TPS9606 Background: The outcome of high risk stage III melanoma patients (pts) is poor, with a 5 year overall survival (OS) rate of < 50%. Adjuvant (adj) high dose IPI significantly improves 5 year progression free survival (PFS) and OS and adj NIVO improves the median PFS even more. In stage IV pts, the combination of IPI and NIVO improves response rates (RR) and PFS compared to monotherapy, but at cost of higher toxicity. Neo-adjuvant (neoadj) 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. The phase Ib OpACIN study compared neoadj versus adj IPI plus NIVO. The pathological RR (pRR) was 80% in the neoadj arm, and to date after a median follow-up of 24 months, none of the responders has relapsed, while 4/10 pts have relapsed in the adj arm. Moreover, pts in the neoadj arm expanded more tumor-resident TCR clones than adj treated pts. Neoadj IPI+NIVO was feasible, but toxicity was high with 90% grade 3/4 immuun-related adverse events (irAE) in both arms. This raises the question whether neoadj IPI plus NIVO can be alternatively scheduled to reduce toxicity but preserve efficacy. Methods: The aim of the multi-center phase 2 OpACIN-neo trial is to identify an optimal neoadj combination scheme of IPI and NIVO. 90 pts with resectable stage III melanoma will be randomized 1:1:1 between three different combination schemes of IPI and NIVO (Arm A: 2x IPI 3mg/kg plus NIVO 1mg/kg q3wks, Arm B: 2x IPI 1mg/kg plus NIVO 3mg/kg q3wks, Arm C: 2x IPI 3mg/kg q3wks directly followed by 2x NIVO 3mg/kg q2wks). All pts will undergo surgery at week 6. Primary endpoints are rate of grade 3 and 4 irAEs, pRR, and radiologic RR according to RECIST 1.1. 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. An interim analysis was planned after 13 pts had been accrued to each arm (according to the Simon stage-2 design). Pre-specified activity goals for the first stage of accrual were met; until now 56 of 90 pts have been enrolled. Clinical trial information: NCT02977052.

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.

Our study highlights significant limitations imposed by inconsistent sensitivity and specificity due to differences in the detection antibodies or aptamers of these widespread biomarker discovery approaches. Our findings emphasize the need to improve these technologies for the accurate identification of biomarkers.

Tumor thickness is the strongest predictor of outcome for clinically localized melanoma. Therefore, accurate assessment is critical for appropriate staging, reliable estimation of prognosis, and management. When melanoma extends alongside skin adnexal structures more deeply than the main tumor mass (periadnexal extension), it is currently unknown whether the prognosis is more accurately reflected by the deepest point of periadnexal tumor extension or the main tumor mass. This study sought to address this question. Survival outcomes of 257 primary cutaneous melanoma patients with periadnexal extension diagnosed between 2005 and 2015 and managed at Melanoma Institute Australia were identified and compared with a control cohort of 514 patients who were matched for tumor thickness, sex, age, mitotic rate, ulceration status, and year of diagnosis but lacked periadnexal extension. The incidence of periadnexal extension at Melanoma Institute Australia was 1.5% (257/16,692 cutaneous melanomas diagnosed between 2005 and 2015). The patient characteristics between the 2 groups were otherwise very similar; median Breslow thickness was 0.9 mm for the periadnexal group and 1.0 mm for the control group. The median extension beyond the Breslow thickness in the tumors with periadnexal extension was 0.45 mm (mean, 0.4 mm). Median follow-up was 46 months for the periadnexal group and 44 months for the control group. Measures of clinical outcomes all showed trends for improved survival in the periadnexal extension group; these were melanoma-specific survival (hazard ratio [HR], 0.78; 95% confidence interval [CI], 0.44, 1.38), overall survival (HR, 0.91; 95% CI, 0.59, 1.41), disease-free survival (HR, 0.68; 95% CI, 0.45, 1.03), and distant disease-free survival (HR, 0.69; 95% CI, 0.4, 1.17), although none were statistically significant. There was a higher rate of sentinel lymph node (SLN) metastasis in the periadnexal group versus the control group in patients whose tumors were >1 mm thick (24/100=24% vs. 23/187=12.3%). Periadnexal extension was significantly associated with SLN metastasis on univariate logistic regression analysis (odds ratio [95% CI], 2.25 [1.20, 4.24], P=0.01). If the periadnexal extension had been included in the measurement of tumor thickness, 42.8% of patients would have been upstaged to a higher American Joint Committee on Cancer T category. The findings of this study indicate that periadnexal involvement that extends more deeply than the thickness of the main tumor mass increases the risk of SLN metastasis in tumours >1 mm thick, however, does not worsen clinical outcomes overall, and tumor thickness measurements should not include deeper foci of periadnexal tumor.

Cutaneous melanoma accounts for at least >10% of all cancers in adolescents and young adults (AYA, 15-30 years of age) in Western countries. To date, little is known about the correlations between germline variants and somatic mutations and mutation signatures in AYA melanoma patients that might explain why they have developed a cancer predominantly affecting those over 65 years of age. We performed genomic analysis of 50 AYA melanoma patients (onset 10-30 years, median 20); 25 underwent whole genome sequencing (WGS) of both tumor and germline DNA, exome data were retrieved from 12 TCGA AYA cases, and targeted DNA sequencing was conducted on 13 cases. The AYA cases were compared with WGS data from 121 adult cutaneous melanomas. Similar to mature adult cutaneous melanomas, AYA melanomas showed a high mutation burden and mutation signatures of ultraviolet radiation (UVR) damage. The frequencies of somatic mutations in BRAF (96%) and PTEN (36%) in the AYA WGS cohort were double the rates observed in adult melanomas (Q < 6.0 × 10^-6 and 0.028, respectively). Furthermore, AYA melanomas contained a higher proportion of non-UVR-related mutation signatures than mature adult melanomas as a proportion of total mutation burden (p = 2.0 × 10^-4 ). Interestingly, these non-UVR mutation signatures relate to APOBEC or mismatch repair pathways, and germline variants in related genes were observed in some of these cases. We conclude that AYA melanomas harbor some of the same molecular aberrations and mutagenic insults occurring in older adults, but in different proportions. Germline variants that may have conferred disease susceptibility correlated with somatic mutation signatures in a subset of AYA melanomas.

BRAF is suppress glycolysis and provide strong clinical benefi t in BRAF V600 melanoma. We show that BRAF inhibition suppresses glycolysis via a network of transcription factors that are critical for complete BRAFi responses. Furthermore, we provide evidence for the clinical potential of therapies that combine BRAFis with glycolysis inhibitors.

In a recent article, Taran and Heenan maintained that metastasis from Clark level 2 cutaneous malignant melanoma (CMM) “is rare, if it occurs at all,” a conclusion based on the examination of all pathology reports of patients diagnosed with a primary malignant melanoma at the Cancer Registry of Western Australia between 1982–1996.1 They found that no level 2 CMM in their data set was > 1 mm thick. There were 1716 patients with a CMM that measured ≤ 1 mm in thickness, 67 of whom developed metastases within a follow-up period of 7–15 years. After excluding 18 patients with multiple CMMs, 5 of the remaining 49 patients with metastases were found to have a level 2 CMM. All five patients displayed focal to extensive regression; therefore the authors concluded that the assessment of level of invasion may have been inaccurate, with these CMMs being invasive to level 3 or 4 before the onset of regression. We would like to present corresponding figures from the Sydney Melanoma Unit (SMU) database, which contains the records of nearly 19,500 melanoma patients treated between January 1950 and July 2001 at Royal Prince Alfred, St. Vincent's, and Sydney Hospitals. All pathology slides were reviewed by specialists from the Department of Anatomical Pathology at the Royal Prince Alfred Hospital who had extensive experience in melanoma pathology. Metastases developed in 164 of 2761 patients with Clark level 2 CMMs who had only a single Stage I or II lesion (American Joint Committee on Cancer/International Union Against Cancer staging system) over a follow-up period of 1–45 years. Approximately 22% of these metastasizing CMMs were > 1 mm thick, with 50% of the latter having a polypoid architectural pattern. Early, intermediate, or late regression was found in 73% of metastasizing level 2 CMMs, a figure that was nearly identical to the proportion with regression in 2597 nonmetastasizing level 2 CMMs (74%). In the majority of metastasizing level 2 CMMs displaying regression (57%), the regression was at the early stage. We previously have shown that in very thin CMMs (< 0.5 mm), evidence of regression did not constitute a risk for recurrence.2 Part of the discrepancy between the current results and those of Taran and Heenan appears to lie in pathologic definitions and interpretation of Clark levels 2 and 3 and the presence or absence of regression. Interobserver variability in reporting the level of invasion is referred to by Taran and Heenan.1 Some of our metastasizing level 2 CMMs were > 1 mm thick and some of these were polypoid. Such CMMs may be associated with expansion but not filling of the papillary dermis by melanoma cells and the cells may not reach the papillary/reticular dermal interface and hence can be designated as level 2. Although we accept that some authors regard such polypoid CMMs as level 3,3, 4 applying the criteria of Clark et al.5 (as used by Taran and Heenan in their study1), such CMMs are level 2. Furthermore, some of our metastasizing level 2 CMMs measuring > 1 mm thick were acral lesions, which may become this thick as a result of their epidermal thickening. The definition of regression used by Taran and Heenan1 encompasses the definitions of intermediate and late regression as used by the SMU. Because our analysis also included CMMs with early regression (defined as the presence of tumor-infiltrating lymphocytes), the proportion of CMMs displaying regression in our metastasizing level 2 CMMs was not comparable to that in the study by Taran and Heenan.1 The SMU is a major referral center for melanoma in New South Wales and therefore is not population-based like the Cancer Registry of Western Australia. However, it appears from our results that metastases from level 2 CMMs are not as uncommon as maintained by Taran and Heenan.1 Although the majority of these metastasizing CMMs displayed regression, this alone could not explain their metastatic potential. We conclude that it is injudicious to dismiss level 2 CMMs as having practically no metastatic potential.

Despite exciting, recent major advances in the development and clinical application of immune checkpoint and signalling pathway targeted therapies for patients with advanced stage disease; melanoma remains a major public health problem. Not only is it one of the commonest malignancies in young adults in many Western countries (Thompson et al., 2009); in contrast to most other cancers, it possesses an innate capacity to metastasis when at a very early clinical stage. In fact, many metastasising melanomas are less than 1 mm thick at the time when the primary tumour is first recognized. For these reasons, much scientific research has focused on the pathogenic mechanisms underlying melanoma metastasis. Metastases develop following a complex series of interactions between the tumour and the host microenvironment. The classical invasion and metastasis paradigm are typically portrayed as commencing with adhesion to and invasion through the basement membrane around the primary tumour, followed by passage through the extracellular matrix, invasion of vessels, lymphatic and/or haematogenous dissemination and ending in extravasation and colonization and growth of the metastatic deposit at a new site separated from the primary tumour. Over the past decade or so, an increasing body of research, much of which has been championed by Barnhill and Lugassy and underpinned by careful and meticulous pathological observations, has recognized that melanoma may also metastasise by utilizing some of the migratory systems employed by neural crest cells, the precursors of melanocytes (reviewed by Lugassy et al., 2013). During embryogenesis, neural crest cells are in part guided along their migratory route from the dorsal most region of the neural tube by a network of nascent vessels (Lugassy et al., 2013). Likewise, angiotropism in melanoma, defined as tropism of cells for small and medium-sized vessels (Figure 1), has been identified as a potential route for both local recurrence and distant spread (Van Es et al., 2008; Wilmott et al., 2012). This process, whereby melanoma cells track along the outside of vessels in a pericytic location, termed extravascular migratory metastasis (EVMM)(Lugassy et al., 2013), has also been associated with the acquisition of embryonic properties promoting melanoma plasticity and invasion (Lugassy et al., 2013). In a recent study published in the journal Nature, Bald et al. identified a mechanism capable of both inducing the migration of melanoma cells into the perivascular compartment and promoting metastasis. They showed that a neutrophil-dependent inflammatory response to repeated ultraviolet (UV)-induced keratinocyte damage results in enhancement of the migratory and static capacity of melanoma (Figure 2). A complex relationship exists between UV radiation and melanoma. The results of the study by Bald et al. encourage distinction between the effects of UV radiation as a major aetiological factor for primary melanoma development on the one hand and its effects as a promotor of invasion and metastasis in established tumours on the other. They discovered that in chemically induced murine melanomas, repeated UV exposure did not affect the growth capacity of the primary tumour at the irradiated cutaneous site. Instead, the observed effect was an increase in the number of lung metastases compared with the non-UV-irradiated melanoma group. The UV-irradiated primary murine melanomas also showed increased co-localization of tumour cells with the abluminal surface of dermal blood vessels, that is, angiotropism. The number of lung metastases was positively correlated with the degree of both neutrophilic inflammation and perivascular invasion. These factors, together with others, including primary tumour ulceration, were also recognized as poor prognostic factors in the authors' concurrent analysis of 178 unselected sentinel lymph node-staged patients with primary cutaneous melanoma. The results of Bald et al. may also provide a biological basis for the adverse prognostic effect of primary melanoma ulceration, via its potent stimulation of an acute inflammatory response. Bald et al.'s study also employed a combination of murine knock-outs of four key effectors of the Toll-like receptor signalling pathway to uncover the mechanism coupling UV irradiation with neutrophil infiltration. The UV-induced recruitment of neutrophils was identified as being highly dependent on the function of the myeloid differentiation primary-response gene 88 (MyD88)-dependent pathway mediated by activation of Toll-like receptor 4 (TLR4), but not the TLR3-TRIF MyD88-independent pathway. In this system, activation of TLR4 is triggered by binding to the high-mobility group box 1 (HMGB1). This HMGB1/TLR4-MyD88 signalling axis was also shown to be a requisite for the UV irradiation-induced increase in both the metastatic potential and angiotropic invasion of skin transplanted melanoma cell lines HCmel12 and HCmel31. The authors then turned their attention to the interaction between melanoma and endothelium under ex vivo inflammatory conditions. Using transwell assays, Bald et al. showed that either neutrophil-conditioned media, or tissue necrosis factor (TNF) alone, could induce the migration of HCmel12 cells towards endothelium. In particular, TNF enhanced migration distance and velocity of melanoma cells along endothelium when compared to spread along keratinocytes or extracellular matrix extracts. These findings were also supported by two other observations: (i) in aortic ring explants, TNF in neutrophil-conditioned media promoted endothelial sprouting and melanoma co-migration; (ii) HCmel12 cells introduced into mouse ear explants showed marked angiotropism, but only in the presence of UV-induced skin inflammation. The human corollary of this was the observation that patient-derived BRAF and RAS mutant cell lines showed the same enhanced migratory properties in the presence of TNF. Taken together, these experiments support the notion that neutrophil-mediated inflammation promotes melanoma angiotropism. Finally, the authors confirmed the presence of a TNF-response gene expression signature in melanoma cell lines, involving regulators of tumour-endothelial cell interactions and angiogenesis. The results of Bald et al.'s study may appear at odds with the clinical studies reporting more favourable outcomes in melanomas associated with cumulative sun exposure and evidence of a lower mortality rate for melanomas diagnosed in summer. However, a similar seasonal effect is observed in patients with other tumours, including colorectal, prostate, lung and breast cancer implicating UV-induced systemic effects, possibly through the anti-tumour properties of enhanced vitamin D production. Interestingly, in their disease model, Bald et al. prove TNF is responsible for enabling the pro-invasive and pro-metastatic effects of UV-induced inflammation. This stands in contrast to recent findings identifying the use of tissue necrosis factor inhibitors, in the context of immunomodulation in patients with rheumatoid arthritis, as being at increased risk of developing invasive melanoma. Still, the identification of Toll-like receptor signalling as being a key mediator of angiotropism and metastasis opens up a potential avenue of research into the use of TLR4 antagonists as a therapeutic strategy for melanoma. “provide further evidence of the central role UV radiation plays not only in the initiation but also the promotion of melanomagenesis”

In patients with metastatic melanoma, the identification and validation of accurate prognostic biomarkers will assist rational treatment planning. Studies based on "-omics" technologies have focussed on a single high-throughput data type such as gene or microRNA transcripts. Occasionally, these features have been evaluated in conjunction with limited clinico-pathologic data. With the increased availability of multiple data types, there is a pressing need to tease apart which of these sources contain the most valuable prognostic information. We evaluated and integrated several data types derived from the same tumor specimens in AJCC stage III melanoma patients-gene, protein, and microRNA expression as well as clinical, pathologic and mutation information-to determine their relative impact on prognosis. We used classification frameworks based on pre-validation and bootstrap multiple imputation to compare the prognostic power of each data source, both individually as well as integratively. We found that the prognostic utility of clinico-pathologic information was not out-performed by any of the various "-omics" platforms. Rather, a combination of clinico-pathologic variables and mRNA expression data performed best. Furthermore, a patient-based classification analysis revealed that the prognostic accuracy of various data types was not the same for different patients. This indicates that ongoing development in the individualized evaluation of melanoma patients must take account of the value of both traditional and novel "-omics" measurements.