutic strategies are underway to identify early detection of melanomas with a high propensity to metastasize. We recently discovered that the loss of cell surface glycan, I-antigen, corresponds with the transition of primary melanoma to metastatic melanoma. I-antigen or I-branched glycans are synthesized by β16, N-acetylglucosaminyltransferase 2 (GCNT2) and inversely correlate with the growth and signaling potential of metastatic melanoma cells. Moreover, compared with high GCNT2 expression in normal melanocytes, nevi, and early-stage primary melanomas, GCNT2 is conspicuously lost in metastatic melanomas. We anticipate the potential utilization of GCNT2 expression as a biomarker to predict melanoma metastasis. Further, metastasis and aggressive disease progression are key phenotypes of tumor-initiating cells (TIC), which are preferentially generated in areas of hypoxia. In the vertical growth phase of primary melanomas and melanoma metastases, the tumor microenvironment is typically hypoxic (1.5% oxygen). We hypothesize that the hypoxic microenvironment aids in metastatic melanoma progression through TIC generation and immune evasion, by downregulating GCNT2 and switching I-branched glycans to linear glycans. In this study, metastatic melanoma cells grown under hypoxic conditions had reduced GCNT2 and MITF with upregulated stem cell marker KLF4 expression. Importantly, in the in vivo TIC assay, we found significantly decreased tumor formation with increased GCNT2 expression while low GCNT2 levels enabled tumor formation even when 103 cells were injected in immunocompromised mice. Since TICs are thought to evade immune clearance, we investigated whether loss of GCNT2 increased TIC characteristics and also enabled immunosuppressive features. In human PBMC - metastatic melanoma co-cultures, there was an increase in T regulatory cell generation associated with low GCNT2 compared to high GCNT2 expression in melanoma cells, suggesting that loss of GCNT2 associates with increased TIC generation, tumor formation, and immunoevasion potential. Using melanoma patient specimens, immunohistochemical analysis of GCNT2 corresponded with a significant increase in mortality with the loss of GCNT2 staining. Altogether, these findings highlight GCNT2/I-branching not only as a biomarker of melanoma virulence but reveal malignancy-associated pathways functioning in parallel with loss of GCNT2/I-branching that could offer additional targets for the treatment of metastatic melanoma. Citation Format: Asmi Chakraborty, Mariana Perez, Norhan B. B Mohammed, Michael Wells, James S. Wilmott, John F. Thompson, Stuart M. Haslam, Wei Wang, Richard A. Scolyer, George F. Murphy, Charles J. Dimitroff. Hypoxia-mediated downregulation of GCNT2/I-antigen in metastatic melanoma accelerates disease progression and mortality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2609.

Clinically significant colitis varies in presentation, response to immunosuppression, and endoscopic/histologic features depending on the immunotherapy type. Infliximab-refractory colitis occurs early, is often high grade, and has adverse endoscopic and histopathologic features.

To increase understanding of the genomic landscape of acral melanoma, a rare form of melanoma occurring on palms, soles or nail beds, whole genome sequencing of 87 tumors with matching transcriptome sequencing for 63 tumors was performed. Here we report that mutational signature analysis reveals a subset of tumors, mostly subungual, with an ultraviolet radiation signature. Significantly mutated genes are BRAF, NRAS, NF1, NOTCH2, PTEN and TYRP1. Mutations and amplification of KIT are also common. Structural rearrangement and copy number signatures show that whole genome duplication, aneuploidy and complex rearrangements are common. Complex rearrangements occur recurrently and are associated with amplification of TERT, CDK4, MDM2, CCND1, PAK1 and GAB2, indicating potential therapeutic options.

Abstract Background: Thyroid toxicity is common following immune checkpoint inhibitor (ICI) treatment. Published studies estimate the incidence at 10-20%, although rates vary widely between different ICIs. The etiology of ICI-associated thyroid immune related adverse events (irAEs) is unknown &amp; not all patients develop a classic thyroiditis-like presentation of transient hyperthyroidism followed by a hypothyroid phase. Only small observational cohorts have been reported &amp; the clinical &amp; biochemical features of thyroid irAEs have not been well characterized. The current study aimed to describe thyroid irAEs in a large cohort of patients with melanoma. Methods: We reviewed outcomes in a prospective cohort of adult patients undergoing ICI treatment for advanced melanoma. Thyroid function was measured at baseline &amp; at regular intervals during treatment. Thyroid irAEs were defined as new biochemical thyroid dysfunction developing over the course of routine follow-up. Results: Thyroid irAEs occurred in 518 of 1246 (42%) patients. Median follow-up was 11.3 months. Multiple patterns of thyroid-irAEs were observed, such as hyperthyroidism (subclinical or overt) in 31%, hypothyroidism in 8%, &amp; euthyroid hyperthyroxinemia, hypothyroxinemia &amp; isolated low FT3 syndrome each in 1% of participants. Thyroid irAEs were more frequent following combination (CTLA-4 + PD-1) ICI treatment (56%) than following PD-1 (38%) or CTLA-4 (25%) based monotherapies (p=0.001). The severity of thyroid irAEs differed by ICI, with higher rates of overt (vs. subclinical) thyroid dysfunction following combination ICI treatment (47%) relative to PD-1 (37%) &amp; CTLA-4 (19%) monotherapies (p=0.001). Younger age (OR 0.88 per 10-yrs; 95% CI 0.81-0.96), female sex (OR 1.62; 95% CI 1.27-2.08) &amp; combination ICI-treatment (vs. CTLA-4, OR 3.76, 95% CI 2.49-5.75; vs. PD-1, OR 1.90, 95% CI 1.45-2.49) were associated with higher odds of thyroid irAE. Time to onset of thyroid dysfunction was shorter in patients with overt hyperthyroidism relative to other types of thyroid irAE (log rank p=0.001). Overt hyperthyroidism was associated with increased irAEs in other organ systems (colitis, hepatitis, etc), increased irAE severity &amp; increased multi-system irAEs than euthyroid patients or patients with other subtypes of thyroid irAE (p=0.003). Overt hyperthyroidism was also associated with improved progression free survival (HR 0.57; 95% CI 0.39-0.84; p=0.005) &amp; overall survival (HR 0.68; 95% CI 0.49-0.94; p=0.02). No benefit to cancer survival was observed with other thyroid irAE subtypes. Conclusions: Thyroid irAEs were common. Combination ICI treatment resulted in more frequent, more severe, &amp; earlier onset thyroid irAEs. Of thyroid irAE subtypes, overt hyperthyroidism was uniquely associated with increased immune responsiveness, as evidenced by higher incidence of extra-thyroidal irAEs &amp; improvements in cancer survival.

Additional file 8. Gene pathways for each cell type across cryopreservation conditions. Shared Gene Ontology (GO) pathways for each cell type and cryopreservation condition across the five tumours analysed. Only pathways detected in the same cell type and cryopreservation condition in more than two tumours were analysed. All DEGs from the comparison of annotated cell types across cryopreservation conditions (Additional file 7) were passed on to the ClusterProfiler package for functional enrichment with the CC sub-ontology under the human org.Hs.eg.db database.

Additional file 5. Differentially expressed genes for all cells across cryopreservation conditions. Differential gene expression was performed using the MAST method within Seurat v3 with the RNA assay and default parameters. Differential gene expression was performed using integrated data. Data from all conditions from each matched tumour datasets were down sampled to the same number of mapped sequencing reads using CellRanger. The number of cells in each condition was randomly down sampled to match the condition with the lowest number of cells.