Targeted DNA Sequencing of Cutaneous Melanoma Identifies Prognostic and Predictive Alterations.

BACKGROUND: Cutaneous melanoma (CM) can be molecularly classified into four groups: BRAF mutant, NRAS mutant, NF1 mutant and triple wild-type (TWT) tumors lacking any of these three alterations. In the era of immune checkpoint inhibition (ICI) and targeted molecular therapy, the clinical significance of these groups remains unclear. Here, we integrate targeted DNA sequencing with comprehensive clinical follow-up in CM patients. METHODS: This was a retrospective cohort study that assessed clinical and molecular features from patients with localized or metastatic CM who underwent targeted next-generation sequencing as part of routine clinical care. A total of 254 patients with CM who had a CLIA-certified targeted sequencing assay performed on their tumor tissue were included. RESULTS: Of the 254 patients with cutaneous melanoma, 77 were BRAF mutant (30.3%), 77 were NRAS mutant (30.3%), 47 were NF1 mutant (18.5%), 33 were TWT (13.0%) and the remaining 20 (7.9%) carried mutations in multiple driver genes (BRAF/NRAS/NF1 co-mutated). The majority of this co-mutation group carried mutations in NF1 (n = 19 or 90%) with co-occurring mutations in BRAF or NRAS, often with a weaker oncogenic variant. Consistently, NF1 mutant tumors harbored numerous significantly co-altered genes compared to BRAF or NRAS mutant tumors. The majority of TWT tumors (n = 29, 87.9%) harbor a pathogenic mutation within a known Ras/MAPK signaling pathway component. Of the 154 cases with available TMB data, the median TMB was 20 (range 0.7-266 mutations/Mb). A total of 14 cases (9.1%) were classified as having a low TMB (≤5 mutations/Mb), 64 of 154 (41.6%) had an intermediate TMB (>5 and ≤20 mutations/Mb), 40 of 154 (26.0%) had a high TMB (>20 and ≤50 mutations/Mb) and 36 of 154 (23.4%) were classified as having a very high TMB (>50 mutations/Mb). NRAS mutant melanoma demonstrated significantly decreased overall survival on multivariable analysis (HR for death 2.95, 95% CI 1.13-7.69, p = 0.027, log-rank test) compared with other TCGA molecular subgroups. Of the 116 patients in our cohort with available treatment data, 36 received a combination of dual ICI with anti-CTLA4 and anti-PD1 inhibition as first-line therapy. Elevated TMB was associated with significantly longer progression-free survival following dual-agent ICI (HR 0.26, 95% CI 0.07-0.90, p = 0.033, log-rank test). CONCLUSIONS: NRAS mutation in CMs correlated with significantly worse overall survival. Elevated TMB was associated with increased progression-free survival for patients treated with a combination of dual ICI, supporting the potential utility of TMB as a predictive biomarker for ICI response in melanoma.

Current Role and Status for Intratumoral Injection Therapies in Metastatic Melanoma.

ABSTRACT: Intratumoral therapies represent a unique avenue for drug development in melanoma as patients often have accessible lesions that are particularly amenable to these approaches. In addition, a majority of intratumoral therapies have focused on stimulating antitumor immune responses, making them a particularly attractive option for use in melanoma. In this review, we describe applications for talimogene laherparepvec, a US Food and Drug Administration-approved intratumoral therapy in melanoma, as well as several classes of intratumoral therapies in development including novel oncolytic viruses, mRNA-based intratumoral injections, and cytokines and other signaling molecules.

Advanced Melanoma: Resistance Mechanisms to Current Therapies.

Novel therapeutic agents introduced over the past decade, including immune checkpoint inhibitors and targeted therapies, have revolutionized the management of metastatic melanoma and significantly improved patient outcomes. Although robust and durable responses have been noted in some cases, treatment is often limited by innate or acquired resistance to these agents. This article provides an overview of known and suspected mechanisms involved with acquired resistance to BRAF/MEK inhibitors as well as developing insights into innate and acquired resistance to checkpoint inhibitors in patients with melanoma.

Neurologic complications of immune checkpoint inhibitors.

Introduction: Immune checkpoint inhibitors (ICI) are associated with a wide spectrum of neurologic immune-related adverse events (irAEs) including meningo-encephalitis, myasthenia gravis and various neuropathies. Although relatively rare, they often present significant diagnostic complexity and may be under-recognized. Permanent neurologic deficits and/or fatality have been described but improvement is noted in most cases with ICI discontinuation and immunosuppressive therapy.Areas covered: This review highlights the most frequently reported ICI-associated neurologic toxicities with a particular focus on those that may be more severe and/or fatal. Data from case series and pharmacovigilance studies is leveraged to provide an overview of associated clinical features, expected outcomes and appropriate management. Various immunobiologic triggers have been proposed to explain why certain patients might develop neurologic irAEs and are also briefly discussed.Expert opinion: All providers who care for patients with cancer should be made aware of common neurologic irAEs and able to recognize when prompt evaluation and consultation with appropriate specialists are indicated. Symptoms suggestive of encephalitis, myasthenia-gravis or an acute polyradiculopathy such as Guillain-Barre Syndrome (GBS) in patients exposed to these agents warrant immediate attention with a low threshold for hospitalization to expedite work-up and monitor for severe and/or life-threatening manifestations.

Management of V600E and V600K BRAF-Mutant Melanoma.

OPINION STATEMENT: The optimal management of advanced stage BRAF-mutated melanoma is widely debated and complicated by the availability of several different regimens that significantly improve outcomes but have not been directly compared. While there are many unanswered questions relevant to this patient population, the major uncertainty in current practice is the choice between BRAF/MEK inhibitors or immunotherapy for those with previously untreated metastatic or high-risk disease. Decisions regarding first line therapy should include consideration of patient preference as well as the presence of symptomatic metastatic disease and degree of comorbidity, particularly secondary to any history of severe auto-immune disorder.BRAF/MEK inhibitors have a high response rate and rapid onset and thus can be quickly introduced when patients are symptomatic. They have also produced long-term responses in a subset of patients with more favorable prognostic indicators. In addition, impressive survival benefits have also been observed in patients with resected stage 3 disease at high risk of recurrence. On the other hand, anti-PD-1 monotherapy is associated with high rates of clinical benefit (~45% response rate in the metastatic setting) and low rates of severe toxicity. In many patients with adverse prognostic features, we use combined anti-PD-1 and anti-CTLA-4 for metastatic disease. While associated with high rates of toxicity, adverse events are largely manageable with corticosteroids and treatment cessation, in which case patients may continue to benefit even after a limited duration of treatment.Multiple treatment options exist for patients with BRAF V600 mutant melanoma. Herein, we review the clinical data for safety and efficacy of these options.

Anti-PD-1-Induced Pneumonitis Is Associated with Persistent Imaging Abnormalities in Melanoma Patients.

Pneumonitis may complicate anti-programmed death-1 (PD-1) therapy, although symptoms usually resolve with steroids. The long-term effects on respiratory function, however, are not well defined. We screened melanoma patients treated with anti-PD-1, with and without ipilimumab (anti-CTLA-4), and identified 31 patients with pneumonitis. Median time to radiographic findings was 4.8 months. Twenty-three patients (74%) presented with respiratory symptoms, whereas 8 (26%) were asymptomatic, and 11 (35%) were hospitalized. With 22.1 months median follow-up, 27 patients (87%) had resolution of symptoms, whereas 4 had persistent cough, dyspnea, and/or wheezing. By contrast, the rate of radiographic resolution was lower: Only 11 (35%) had complete radiographic resolution, whereas 14 (45%) had improvement of pneumonitis with persistent scarring or opacities, and 6 (19%) had persistent or worsened ground-glass opacities and/or nodular densities. Persistence (vs. resolution) of radiographic findings was associated with older age and initial need for steroids but not with need for hospitalization, timing of onset, or treatment regimen (combination vs. monotherapy). Among patients with serial pulmonary function tests, lung function improved with time. Although symptoms of anti-PD-1-induced pneumonitis resolved quickly, scarring or inflammation frequently persisted on computerized tomography. Therefore, further study of subclinical pulmonary effects of anti-PD-1 is needed.

Neurologic toxicity associated with immune checkpoint inhibitors: a pharmacovigilance study.

BACKGROUND: Immune checkpoint inhibitors (ICI) produce durable antitumor responses but provoke autoimmune toxicities, including uncommon but potentially devastating neurologic toxicities. The clinical features, including the spectrum, timing, and outcomes, of ICI-induced neurologic toxicities are not well characterized. METHODS: We performed disproportionality analysis using Vigibase, the World Health Organization pharmacovigilance database, comparing neurologic adverse event (AE) reporting in patients receiving ICIs vs. the full database. Neurologic AEs were classified by group queries using Medical Dictionary for Regulatory Activities, between database inception to September 28, 2018. Associations between ICIs and neurologic AEs were assessed using reporting odds ratios (ROR) and information component (IC). IC compares observed and expected values to find associations between drugs and AEs using disproportionate Bayesian reporting; IC025 (lower end of the IC 95% credibility interval) > 0 is considered statistically significant. RESULTS: Among the full database, 18,518,994 AEs were reported, including 48,653 with ICIs. ICIs were associated with higher incidence of myasthenia gravis (0.47% of ICI reports vs. 0.04% of the full database, ROR 16.5 [95% CI 14.5-18.9]; IC025 3.31), encephalitis (0.51% vs. 0.05%, ROR 10.4 [95% CI 9.2-11.8]; IC025 3.15), peripheral neuropathy (1.16% vs. 0.67%, IC025 0.68), and meningitis (0.15% vs. 0.06%, ROR 3.1 [95% CI 2.5-3.9]; IC025 1.01). Myasthenia gravis and encephalitis were associated with anti-PD-1 whereas other neurologic AEs were associated with anti-CTLA-4. Myasthenia gravis was characterized by high fatality rates (~ 20%), early onset (median 29 days), and frequent concurrent myocarditis and myositis; whereas other neurologic AEs had lower fatality rates (6-12%), later onset (median 61-80 days), and were non-overlapping. CONCLUSIONS: ICIs produce a spectrum of distinct classes of neurologic AEs that can cause significant morbidity and mortality and tend to occur early and with class-specific associations.

The role of TERT promoter mutations in differentiating recurrent nevi from recurrent melanomas: A retrospective, case-control study.

BACKGROUND: Repigmentation at previous biopsy sites pose a significant diagnostic dilemma given clinical and histologic similarities between recurrent nevi and locally recurrent melanoma. Though common in melanoma, the role of TERT promoter mutations (TPMs) in recurrent nevi is unknown. OBJECTIVE: We investigated the role of TPMs in recurrent nevi and whether the presence of hotspot TPM distinguishes recurrent nevi from locally recurrent melanoma. We also characterized clinical and histologic features differentiating these lesions. METHODS: We analyzed 11 locally recurrent melanomas, 17 recurrent nevi, and melanoma and nevus controls to determine TPM status. We also assessed clinical and histologic features of the recurrent groups. RESULTS: Hotspot TPMs were more common in recurrent melanomas than recurrent nevi (P = .008). Recurrent melanomas were more likely to have solar elastosis (P = .0047), multilayering of melanocytes in the epidermis (P = .0221), adnexal involvement (P = .0069), and epidermal consumption (P = .0204). Recurrent nevi had intra-epidermal atypia limited to the area above the scar (P < .0001) and occurred earlier after the original biopsy (P < .0008). Solar elastosis, months to recurrence, and hotspot TPMs were independently associated with recurrent melanoma in multivariate analysis. LIMITATIONS: This was a retrospective study. CONCLUSION: Hotspot TPMs are significantly more frequent in recurrent melanomas and could serve as a diagnostic clue in histologically ambiguous cases.

Genomic Assessment of Blitz Nevi Suggests Classification as a Subset of Blue Nevus Rather Than Spitz Nevus: Clinical, Histopathologic, and Molecular Analysis of 18 Cases.

Blitz nevi/tumors are a distinct subset of melanocytic neoplasia which show mixed morphologic features of Spitz and blue nevus. Genomically, most blue nevi have GNAQ or GNA11 mutations while most Spitzoid neoplasms have either an HRAS mutation or translocations involving MET, ROS, BRAF, ALK1, NTRK1, and RET. The criteria used for the assessment of malignancy in blue and Spitzoid lesions are different, and these lesions have different prognostic markers. In this study, we assess the clinical, morphological, and genomic changes in 18 cases of Blitz nevi/tumors to better characterize this subset of neoplasms and determine their optimal genomic classification. Most lesions occurred on the extremities followed by the head and neck region typical of blue nevi. Histology showed most cases having a prominent plexiform growth pattern with cells aggregating around the adnexal structures and neurovascular bundles also typical of blue nevi. Using next generation sequencing, we detected the presence of somatic mutations in GNAQ or GNA11 in 4 of 7 cases (57%) of Blitz nevi with sufficient DNA available for sequencing. Normal skin samples in these 4 cases were sequenced to confirm that the GNAQ or GNA11 mutations were somatic mutations. All 4 cases were negative for immunohistochemical assessment for wild-type BRAF, RET, ALK, and NTRK1 and mutational analysis of HRAS was also negative in all cases. Hence, our study suggests that Blitz nevi/tumors are a distinct subset which genomically are best classified as a subset of blue nevi.

Distinct Patterns of Acral Melanoma Based on Site and Relative Sun Exposure.

Acral melanoma is distinct from melanoma of other cutaneous sites, yet there is considerable variation within this category. To better define this variation, we assessed melanomas occurring on dorsal (n = 21), volar (n = 9), and subungual/interdigital (n = 13) acral skin as well as acral nevi (n = 24) for clinical, histologic, and molecular features. Melanomas on dorsal acral surfaces demonstrated clear differences compared with volar and subungual/interdigital melanomas. The latter two groups exhibited significantly less frequent BRAF mutations (P = 0.01), were significantly less likely to have the superficial spreading histologic subtype (P = 0.01), occurred in older patients (P = 0.05), and had more frequent involvement in non-Caucasians (P = 0.01). These differences can be explained by differing levels of UV exposure. Subungual/interdigital melanomas had the most diverse group of oncogenic mutations including PIK3CA (2/13), STK11 (2/13), EGFR (1/13), FGFR3 (1/13), and PTPN11 (1/13). In addition, subungual/interdigital melanomas had a significantly higher frequency of copy number aberrations (67%) than other subgroups (P = 0.02), particularly in CDK4 and cyclin D1, and were less likely to have BRAF mutations or a superficial spreading histologic subtype (P = 0.05) compared with volar acral melanomas. Although based on a limited sample size, differences between volar and subungual/interdigital melanomas in our study may be the result of differing levels of UV exposure.

Genotypic and Phenotypic Features of BAP1 Cancer Syndrome: A Report of 8 New Families and Review of Cases in the Literature.

Importance: Patients with germline mutations in BAP1 may develop several flesh-colored melanocytic BAP1-mutated atypical intradermal tumors (MBAITs). These tumors generally develop earlier than other BAP1-associated tumors, highlighting an important role for dermatologists in identifying and screening patients with a history suggestive of a germline mutation. Objective: To describe 8 new families with germline mutations in BAP1 and provide a comprehensive review of reported cases. Design, Settings and Participants: Patients were identified in an outpatient dermatology clinical setting over a 6-month period (10 mutation carriers from 8 families) and through a literature review using PubMed (205 patients). Exposures: Mutations were identified through next-generation sequencing of saliva or blood samples, and RNA was extracted from fibroblasts cultured from a patient with an intronic variant to determine the impact of the mutation on the coding sequence. Main Outcomes and Measures: All 215 patients were assessed for personal and/or family history and genotype. These findings were compiled and assessed for any association between genotype and phenotype. Results: Overall, this study included 215 patients (108 women, 91 men, and 16 gender unspecified; median [range] age, 46.5 [10.0-79.0] years). Nine of the 10 patients who were identified in the outpatient dermatology setting were found to have MBAITs on clinical examination. Forty of 53 patients (75%) identified in the literature review who underwent total-body skin examinations (TBSE) were found to have MBAITs, suggesting a high penetrance in patients who have undergone TBSE. The most prevalent malignancies among BAP1 mutation carriers were uveal melanoma (n = 60 [28%]), mesothelioma (n = 48 [22%]), cutaneous melanoma (n = 38 [18%]), and renal cell carcinoma (n = 20 [9%]). A total of 71 unique mutations in BAP1 have been reported. Conclusions and Relevance: Our results indicate that germline mutations in both coding and noncoding regions throughout the BAP1 gene can impair protein function, leading to an increased risk for several associated malignancies. Four of the 8 probands we present had no history of BAP1-associated malignancies and were assessed for germline mutations when found to have MBAITs on dermatologic examination. Dermatologists can identify patients with a high likelihood of the BAP1 cancer syndrome through personal and family history and TBSE for the presence of possible MBAITs.

A single-institution assessment of superficial spreading melanoma (SSM) in the pediatric population: Molecular and histopathologic features compared with adult SSM.

BACKGROUND: The epidemiology of pediatric melanoma is distinct from that seen in adults. This is more distinguishable when pediatric patients are separated into prepubertal and adolescent groups. OBJECTIVE: In this study, we compared epidemiologic, clinical, histologic, and molecular characteristics of pediatric superficial spreading melanoma (SSM) in prepubertal and adolescent patients to that in adults. METHOD: We reviewed our database for pediatric melanomas, comparing SSM data between pediatric and adult cases for pathologic stage at presentation, ratio of radial to vertical growth phase, average Breslow depth and mitotic index, and frequency of fluorescence in situ hybridization (FISH) positivity. RESULTS: Of 84 pediatric melanomas, 38 were SSM, and 5 of the latter (6%) were prepubertal. There were no significant differences when pediatric and adult SSM were compared for stage at presentation, ratio of radial to vertical growth phase, average Breslow depth and mitotic count, or frequency of FISH positivity. A significant difference was detected for SSM arising from a precursor nevus (80% of pediatric cases versus 30% of adult cases). LIMITATIONS: Follow-up time was limited for both cohorts. CONCLUSIONS: SSM melanoma is infrequent in childhood, particularly in the prepubertal years. Features such as tumor stage, Breslow depth, mitotic activity, and FISH positivity suggest morphologic and molecular characteristics similar to those of adult SSM.

Amnion Membrane in Diabetic Foot Wounds: A Meta-analysis.

BACKGROUND: Amniotic membrane is tissue obtained from human placenta rich in cytokines, growth factors, and stem cells that possess the ability to inhibit infection, improve healing, and stimulate regeneration. METHODS: A meta-analysis was performed examining randomized controlled trials comparing amniotic tissue products with standard of care in nonhealing diabetic foot ulcers including PubMed, Cochrane Central Register of Controlled Trials, and the Cochrane Database of Systematic Reviews. RESULTS: A search of 3 databases identified 596 potentially relevant articles. Application of selection criteria led to the selection of 5 randomized controlled trials. The 5 selected randomized controlled trials represented a total of 311 patients. The pooled relative risk of healing with amniotic products compared with control was 2.7496 (2.05725-3.66524, P < 0.001). CONCLUSIONS: The current meta-analysis indicates that the treatment of diabetic foot ulcers with amniotic membrane improves healing rates in diabetic foot ulcers. Further studies are needed to determine whether these products also decrease the incidence of subsequent complications, such as amputation or death, in diabetic patients.

A clinical, histologic, and follow-up study of genital melanosis in men and women.

BACKGROUND: Genital melanosis may clinically mimic melanoma. Little is known about the potential risk for genital and nongenital melanoma in these patients. OBJECTIVE: In this retrospective cohort study, we analyzed clinical and histologic data from patients with genital melanosis to better characterize these lesions and the risk they confer for genital and nongenital melanoma. METHODS: In all, 41 patients were identified for a retrospective chart review and histologic analysis. RESULTS: Genital melanosis can clinically mimic melanoma but the typical age of onset is younger than for genital melanoma. A majority of lesions were found to stabilize or regress over time. Five patients were found to have a history of melanoma, only 1 of which was in the genital region. Lesions from these patients were more likely to show melanocytes with suprabasal movement (P = .0101) and to have a higher melanocyte count (P < .0462). LIMITATIONS: We present a relatively small cohort of patients with an average follow-up of only 30.5 months. CONCLUSION: Patients with genital melanosis, and in particular those with any level of histologic atypia in the genital melanosis lesion, may require careful total body skin examinations for the possibility of melanoma in any body site.

A Comparison of Morphologic and Molecular Features of BRAF, ALK, and NTRK1 Fusion Spitzoid Neoplasms.

Recent studies have identified translocations involving the kinase domains of ALK, NTRK1, BRAF, RET, and ROS in spitzoid neoplasms. Subsequent studies have also characterized morphologic features corresponding to ALK and NTRK1 translocations. In this study, we sought to further compare morphologic features across a range of 49 genetically defined spitzoid neoplasms with ALK, NTRK1, BRAF, or RET fusions to determine discriminating features. We also compared them with a group of 22 spitzoid neoplasms, which were confirmed to be negative for fusions in ALK, NTRK1, BRAF, and RET. Features with the highest discriminatory value included diameter of the lesion, dermal architecture, and certain cytomorphologic features. Specifically, cases with a large diameter (≥9 mm) and wedge-shaped, plexiform dermal architecture of nests of large, spindle-shaped cells were most likely to have an ALK fusion. NTRK1-fused cases were most likely of the fusions to have Kamino bodies and were typically arranged in smaller nests with smaller predominantly spindle-shaped cells, occasionally forming rosettes. BRAF fusion cases were the only fusion subtype to have a predominance of epithelioid cells, were less organized in nests, and commonly had a sheet-like growth pattern or dysplastic Spitz architecture. BRAF fusion cases were most likely to have high-grade nuclear atypia, to be diagnosed as spitzoid melanoma, to have a positive result by melanoma fluorescence in situ hybridization assay, and to develop copy number gains in the kinase domain of the fusion protein. On the basis of experience from this cohort, BRAF-fused cases appear most likely to progress to melanoma.