Prognostic significance of tumor iNOS and COX-2 in stage III malignant cutaneous melanoma

Purpose  New prognostic markers are needed for malignant melanoma. Inducible nitric oxide synthase (iNOS) and cyclooxygenase type 2 (COX-2) have been described to correlate with progression of melanoma. Moreover, activating mutations in BRAF/NRAS oncogenes are often detected in melanoma. The BRAF/NRAS mutation status and expression of COX-2 and iNOS were examined to compare their prognostic value for overall survival (OS) in stage III malignant cutaneous melanoma. Experimental design  The expression of iNOS and COX-2 in metastatic lymph nodes from 21 rapidly progressing (OS from date of diagnosis of stage III disease ≤14 months) and 17 slowly progressing (OS ≥60 months) stage III cutaneous melanoma patients was examined by immunohistochemistry. The presence of BRAF/NRAS mutations was analyzed using direct DNA sequencing. χ² exact trend test and logistic regression analysis were used for statistical analysis. Results  Both iNOS (P = 0.002) and COX-2 (P = 0.048) alone significantly predicted OS. The BRAF/NRAS mutation status did not significantly differ between patient groups, although iNOS significantly (P = 0.013) correlated with BRAF mutation frequency. Furthermore, the odds ratio (OR) with respect to OS of iNOS (OR = 10.4) was higher than that of COX-2 (OR = 5.6) and was stable in the multivariate analysis of OS together with disease stage IIIB/C, ulceration, number of metastatic lymph nodes, and Breslow tumor thickness. Conclusion  Our data show that iNOS is an independent and stronger prognostic factor for OS in stage III malignant cutaneous melanoma than COX-2.     

Neuroblastoma rat sarcoma mutated melanoma: That’s what we got so far

Neuroblastoma rat sarcoma (NRAS) mutation, occurring in about 20%–30% of cutaneous melanomas, leads to activation of RAS‐RAF‐MAPK cascade and represents a clear distinct clinicopathological entity in melanoma. In contrast with BRAF mutant melanoma, no specific target therapies are available outside the setting of clinical trials. In the field of immunoncology, the predictive role of NRAS mutation with respect to checkpoint inhibitors treatment has not clearly established and deserves further investigation. At present, the standard treatment is the same as for BRAF wild type melanoma. Ongoing trials are exploring novel combination strategies among patients with advanced NRAS mutant melanoma.     

Clinicopathological characteristics associated with BRAFK601E and BRAFL597 mutations in melanoma

BRAF mutations at codons L597 and K601 occur uncommonly in melanoma. Clinical and pathological associations of these mutations were investigated in a cohort of 1119 patients with known BRAF mutation status. A BRAF mutation was identified in 435 patients; Mutations at L597 and the K601E mutation were seen in 3.4 and 3.2% of these, respectively. K601E melanomas tended to occur in male patients, a median age of 58 yr, were generally found on the trunk (64%) and uncommonly associated with chronically sun‐damaged (CSD) skin. BRAF L597 melanomas occurred in older patients (median 66 yr), but were associated with CSD skin (extremities or head and neck location – 73.3%, P = 0.001). Twenty‐three percent of patients with V600E‐ and 43% of patients with K601E‐mutant melanomas presented with nodal disease at diagnosis compared to just 14% of patients with BRAF wild‐type tumors (P = 0.001 and 0.006, respectively). Overall, these mutations represent a significant minority of BRAF mutations, but have distinct clinicopathological phenotypes and clinical behaviors.     

Distinct genomic traits of acral and mucosal melanomas revealed by targeted mutational profiling

The incidence of melanoma is rising globally including China. Comparing to Caucasians, the incidence of non‐cutaneous melanomas is significantly higher in Chinese. Herein, we performed genomic profiling of 89 Chinese surgically resected primary melanomas, including acral (n = 54), cutaneous (n = 22), and mucosal (n = 13), by hybrid capture‐based next‐generation sequencing. We show that mucosal melanomas tended to harbor more pathogenic mutations than other types of melanoma, though the biological significance of this finding remains uncertain. Chromosomal arm‐level alterations including 6q, 9p, and 10p/q loss were highly recurrent in all subtypes, but mucosal melanoma was significantly associated with increased genomic instability. Importantly, 7p gain significantly correlated with unfavorable clinical outcomes in non‐cutaneous melanomas, representing an intriguing prognostic biomarker of those subtypes. Furthermore, focal amplification of 4q12 (KIT, KDR, and PDGFRα) and RAD51 deletion were more abundant in mucosal melanoma, while NOTCH2 amplification was enriched in acral melanoma. Additionally, cutaneous melanomas had higher mutation load than acral melanomas, while mucosal melanomas did not differ from other subtypes in mutation burden. Together, our data revealed important features of acral and mucosal melanomas in Chinese including distinctive driver mutation pattern and increased genomic instability. These findings highlight the possibilities of combination therapies in the clinical management of melanoma.     

Genetic and morphologic features for melanoma classification

Melanoma is comprised of biologically distinct subtypes. The defining clinical, histomorphologic, and molecular features are not fully established. This study sought to validate the association between genetic and histomorphologic features previously described and to determine their reproducibility and association with important clinical variables. Detailed clinical and histomorphologic features of 365 primary cutaneous melanomas were assessed by 11 pathologists and correlated with mutation status of BRAF and NRAS. There was substantial agreement in the quantitative assessment of histomorphologic features showing similar or better interobserver reproducibility than the established World Health Organization classification scheme. We confirmed that melanomas with BRAF mutations showed characteristic morphologic features (P < 0.0001) and metastasized more frequently to regional lymph nodes (P = 0.046). Importantly, melanomas without mutations were a heterogeneous group, with a subset having very similar clinical and morphological features as those with BRAF mutation raising the possibility that they are biologically related. Our study confirms an association between histomorphologic features, mutation status, and pattern of metastasis, providing criteria for a refined melanoma classification aimed at defining biologically homogeneous disease subgroups.     

NRAS and BRAF Mutations in Melanoma-Associated Nevi and Uninvolved Nevi

According to the prevailing multistep model of melanoma development, oncogenic BRAF or NRAS mutations are crucial initial events in melanoma development. It is not known whether melanocytic nevi that are found in association with a melanoma are more likely to carry BRAF or NRAS mutations than uninvolved nevi. By laser microdissection we were able to selectively dissect and genotype cells either from the nevus or from the melanoma part of 46 melanomas that developed in association with a nevus. In 25 cases we also genotyped a control nevus of the same patients. Available tissue was also immunostained using the BRAF^V600E-mutation specific antibody VE1. The BRAF^V600E mutation was found in 63.0% of melanomas, 65.2% of associated nevi and 50.0% of control nevi. No significant differences in the distribution of BRAF or NRAS mutations could be found between melanoma and associated nevi or between melanoma associated nevi and control nevi. In concordant cases immunohistochemistry showed a higher expression (intensity of immunohistochemistry) of the mutated BRAF^V600E-protein in melanomas compared to their associated nevi. In this series the presence of a BRAF- or NRAS mutation in a nevus was not associated with the risk of malignant transformation. Our findings do not support the current traditional model of stepwise tumor progression.     

Genomic analysis reveals low tumor mutation burden which may be associated with GNAQ/11 alteration in a series of primary leptomeningeal melanomas

Primary central nervous system melanoma is rare and characterized by a variable prognosis, and no current treatment guidelines exist. We describe the clinical course of a 70‐year‐old female patient diagnosed with primary leptomeningeal melanoma (LMN) whose case represents the diagnostic and management challenges of this tumor. Targeted genomic sequencing of 315 genes from this tumor revealed GNAQ Q209L mutation and low (4 mutations/Megabase) tumor mutation burden (TMB). Wild‐type NRAS, KIT, and BRAF were also observed. A cohort of 4,787 melanomas was subsequently analyzed to identify additional primary central nervous system melanomas, of which 10 additional tumors met pathologic criteria (0.21% of total melanoma cohort). These tumors were genomically assessed according to the same targeted sequencing panel, and 6 of the tumors were also found to harbor a GNAQ mutation. All 10 tumors had low (less than or equal to 2 mutations/Megabase) TMB indicating a potential trend between G‐protein‐coupled receptor (GPCR) alterations and low TMB in LMNs. GPCR alterations were found to significantly correlate with TMB across the cohort of 4,787 melanomas, supporting this potential finding in the limited LMN subset.     

Genomic characterisation of acral melanoma cell lines

Acral melanoma is a rare melanoma subtype with distinct epidemiological, clinical and genetic features. To determine if acral melanoma cell lines are representative of this melanoma subtype, six lines were analysed by whole-exome sequencing and array comparative genomic hybridisation. We demonstrate that the cell lines display a mutation rate that is comparable to that of published primary and metastatic acral melanomas and observe a mutational signature suggestive of UV-induced mutagenesis in two of the cell lines. Mutations were identified in oncogenes and tumour suppressors previously linked to melanoma including BRAF, NRAS, KIT, PTEN and TP53, in cancer genes not previously linked to melanoma and in genes linked to DNA repair such as BRCA1 and BRCA2. Our findings provide strong circumstantial evidence to suggest that acral melanoma cell lines and acral tumours share genetic features in common and that these cells are therefore valuable tools to investigate the biology of this aggressive melanoma subtype. Data are available at: http://rock.icr.ac.uk/collaborations/Furney_et_al_2012/.     

Mutations in KIT occur at low frequency in melanomas arising from anatomical sites associated with chronic and intermittent sun exposure

In melanoma, mutations in KIT are most frequent in acral and mucosal subtypes and rarely reported in cutaneous melanomas particularly those associated with intermittent UV exposure. Conversely melanomas arising within chronic sun damaged skin are considered to harbour KIT mutations at higher rates. To characterize the frequency of KIT mutations in a representative melanoma population, 261 patients from two Australian melanoma centres were prospectively screened for mutations in exons 11, 13 and 17 of the KIT gene. A total of 257 patients had cutaneous melanoma arising from non-acral sites and four were acral melanomas. No mucosal or ocular melanomas were analysed. KIT mutations were identified in five tumours (2% of the entire cohort) including two acral melanomas. Two of the three non-acral melanomas with KIT mutations were associated with markers of chronic sun damage as assessed by the degree of skin elastosis. In the remaining cohort, 43% had chronically sun damaged skin. This report confirms that within an Australian population, KIT mutations are infrequent in cutaneous melanomas associated with both intermittent and chronic sun exposed skin.     

CDKN2A mutations and MC1R variants in Italian patients with single or multiple primary melanoma

We evaluated the contribution of germline CDKN2A mutations and MC1R variants to the development of melanoma in a hospital-based study of single (SPM, n = 398) and multiple primary melanoma (MPM, n = 95). The overall frequency of CDKN2A mutations was 15.2%, and four-fold higher in MPM than in SPM cases (OR = 4.27; 95% CI 2.43-7.53). The likelihood of identifying a CDKN2A mutation increased with family history of melanoma and younger age at diagnosis in MPM cases. Compared to SPM patients, the risk of harboring a CDKN2A mutation rose as the number of primary melanomas increased and was not influenced by family history. The G101W and E27X founder mutations were the most common. Several other mutations (W15X, Q50X, R58X, A68L, A127P and H142R) were detected for the first time in Italian patients. One novel mutation, T77A, was identified. Several non-coding variants with unknown functional significance were also found (5'UTR -25C > T, -21C > T, -67G > C, IVS1 +37G > C); the novel 5'UTR -21C > T variant was not detected in controls. The CDKN2A A148T polymorphism was more frequent in MPM patients than in the control population (15.7% versus 6.6%). Compared to the SPM patients, MPM cases had a 2-fold increased probability of being MC1R variant carriers and a higher probability of carrying two or more variants. No specific association was observed between the type of variant and the number of melanomas, suggesting that the number rather than the type of MC1R variant increases the risk of MPM. We observed no interaction between CDKN2A status and the presence of MC1R variants. The high frequency of CDKN2A mutations in our MPM cases, independent of their family history, is of relevance to genetic counseling and testing in our population.     

Genomic analysis and clinical management of adolescent cutaneous melanoma

Melanoma in young children is rare; however, its incidence in adolescents and young adults is rising. We describe the clinical course of a 15‐year‐old female diagnosed with AJCC stage IB non‐ulcerated primary melanoma, who died from metastatic disease 4 years after diagnosis despite three lines of modern systemic therapy. We also present the complete genomic profile of her tumour and compare this to a further series of 13 adolescent melanomas and 275 adult cutaneous melanomas. A somatic BRAF^V600E mutation and a high mutational load equivalent to that found in adult melanoma and composed primarily of C>T mutations were observed. A germline genomic analysis alongside a series of 23 children and adolescents with melanoma revealed no mutations in known germline melanoma‐predisposing genes. Adolescent melanomas appear to have genomes that are as complex as those arising in adulthood and their clinical course can, as with adults, be unpredictable.     

Analysis of BRAF and NRAS Mutation Status in Advanced Melanoma Patients Treated with Anti-CTLA-4 Antibodies: Association with Overall Survival?

Ipilimumab and tremelimumab are human monoclonal antibodies (Abs) against cytotoxic T-lymphocyte antigen-4 (CTLA-4). Ipilimumab was the first agent to show a statistically significant benefit in overall survival in advanced melanoma patients. Currently, there is no proven association between the BRAFV600 mutation and the disease control rate in response to ipilimumab. This analysis was carried out to assess if BRAFV600 and NRAS mutation status affects the clinical outcome of anti-CTLA-4-treated melanoma patients. This is a retrospective multi-center analysis of 101 patients, with confirmed BRAF and NRAS mutation status, treated with anti-CTLA-4 antibodies from December 2006 until August 2012. The median overall survival, defined from the treatment start date with the anti-CTLA-4. Abs-treatment to death or till last follow up, of BRAFV600 or NRAS mutant patients (n = 62) was 10.12 months (95% CI 6.78–13.2) compared to 8.26 months (95% CI 6.02–19.9) in BRAFV600/NRASwt subpopulation (n = 39) (p = 0.67). The median OS of NRAS mutated patients (n = 24) was 12.1 months and although was prolonged compared to the median OS of BRAF mutated patients (n = 38, mOS = 8.03 months) or BRAFV600/NRASwt patients (n = 39, mOS = 8.26 months) the difference didn’t reach statistical significance (p = 0.56). 69 patients were able to complete 4 cycles of anti-CTLA-4 treatment. Of the 24 patients treated with selected BRAF- or MEK-inhibitors, 16 patients received anti-CTLA 4 Abs following either a BRAF or MEK inhibitor with only 8 of them being able to finish 4 cycles of treatment. Based on our results, there is no difference in the median OS in patients treated with anti-CTLA-4 Abs implying that the BRAF/NRAS mutation status alone is not sufficient to predict the outcome of patients treated with anti-CTLA-4 Abs.     

Melanomas of unknown primary have a mutation profile consistent with cutaneous sun‐exposed melanoma

Melanoma of unknown primary (MUP) is an uncommon phenomenon whereby patients present with metastatic disease without an evident primary site. To determine their likely site of origin, we combined exome sequencing from 33 MUPs to assess the total rate of somatic mutations and degree of UV mutagenesis. An independent cohort of 91 archival MUPs was also screened for 46 hot spot mutations highly prevalent in melanoma including BRAF, NRAS, KIT, GNAQ, and GNA11. Results showed that the majority of MUPs exhibited high somatic mutation rates, high ratios of C>T/G>A transitions, and a high rate of BRAF (45 of 101, 45%) and NRAS (32 of 101, 32%) mutations, collectively indicating a mutation profile consistent with cutaneous sun‐exposed melanomas. These data suggest that a significant proportion of MUPs arise from regressed or unrecognized primary cutaneous melanomas or arise de novo in lymph nodes from nevus cells that have migrated from the skin.     

Clinical and pathological associations of the activating RAC1 P29S mutation in primary cutaneous melanoma

Activating mutations in the GTPase RAC1 are a recurrent event in cutaneous melanoma. We investigated the clinical and pathological associations of RAC1^P29S in a cohort of 814 primary cutaneous melanomas with known BRAF and NRAS mutation status. The RAC1^P29S mutation had a prevalence of 3.3% and was associated with increased thickness (OR=1.6 P = 0.001), increased mitotic rate (OR=1.3 P = 0.03), ulceration (OR=2.4 P = 0.04), nodular subtype (OR=3.4 P = 0.004), and nodal disease at diagnosis (OR=3.3 P = 0.006). BRAF mutant tumors were also associated with nodal metastases (OR=1.9 P = 0.004), despite being thinner at diagnosis than BRAF WT (median 1.2 mm versus 1.6 mm, P < 0.001). Immunohistochemical analysis of 51 melanomas revealed that 47% were immunoreactive for RAC1. Melanomas were more likely to show RAC1 immunoreactivity if they were BRAF mutant (OR=5.2 P = 0.01). RAC1 may therefore be important in regulating the early migration of BRAF mutant tumors. RAC1 mutations are infrequent in primary melanomas but may accelerate disease progression.     

AEBP1 upregulation confers acquired resistance to BRAF (V600E) inhibition in melanoma

An activating BRAF (V600E) kinase mutation occurs in approximately half of melanomas. Recent clinical studies have demonstrated that vemurafenib (PLX4032) and dabrafenib, potent and selective inhibitors of mutant v-raf murine sarcoma viral oncogene homolog B1 (BRAF), exhibit remarkable activities in patients with V600 BRAF mutant melanomas. However, acquired drug resistance invariably develops after the initial treatment. Identification of acquired resistance mechanisms may inform the development of new therapies that elicit long-term responses of melanomas to BRAF inhibitors. Here we report that increased expression of AEBP1 (adipocyte enhancer-binding protein 1) confers acquired resistance to BRAF inhibition in melanoma. AEBP1 is shown to be highly upregulated in PLX4032-resistant melanoma cells because of the hyperactivation of the PI3K/Akt-cAMP response element-binding protein (CREB) signaling pathway. This upregulates AEBP1 expression and thus leads to the activation of NF-κB via accelerating IκBa degradation. In addition, inhibition of the PI3K/Akt-CREB-AEBP1-NF-κB pathway greatly reverses the PLX4032-resistant phenotype of melanoma cells. Furthermore, increased expression of AEBP1 is validated in post-treatment tumors in patients with acquired resistance to BRAF inhibitor. Therefore, these results reveal a novel PI3K/Akt-CREB-AEBP1-NF-κB pathway whose activation contributes to acquired resistance to BRAF inhibition, and suggest that this pathway, particularly AEBP1, may represent a novel therapeutic target for treating BRAF inhibitor-resistant melanoma.     

Sporadic naturally occurring melanoma in dogs as a preclinical model for human melanoma

Melanoma represents a significant malignancy in humans and dogs. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant preclinical model. Canine melanomas rarely arise in sun‐exposed sites. Most occur in the oral cavity, with a subset having intra‐epithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma. The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ‐line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs. Canine and human mucosal melanomas appear to harbor BRAF, NRAS, and c‐kit mutations uncommonly, compared with human cutaneous melanomas, although both species share AKT and MAPK signaling activation. We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas. This represents opportunity to explore canine oral cavity melanoma as a preclinical model.     

Prognostic biomarkers for survival in mucosal melanoma

Mucosal melanoma (MM) is a rare subtype of melanoma with an aggressive clinical course. In cutaneous melanoma (CM), the absence of pigmentation and presence of NRAS/KRAS mutations are biomarkers indicating an aggressive clinical course with shorter overall survival. Similar data for MM are missing. We present the real-world outcome data in a cohort of genotyped MM patients and assessed the prognostic relevance of pigmentation- and NRAS/KRAS mutation status. We correlated pathological reports and clinical data with overall survival of patients with MM. Furthermore, we performed clinically integrated molecular genotyping and analyzed real world treatment regimens for covariates associated with clinical outcome. We identified 39 patients with available clinical and molecular data. Patients with amelanotic MM had a significantly shorter overall survival (p = .003). In addition, the presence of a NRAS or KRAS mutation was significantly associated with poor overall survival (NRAS or KRAS p = .024). Currently, it is unknown if the same prognostic relevance for the lack of pigmentation and RAS mutations in CM, exists in MM. Here we analyzed a cohort of MM for outcome measures and determined that two known prognostic biomarkers for CM are in fact novel prognosticators for MM.     

Mitogen-activated Protein Kinase (MAPK) Hyperactivation and Enhanced NRAS Expression Drive Acquired Vemurafenib Resistance in V600E BRAF Melanoma Cells

Although targeting the V600E activating mutation in the BRAF gene, the most common genetic abnormality in melanoma, has shown clinical efficacy in melanoma patients, response is, invariably, short lived. To better understand mechanisms underlying this acquisition of resistance to BRAF-targeted therapy in previously responsive melanomas, we induced vemurafenib resistance in two V600E BRAF+ve melanoma cell lines, A375 and DM443, by serial in vitro vemurafenib exposure. The resulting approximately 10-fold more vemurafenib-resistant cell lines, A375rVem and D443rVem, had higher growth rates and showed differential collateral resistance to cisplatin, melphalan, and temozolomide. The acquisition of vemurafenib resistance was associated with significantly increased NRAS levels in A375rVem and D443rVem, increased activation of the prosurvival protein, AKT, and the MAPKs, ERK, JNK, and P38, which correlated with decreased levels of the MAPK inhibitor protein, GSTP1. Despite the increased NRAS, whole exome sequencing showed no NRAS gene mutations. Inhibition of all three MAPKs and siRNA-mediated NRAS suppression both reversed vemurafenib resistance significantly in A375rVem and DM443rVem. Together, the results indicate a mechanism of acquired vemurafenib resistance in V600E BRAF+ve melanoma cells that involves increased activation of all three human MAPKs and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated with mutations in the NRAS gene. The data highlight the complexity of the acquired vemurafenib resistance phenotype and the challenge of optimizing BRAF-targeted therapy in this disease. They also suggest that targeting the MAPKs and/or NRAS may provide a strategy to mitigate such resistance in V600E BRAF+ve melanoma.