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Background: Mucosal melanomas in the head and neck region are most frequently located in the nasal cavity and paranasal sinuses. Sinonasal mucosal melanoma (SNMM) comprises

Sinonasal malignant melanoma (SNMM) comprises less than 1% of all melanomas and is located in the nasal cavity and the paranasal sinuses. The majority of SNMMs have unknown underlying oncogenic driver mutations. The recent identification of a high frequency of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene in cutaneous melanoma led us to investigate whether these mutations also occur in SNMM. Our aim was to determine the TERT promoter mutation frequencies in primary SNMMs. Laser capture microdissection and manual dissection were used to isolate tumour cells from 49 formalin-fixed paraffin-embedded tissues. The tumours were screened for TERT promoter mutations by direct Sanger sequencing. Information on NRAS, BRAF and KIT mutation was available from an earlier study. Overall, 8% (4/49) of SNMMs harboured TERT promoter mutations. One of these mutated tumours had a coexistent NRAS mutation and one had a BRAF mutation. Our findings show that TERT promoter mutations are present in a moderate proportion of SNMM. No conclusion can be drawn on their potential influence on the clinical outcome or tumour progression.

Importance BRAF V600E mutations are present in approximately 50% of cutaneous malignant melanomas (CMMs). The use of BRAF V600E mutation–specific monoclonal antibody VE1 immunohistochemical analysis may facilitate rapid detection of BRAF V600E mutations in CMMs and demonstrate heterogeneity among tumors. Objectives To characterize the pattern of BRAF V600E protein expression in primary CMMs with matched metastases and to analyze the use of VE1 immunohistochemical analysis in clinical practice using formalin-fixed, paraffin-embedded tumor tissue. Design, Setting, and Participants In this retrospective cohort study performed at Karolinska University Hospital from September 2012 to September 2013, we examined CMMs (124 primary tumors and 76 metastases) with VE1 immunohistochemical analysis, and results were compared with DNA mutation analyses. Main Outcomes and Measures Determination of intratumoral and intertumoral heterogeneity as well as the sensitivity and specificity of VE1 immunohistochemical analysis. Results Positive staining results with the VE1 antibody were detected in 94 of 200 tumors (47.0%). In general, VE1 staining was homogeneous. However, VE1 staining intensity varied among the primary tumors and corresponding metastases in 63 of 135 tumors (46.7%), but a change of mutational status based on DNA analysis was found in only 4 matched tumors (3.0%). Discordant findings between DNA mutation analysis and immunohistochemical analysis were observed in 12 tumors. The overall sensitivity and specificity of VE1 immunohistochemical analysis were 96.7% and 94.5%, respectively. A comparable sensitivity was obtained for primary and metastatic CMMs. The specificity was lower among primary CMMs (92.4%) compared with metastases (98.0%). Conclusions and Relevance We found VE1 immunohistochemical analysis to be a useful and rapid assay for BRAF V600E mutations that may contribute to the detection of intratumoral and intertumoral heterogenetic subclones. Tumors with positive results, including strong staining, should be expedited for confirmatory BRAF mutation testing. If this test result is negative, a false-negative result of the mutation analysis should be considered. Validation of VE1 immunohistochemical analysis in clinical practice is needed.

8588 Background: Acral lentiginous melanoma (ALM) accounts for ~5% of all melanomas. ALMs are often located on palms, soles and under nails. Patients with ALM are significantly older at diagnosis and have a poorer prognosis than patients with other types of cutaneous melanoma. We analyzed a large series of ALMs from Swedish patients to better define the frequency of KIT, BRAF, NRAS, and PTEN mutations in ALM. The effect of mutation status on tumor and patients characteristics was also studied. Methods: A total of 77 primary ALMs and 8 paired metastases were analyzed. Laser capture microdissection was used to dissect tumor cells from formalin-fixed paraffin-embedded tissue. Tumors were screened for mutations in the KIT (exons 11, 13, 17 and 18), NRAS (exons 1 and 2), BRAF (exons 11 and 15) and PTEN (exons 1, 3-6, 10, 11 and 12) genes by direct sequencing. Results: There were 42 females and 35 males with a median age at diagnosis of 71 years. The most common location was the feet (60.5%), followed by subungual sites (34.2%). Overall, mutations in KIT, NRAS and BRAF were detected in 11.8%, 14.5% and 16.0% of the ALMs, respectively. In no case were KIT, NRAS and BRAF mutations detected in the same tumor. For 8 primary ALMs the corresponding metastases were available for analysis. In five metastases the same KIT (n=3) or BRAF (n=2) mutation was detected as in the primary ALMs, suggesting that the KIT and BRAF mutations had occurred before metastasis. BRAF and NRAS mutations were significantly more common in females (P=0.044). ALMs with NRAS and BRAF mutations were more commonly located on the feet compared with KIT mutated ALMs which were more frequent on subungual sites (P=0.041). Other clinicopathological features such as age at diagnosis, thickness, ulceration, histological subtype and Clark’s level showed no significant correlation with the mutation status. A subset of 25 tumors was evaluated for mutations in the PTEN tumor suppressor gene. One tumor was found to carry a nonsense mutation (W111X). To our knowledge, this is the first description of a PTEN mutation in ALM. Conclusions: Our results confirm the presence of KIT, NRAS and BRAF mutations in ALM. KIT, NRAS and BRAF mutation status associated significantly with anatomical site. PTEN mutations seem to be rare in ALMs.

Up to 10% of all melanoma patients show a family history of disease. One main predisposing gene for familial melanoma development is CDKN2A, a tumor suppressor gene that encodes two distinct proteins, p16INK4A and p14ARF. CDKN2A germline mutations have been found in 20-40% of melanoma-prone families. So far, BRAF and NRAS are the most frequently altered oncogenes described in cutaneous melanoma. To date, little is known about the somatic changes that occur in familial melanoma. The aim of this study was to determine the prevalence of BRAF and NRAS somatic mutations in familial melanomas and to evaluate the associations between these mutations and clinicopathological factors. The study was performed as a collaboration within GenoMEL (The Melanoma Genetics Consortium). A total of 135 primary familial cutaneous melanomas (88 tumors from CDKN2A mutation-carriers and 47 from CDKN2A non-carriers) were investigated. Paraffin-blocks of tumors were collected from 8 GenoMEL centers in Australia, Italy, Latvia, the Netherlands, Spain, Sweden, and UK. Tumor cells were isolated by laser capture microdissection and mutation screening (BRAF exon 15 and NRAS exon 3) was performed by direct sequencing. Mutation analysis revealed BRAF and NRAS mutations in 41.6% and 11.4% of tumors, respectively. In no case were BRAF and NRAS mutations found to coexist in the same tumor. There was no difference in BRAF mutation frequency between tumors from patients with CDKN2A alterations and tumors from patients wild-type for CDKN2A (41.0% vs. 42.9%). Similarly, the frequency of NRAS mutations did not differ markedly when tumors from CDKN2A mutation-carriers and non-carriers were compared (9.0% vs. 15.6%). There was also no difference in the frequency of BRAF mutations between tumors with CDKN2A mutations disrupting both p16INK4A and p14ARF and tumors with CDKN2A mutations affecting p16INK4A only (40.6% vs. 36.4%). When tumors with different CDKN2A founder mutations were compared, BRAF mutations were seen in 50.0%, 45.8%, 37.5% and 35.0% of p.M53I, p.R112_L113insR, p.G101W and c.225_243del19 altered tumors, respectively. NRAS mutations were observed in 14.3% 12.5% and 10.5% of p.R112_L113insR, p.M53I and c.225_243del19 altered tumors, respectively, whereas no NRAS mutations were seen in tumors with the p.G101W founder mutation. Patients with BRAF-mutated tumors had a significantly lower median age at diagnosis compared to patients with NRAS-mutated or BRAF/NRAS wild-type tumors (43.5, 54 and 51.5 years, respectively). Moreover, melanomas with mutated BRAF or NRAS were thicker than BRAF/NRAS wild-type cases (median thickness 1.0, 1.4 and 0.7mm, respectively). Our results suggest a role for the BRAF and NRAS oncogenes in familial melanoma. Somatic changes of these genes occur in tumors from both CDKN2A mutation carriers and non-carriers. Presence of BRAF or NRAS mutations associated with patient and tumor characteristics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 867. doi:10.1158/1538-7445.AM2011-867