Genetic and Methylation Analysis of CTNNB1 in Benign and Malignant Melanocytic Lesions.

Melanocytic neoplasms have been genetically characterized in detail during the last decade. Recurrent CTNNB1 exon 3 mutations have been recognized in the distinct group of melanocytic tumors showing deep penetrating nevus-like morphology. In addition, they have been identified in 1-2% of advanced melanoma. Performing a detailed genetic analysis of difficult-to-classify nevi and melanomas with CTNNB1 mutations, we found that benign tumors (nevi) show characteristic morphological, genetic and epigenetic traits, which distinguish them from other nevi and melanoma. Malignant CTNNB1-mutant tumors (melanomas) demonstrated a different genetic profile, instead grouping clearly with other non-CTNNB1 melanomas in methylation assays. To further evaluate the role of CTNNB1 mutations in melanoma, we assessed a large cohort of clinically sequenced melanomas, identifying 38 tumors with CTNNB1 exon 3 mutations, including recurrent S45 (n = 13, 34%), G34 (n = 5, 13%), and S27 (n = 5, 13%) mutations. Locations and histological subtype of CTNNB1-mutated melanoma varied; none were reported as showing deep penetrating nevus-like morphology. The most frequent concurrent activating mutations were BRAF V600 (n = 21, 55%) and NRAS Q61 (n = 13, 34%). In our cohort, four of seven (58%) and one of nine (11%) patients treated with targeted therapy (BRAF and MEK Inhibitors) or immune-checkpoint therapy, respectively, showed disease control (partial response or stable disease). In summary, CTNNB1 mutations are associated with a unique melanocytic tumor type in benign tumors (nevi), which can be applied in a diagnostic setting. In advanced disease, no clear characteristics distinguishing CTNNB1-mutant from other melanomas were observed; however, studies of larger, optimally prospective, cohorts are warranted.

Clinical and pathological characteristics of familial melanoma with germline TERT promoter variants.

Around 10% of melanoma occurs in patients with a suspected familial predisposition. TERT promoter mutations are the most common somatic hotspot mutations in human cancers. However, only two families with germline mutations have been identified to date. We present detailed histological, clinical, and molecular pathologic analyses of affected patients and details of newly identified individuals in one of these previously reported families. TERT (NM_198253.3) Chr.5:1,295,161T>C (c.-57 T>C) promoter variants were detected in all melanoma-affected (n = 18) and one non-diseased family member. The median age at diagnosis was 30 years (n = 18, range 16-46 years, 2 unknown). While most primary melanomas arose on the upper extremities (n = 7, 21%) and were superficial spreading melanoma (SSM, n = 8, 24%), many primary melanomas also originated from non-UV-exposed mucosal (n = 2, 6%) and acral (n = 4, 12%) locations. One SSM sample harbored a Chr.5:1,295,228C>T TERT promoter mutation in addition to the germline Chr.5:1,295,161T>C variant, arguing additional pathway activation can support tumor pathogenesis. Patients treated with BRAF inhibitor and/or immune checkpoint inhibition (ICI) showed responses, although of limited duration. One mucosal melanoma harbored both a KIT copy number gain and an activating c.1727 p.Leu576Pro mutation. Following the modest response to ICI, subsequent KIT inhibitor (imatinib) therapy demonstrated an ongoing complete pathological response (currently 7 months).

Genetic and methylation profiles distinguish benign, malignant and spitzoid melanocytic tumors.

Accurate classification of melanocytic tumors is important for prognostic evaluation, treatment and follow-up protocols of patients. The majority of melanocytic proliferations can be classified solely based on clinical and pathological criteria, however in select cases a definitive diagnostic assessment remains challenging and additional diagnostic biomarkers would be advantageous. We analyzed melanomas, nevi, Spitz nevi and atypical spitzoid tumors using parallel sequencing (exons of 611 genes and 507 gene translocation analysis) and methylation arrays (850k Illumina EPIC). By combining detailed genetic and epigenetic analysis with reference-based and reference-free DNA methylome deconvolution we compared Spitz nevi to nevi and melanoma and assessed the potential for these methods in classifying challenging spitzoid tumors. Results were correlated with clinical and histologic features. Spitz nevi were found to cluster independently of nevi and melanoma and demonstrated a different mutation profile. Multiple copy number alterations and TERT promoter mutations were identified only in melanomas. Genome-wide methylation in Spitz nevi was comparable to benign nevi while the Leukocytes UnMethylation for Purity (LUMP) algorithm in Spitz nevi was comparable to melanoma. Histologically difficult to classify Spitz tumor cases were assessed which, based on methylation arrays, clustered between Spitz nevi and melanoma and in terms of genetic profile or copy number variations demonstrated worrisome features suggesting a malignant neoplasm. Comprehensive sequencing and methylation analysis verify Spitz nevi as an independent melanocytic entity distinct from both nevi and melanoma. Combined genetic and methylation assays can offer additional insights in diagnosing difficult to classify Spitzoid tumors.

Genetic and Clinical Characteristics of ARID1A Mutated Melanoma Reveal High Tumor Mutational Load without Implications on Patient Survival.

(1) Background: Melanoma has the highest mortality of all cutaneous tumors, despite recent treatment advances. Many relevant genetic events have been identified in the last decade, including recurrent ARID1A mutations, which in various tumors have been associated with improved outcomes to immunotherapy. (2) Methods: Retrospective analysis of 116 melanoma samples harboring ARID1A mutations. Assessment of clinical and genetic characteristics was performed as well as correlations with treatment outcome applying Kaplan-Meier (log-rank test), Fisher's exact and Chi-squared tests. (3) Results: The majority of ARID1A mutations were in cutaneous and occult melanoma. ARID1A mutated samples had a higher number of mutations than ARID1A wild-type samples and harbored UV-mutations. A male predominance was observed. Many samples also harbored NF1 mutations. No apparent differences were noted between samples harboring genetically inactivating (frame-shift or nonsense) mutations and samples with other mutations. No differences in survival or response to immunotherapy of patients with ARID1A mutant melanoma were observed. (4) Conclusions: ARID1A mutations primarily occur in cutaneous melanomas with a higher mutation burden. In contrast to findings in other tumors, our data does not support ARID1A mutations being a biomarker of favorable response to immunotherapies in melanoma. Larger prospective studies would still be warranted.

Genetic characterization of advanced conjunctival melanoma and response to systemic treatment.

BACKGROUND: Conjunctival melanoma is a rare type of ocular melanoma, which is prone to local recurrence and metastasis and can lead to patient death. Novel therapeutic strategies have revolutionized cutaneous melanoma management. The efficacy of these therapies in conjunctival melanoma, however, has not been evaluated in larger patient cohorts. METHODS: In this multi-center retrospective cohort study with additional screening of the ADOREG database, data were collected from 34 patients with metastatic conjunctival melanoma who received targeted therapy (TT) (BRAF ± MEK inhibitors) or immune checkpoint inhibitors (ICI) (anti-PD-1 ± anti-CTLA4). In 15 cases, tissue was available for targeted next-generation-sequencing (611 genes) and RNA sequencing. Driver mutations, tumor mutational burden, copy number variations and inflammatory/IFNγ gene expression signatures were determined. RESULTS: Genetic characterization identified frequent BRAF (46.7%, 7/15), NRAS (26.7%, 4/15), NF1 (20%, 3/15), and TERT promoter (46.7%, 7/15) mutations. UV associated C>T and CC>TT mutations were common. Median follow-up time after start of first TT or ICI therapy was 13.2 months. In 26 patients receiving first-line ICI, estimated one-year progression-free survival (PFS) rate was 42.0%, PFS and overall survival (OS) 6.2 and 18.0 months, respectively. First-line TT was given to 8 patients, estimated one-year PFS rate was 54.7%, median PFS and OS 12.6 and 29.1 months, respectively. CONCLUSIONS: Our findings support the role of UV irradiation in conjunctival melanoma and the genetic similarity with cutaneous melanoma. Conjunctival melanoma patients with advanced disease benefit from both targeted therapies (BRAF ± MEK inhibitors) and immune checkpoint inhibitors.

TERT promoter mutations are associated with longer progression-free and overall survival in patients with BRAF-mutant melanoma receiving BRAF and MEK inhibitor therapy.

BACKGROUND: Around 50% of cutaneous melanomas harbour therapeutically targetable BRAF V600 mutations. Reliable clinical biomarkers predicting duration of response to BRAF-targeted therapies are still lacking. Recent in vitro studies demonstrated that BRAF-MEK inhibitor therapy response is associated with tumour TERT promoter mutation status. We assessed this potential association in a clinical setting. METHODS: The study cohort comprised 232 patients with metastatic or unresectable BRAF V600-mutated melanoma receiving combined BRAF/MEK inhibitor treatment, including a single-centre retrospective discovery cohort (N = 120) and a prospectively collected multicenter validation cohort (N = 112). Patients were excluded if they received BRAF or MEK inhibitors in an adjuvant setting, as monotherapy, or in combination with immunotherapy. Kaplan-Meier and univariate/multivariate Cox regression analyses were performed as appropriate. RESULTS: median age at first diagnosis was 54 years (range 16-84 years). The majority of patients were men 147/232 (63.4%). Most tumours harboured TERT promoter mutations (72%, N = 167). A survival advantage was observed in both progression-free survival (PFS) and overall survival (OS) for patients with TERT promoter-mutant versus wild-type tumours in both the discovery cohort (mPFS of 9.6 months [N = 87] vs 5.0 months [N = 33]; hazard ratio [HR] = 0.56 [95% confidence interval {CI} 0.33-0.96] and mOS of 33.6 months vs 15.0 months; HR = 0.47 [95%CI 0.32-0.70]) as well as the validation cohort (mPFS of 7.3 months [N = 80] vs 5.8 months [N = 32]; HR = 0.67 [95%CI 0.41-1.10] and mOS of 51.1 months vs 15.0 months; HR = 0.33 [95%CI 0.18-0.63]). In the pooled cohort of TERT promoter-mutant (N = 167) versus wild-type (N = 65) tumours, respectively, PFS was 8.9 versus 5.5 months, (HR = 0.62; 95%CI 0.45-0.87; P = 0.004), and OS was 33.6 versus 17.0 months, (HR = 0.51; 95%CI 0.35-0.75, P = 0.0001). CONCLUSIONS: In patients with melanoma receiving BRAF/MEK-targeted therapies, TERT promoter mutations are associated with longer survival. If validated in larger studies, TERT promoter mutation status should be included as a predictive biomarker in treatment algorithms for advanced melanoma.

NF1-mutated melanomas reveal distinct clinical characteristics depending on tumour origin and respond favourably to immune checkpoint inhibitors.

BACKGROUND: NF1-mutated tumours represent a small subset (10-15%) of melanomas, not sufficiently analysed in large clinical cohorts. This study investigated the largest multicentre collection of NF1-mutated melanomas to date. METHODS: This study analysed a multicentre tumour tissue sample cohort from 266 patients with NF1-mutated melanoma. Targeted next-generation sequencing of the TERT promoter and 29 relevant melanoma genes was performed. Survival was compared with NF1 wild-type cohorts from the Tissue Registry in Melanoma project (n = 432). RESULTS: Most NF1-mutated melanoma arose in the head-and-neck region of patients >60 years. NF1 alterations were frequently inactivating, primarily non-sense, less frequently truncating mutations. Non-inactivating NF1 mutations more frequently co-occurred with activating BRAF and RAS mutations. NF1-mutated tumours had higher numbers of gene mutations and UV signature C>T and CC>TT transitions than BRAF, RAS and triple wild-type melanomas. NF1-mutated acral and mucosal melanomas harboured a different mutation signature and were frequent in women (69% and 83%, respectively), differing from non-acral cutaneous NF1-mutated melanomas (men 73%, women 27%). Overall survival in stage IV disease was comparable for patients with NF1-mutated or wild-type melanoma. However, in patients receiving first-line immune checkpoint inhibitor treatment, better median overall survival (mOS) was observed for NF1-mutated than wild-type tumours (mOS = not reached vs mOS = 25.82, p = 0.0154, n = 80 and 432, respectively). CONCLUSIONS: Cutaneous, acral and mucosal NF1-mutated melanomas vary in clinical and genetic characteristics and demonstrate a favourable outcome on immune checkpoint inhibition therapy.

GNA14, GNA11, and GNAQ Mutations Are Frequent in Benign but Not Malignant Cutaneous Vascular Tumors.

Cutaneous vascular tumors consist of a heterogeneous group of benign proliferations, including a range of hemangiomas and vascular malformations, as well as heterogeneous groups of both borderline and malignant neoplasms such as Kaposi's sarcoma and angiosarcomas. The genetics of these tumors have been assessed independently in smaller individual cohorts making comparisons difficult. In our study, we analyzed a representative cohort of benign vascular proliferations observed in a clinical routine setting as well as a selection of malignant vascular proliferations. Our cohort of 104 vascular proliferations including hemangiomas, malformations, angiosarcomas and Kaposi's sarcoma were screened by targeted next-generation sequencing for activating genetic mutations known or assumed to be potentially relevant in vascular proliferations. An association analysis was performed for mutation status and clinico-pathological parameters. Frequent activating hotspot mutations in GNA genes, including GNA14 Q205, GNA11 and GNAQ Q209 were identified in 16 of 64 benign vascular tumors (25%). GNA gene mutations were particularly frequent (52%) in cherry (senile) hemangiomas (13 of 25). In angiosarcomas, activating RAS mutations (HRAS and NRAS) were identified in three samples (16%). No activating GNA or RAS gene mutations were identified in Kaposi's sarcomas. Our study identifies GNA14 Q205, GNA11 and GNAQ Q209 mutations as being the most common and mutually exclusive mutations in benign hemangiomas. These mutations were not identified in malignant vascular tumors, which could be of potential diagnostic value in distinguishing these entities.

Molecular pathology as a diagnostic aid in difficult-to-classify melanocytic tumours with spitzoid morphology.

Accurate classification of melanocytic proliferations has important implications for prognostic prediction, treatment and follow-up. Although most melanocytic proliferations can be accurately classified using clinical and pathological criteria, classification (specifically distinction between nevus and melanoma) can be challenging in a subset of cases, including those with spitzoid morphology. Genetic studies have shown that mutation profiles differ between primary melanoma subtypes and Spitz nevi. These differences may aid in distinguishing benign from malignant in some melanocytic tumours. Here, we present a selection of melanocytic proliferations with equivocal histopathological criteria, wherein genetic analysis was requested to help guide classification. In two of four cases, the genetic results offered valuable insights, allowing a definitive diagnosis, indicating the diagnostic value of mutation profiling in a real-world routine clinical setting. Although histopathological assessment remains decisive in melanocytic proliferation classification, we recommend including genetic profiling in cases of borderline or atypical lesion to support accurate classification.

Clinical and genetic analysis of melanomas arising in acral sites.

STUDY AIM: Melanomas arising in acral sites are associated with a poorer prognosis than other melanoma subtypes. The aim of this study was to evaluate clinical-pathological and genetic characteristics as well as therapeutic responses of a larger cohort of patients with melanomas arising in acral sites. METHODS: Clinical data of 134 patients with melanomas arising in acral sites from the Dept. of Dermatology Essen were collected and analysed with regard to clinicopathological characteristics and treatment responses. Genetic analysis with targeted next-generation sequencing was done on 50 samples. RESULTS: In our cohort, BRAF (30%), NRAS (28%), TERT promoter (26%), NF1 (14%) and KIT (6%) were frequently identified mutations. Comparing tumours situated on palms and soles with melanomas arising on dorsal acral sites, a higher frequency of NRAS (39.1% versus 25%) and NF1 (17.3% versus 0%) and lower frequencies of BRAF (21.7% versus 75%) and TERT promoter (8.6% versus 50%) mutations were observed. MAPK activating mutations were identified in 64% of tumours. Overall survival was longer in patients treated with immune checkpoint inhibitors as first-line treatment than in patients receiving other systemic therapies (i.e. BRAF/MEK inhibitors and chemotherapy). CONCLUSION: Our data suggest that the genetics of melanomas arising in acral sites varies by tumour location and may influence biological behaviour.

Frequent Occurrence of NRAS and BRAF Mutations in Human Acral Naevi.

Acral naevi are benign melanocytic tumors occurring at acral sites. Occasionally they can progress to become malignant tumors (melanomas). The genetics of acral naevi have not been assessed in larger studies. In our study, a large cohort of 130 acral naevi was screened for gene mutations known to be important in other naevi and melanoma subtypes by targeted next-generation sequencing. Mutation status was correlated with clinicopathological parameters. Frequent mutations in genes activating the MAP kinase pathway were identified, including n = 87 (67%) BRAF, n = 24 (18%) NRAS, and one (1%) MAP2K1 mutations. BRAF mutations were almost exclusively V600E (n = 86, 99%) and primarily found in junctional and compound naevi. NRAS mutations were either Q61K or Q61R and frequently identified in dermal naevi. Recurrent non-V600E BRAF, KIT, NF1, and TERT promoter mutations, present in acral melanoma, were not identified. Our study identifies BRAF and NRAS mutations as the primary pathogenic event in acral naevi, however, distributed differently to those in non-acral naevi. The mutational profile of acral naevi is distinct from acral melanoma, which may be of diagnostic value in distinguishing these entities.

Integrated Genomic Classification of Melanocytic Tumors of the Central Nervous System Using Mutation Analysis, Copy Number Alterations, and DNA Methylation Profiling.

Purpose: In the central nervous system, distinguishing primary leptomeningeal melanocytic tumors from melanoma metastases and predicting their biological behavior solely using histopathologic criteria may be challenging. We aimed to assess the diagnostic and prognostic value of integrated molecular analysis.Experimental Design: Targeted next-generation sequencing, array-based genome-wide methylation analysis, and BAP1 IHC were performed on the largest cohort of central nervous system melanocytic tumors analyzed to date, including 47 primary tumors of the central nervous system, 16 uveal melanomas, 13 cutaneous melanoma metastases, and 2 blue nevus-like melanomas. Gene mutation, DNA-methylation, and copy-number profiles were correlated with clinicopathologic features.Results: Combining mutation, copy-number, and DNA-methylation profiles clearly distinguished cutaneous melanoma metastases from other melanocytic tumors. Primary leptomeningeal melanocytic tumors, uveal melanomas, and blue nevus-like melanoma showed common DNA-methylation, copy-number alteration, and gene mutation signatures. Notably, tumors demonstrating chromosome 3 monosomy and BAP1 alterations formed a homogeneous subset within this group.Conclusions: Integrated molecular profiling aids in distinguishing primary from metastatic melanocytic tumors of the central nervous system. Primary leptomeningeal melanocytic tumors, uveal melanoma, and blue nevus-like melanoma share molecular similarity with chromosome 3 and BAP1 alterations, markers of poor prognosis. Clin Cancer Res; 24(18); 4494-504. ©2018 AACR.

Intraventricular melanocytoma diagnosis confirmed by gene mutation profile.

Primary leptomeningeal melanocytic tumors (PLMTs) are rare. They usually arise along the spinal cord and at the skull base. Here we report on a patient with a very rare intraventricular melanocytoma. Histologically, a melanocytic tumor was clearly diagnosed. However, to make the uncommon diagnosis of an intraventricular melanocytoma, metastatic melanoma needed to be excluded. Next generation sequencing covering gene mutations that may occur in PLMTs and cutaneous melanoma was performed. The unique gene mutation profile detected, consisting of an activating CYSLTR2 L129Q mutation and EIF1AX G9R mutation and a lack of mutations in genes known to occur in metastatic melanoma (i.e. BRAF or NRAS) confirmed the diagnosis of an intraventricular melanocytoma. This case report is the second intraventricular melanocytoma published to date and demonstrates the value of applying novel genetic assays to make this diagnosis.

Atypical fibroxanthoma and pleomorphic dermal sarcoma harbor frequent NOTCH1/2 and FAT1 mutations and similar DNA copy number alteration profiles.

Atypical fibroxanthomas and pleomorphic dermal sarcomas are tumors arising in sun-damaged skin of elderly patients. They have differing prognoses and are currently distinguished using histological criteria, such as invasion of deeper tissue structures, necrosis and lymphovascular or perineural invasion. To investigate the as-yet poorly understood genetics of these tumors, 41 atypical fibroxanthomas and 40 pleomorphic dermal sarcomas were subjected to targeted next-generation sequencing approaches as well as DNA copy number analysis by comparative genomic hybridization. In an analysis of the entire coding region of 341 oncogenes and tumor suppressor genes in 13 atypical fibroxanthomas using an established hybridization-based next-generation sequencing approach, we found that these tumors harbor a large number of mutations. Gene alterations were identified in more than half of the analyzed samples in FAT1, NOTCH1/2, CDKN2A, TP53, and the TERT promoter. The presence of these alterations was verified in 26 atypical fibroxanthoma and 35 pleomorphic dermal sarcoma samples by targeted amplicon-based next-generation sequencing. Similar mutation profiles in FAT1, NOTCH1/2, CDKN2A, TP53, and the TERT promoter were identified in both atypical fibroxanthoma and pleomorphic dermal sarcoma. Activating RAS mutations (G12 and G13) identified in 3 pleomorphic dermal sarcoma were not found in atypical fibroxanthoma. Comprehensive DNA copy number analysis demonstrated a wide array of different copy number gains and losses, with similar profiles in atypical fibroxanthoma and pleomorphic dermal sarcoma. In summary, atypical fibroxanthoma and pleomorphic dermal sarcoma are highly mutated tumors with recurrent mutations in FAT1, NOTCH1/2, CDKN2A, TP53, and the TERT promoter, and a range of DNA copy number alterations. These findings suggest that atypical fibroxanthomas and pleomorphic dermal sarcomas are genetically related, potentially representing two ends of a common tumor spectrum and distinguishing these entities is at present still best performed using histological criteria.

Osteosynthesis of a cranio-osteoplasty with a biodegradable magnesium plate system in miniature pigs.

Biodegradable magnesium alloys are a new class of implant material suitable for bone surgery. The aim of this study was to investigate plates and screws made of magnesium for osteosynthesis in comparison to titanium in a cranial fracture model. Implants were used for internal fixation of a cranio-osteoplasty in nine minipigs. Computed tomography was conducted repeatedly after surgery. The implants and the adjacent tissues were harvested 10, 20 and 30weeks after surgery and investigated by micro-computed tomography and histological analysis. The surgical procedure and the inserted osteosynthesis material were well tolerated by the animals, and the bone healing of the osteoplasty was undisturbed at all times. The adjacent bone showed formation of lacunas in the magnesium group, resulting in a lower bone-to-implant contact ratio than that of titanium (72 vs. 94% at week 30), but this did not lead to clinical side effects. Radiological measurements showed no reduction in osteosynthesis material volume, but indicated signs of degradation: distinct volumes within the magnesium osteosynthesis group had lower density in micro-computed tomography, and these volumes increased up to 9% at week 30. The histological preparations showed areas of translucency and porosity inside the magnesium, but the outer shape of the osteosynthesis material remained unchanged. No fracture or loosening of the osteosynthesis devices appeared. Soft tissue probes confirmed sufficient biocompatibility. Given their biodegradable capacity, biocompatibility, mechanical strength and visibility on radiographs, osteosynthesis plates made of magnesium alloys are suitable for internal fixation procedures. STATEMENT OF SIGNIFICANCE: To the best of our knowledge this is the first study that used biodegradable magnesium implants for osteosynthesis in a cranial fracture model. The cranio-osteoplasty in miniature pigs allowed in vivo application of plate and screw osteosynthesis of standard-sized implants and the implementation of surgical procedures similar to those conducted on human beings. The osteosynthesis configuration, size, and mechanical properties of the magnesium implants within this study were comparable to those of titanium-based osteosynthesis materials. The results clearly show that bone healing was undisturbed in all cases and that the biocompatibility to hard- and soft tissue was sufficient. Magnesium implants might help to avoid long-term complications and secondary removal procedures due to their biodegradable properties.

Frequent GNAQ, GNA11, and EIF1AX Mutations in Iris Melanoma.

Purpose: The most common malignant intraocular tumors with a high mortality in adults are uveal melanomas. Uveal melanomas arise most frequently in the choroid or ciliary body (97%) and rarely in the iris (3%). Whereas conjunctival and posterior uveal (ciliary body and choroidal) melanomas have been studied in more detail genetically, little data exist regarding iris melanomas. Methods: In our study, we genetically analyzed 19 iris melanomas, 8 ciliary body melanomas, 3 ring melanomas, and 4 iris nevi. A targeted next-generation sequencing approach was applied, covering the mutational hotspot regions of nine genes known to be mutated in conjunctival and uveal melanoma (BRAF, NRAS, KIT, GNAQ, GNA11, CYSLTR2, SF3B1, EIF1AX, and BAP1). Results: Activating GNAQ or GNA11 hotspot mutations were detected in a mutually exclusive fashion in 84% (16/19) of iris melanomas. EIF1AX gene mutations also were frequent, detected in 42% (8/19) of iris melanomas. In 4 iris nevi, one GNAQ mutation was identified. GNAQ, GNA11, EIF1AX, and BAP1 mutations were identified at varying frequencies in ciliary body and ring melanomas. Conclusions: In this most comprehensive genetic analysis of iris melanomas published to date, we find iris melanomas to be related genetically to choroidal and ciliary body melanomas, frequently harboring GNAQ, GNA11, and EIF1AX mutations. Future studies will need to assess if screening mutation profiles in iris melanomas may be of diagnostic or prognostic value.

SF3B1 and BAP1 mutations in blue nevus-like melanoma.

Blue nevi are melanocytic tumors originating in the cutaneous dermis. Malignant tumors may arise in association with or resembling blue nevi, so called 'blue nevus-like melanoma', which can metastasize and result in patient death. Identifying which tumors will behave in a clinically aggressive manner can be challenging. Identifying genetic alterations in such tumors may assist in their diagnosis and prognostication. Blue nevi are known to be genetically related to uveal melanomas (eg, both harboring GNAQ and GNA11 mutations). In this study, we analyzed a large cohort (n=301) of various morphologic variants of blue nevi and related tumors including tumors diagnosed as atypical blue nevi (n=21), and blue nevus-like melanoma (n=12), screening for all gene mutations known to occur in uveal melanoma. Similar to published reports, we found the majority of blue nevi harbored activating mutations in GNAQ (53%) or GNA11 (15%). In addition, rare CYSLTR2 (1%) and PLCB4 (1%) mutations were identified. EIF1AX, SF3B1, and BAP1 mutations were also detected, with BAP1 and SF3B1 R625 mutations being present only in clearly malignant tumors (17% (n=2) and 25% (n=3) of blue nevus-like melanoma, respectively). In sequencing data from a larger cohort of cutaneous melanomas, this genetic profile was also identified in tumors not originally diagnosed as blue nevus-like melanoma. Our findings suggest that the genetic profile of coexistent GNAQ or GNA11 mutations with BAP1 or SF3B1 mutations can aid the histopathological diagnosis of blue nevus-like melanoma and distinguish blue nevus-like melanoma from conventional epidermal-derived melanomas. Future studies will need to further elucidate the prognostic implications and appropriate clinical management for patients with tumors harboring these mutation profiles.

Activating cysteinyl leukotriene receptor 2 (CYSLTR2) mutations in blue nevi.

Blue nevi are common melanocytic tumors arising in the dermal layer of the skin. Similar to uveal melanomas, blue nevi frequently harbor GNAQ and GNA11 mutations. Recently, recurrent CYSLTR2 and PLCB4 mutations were identified in uveal melanomas not harboring GNAQ or GNA11 mutations. All four genes (GNAQ, GNA11, CYSLTR2, and PLCB4) code for proteins involved in the same signaling pathway, which is activated by mutations in these genes. Given the related functional consequences of these mutations and the known genetic similarities between uveal melanoma and blue nevi, we analyzed a cohort of blue nevi to investigate whether CYSLTR2 and PLCB4 mutations occur in tumors lacking GNAQ or GNA11 mutations (as in uveal melanoma). A targeted next-generation sequencing assay covering known activating mutations in GNAQ, GNA11, CYSLTR2, PLCB4, KIT, NRAS, and BRAF was applied to 103 blue nevi. As previously reported, most blue nevi were found to harbor activating mutations in GNAQ (59%, n=61), followed by less frequent mutations in GNA11 (16%, n=17). Additionally, one BRAF (1%) and three NRAS (3%) mutations were detected. In three tumors (3%) harboring none of the aforementioned gene alterations, CYSLTR2 mutations were identified. All three CYSLTR2 mutations were the same c.386T>A, L129Q mutation previously identified in uveal melanoma that has been shown to lead to increased receptor activation and signaling. In summary, our study identifies CYSLTR2 L129Q alterations as a previously unrecognized activating mutation in blue nevi, occuring in a mutually exclusive fashion with known GNAQ and GNA11 mutations. Similar to GNAQ and GNA11 mutations, CYSLTR2 mutations, when present, are likely defining pathogenetic events in blue nevi.