Smooth muscle hyperplasia in protein kinase C-fused blue naevi: Report of 12 cases.

BACKGROUND AND AIMS: PKC-fused blue naevi are a recently described group of melanocytic tumours that have distinctive morphological features, including a pigmented epithelioid melanocytoma-like junctional component or a dermal biphasic architecture associating with naevocytoid nests surrounded by dendritic and spindled pigmented melanocytes (so-called 'combined common and blue naevus'). There have been reports of smooth muscle hyperplasia in a hamartoma-like pattern in cases of combined blue naevi without genetic exploration. MATERIALS AND METHODS: Herein, we describe 12 cases of protein kinase C (PKC)-fused blue tumours associated with a co-existing smooth muscle hyperplasia, identified from a total of 98 PKC-fused melanocytic tumours. Archived slides of PKC-fused blue naevi with haematoxylin, eosin and phloxin staining, immunohistochemistry and molecular confirmation of a PKC-fusion by fluorescence in-situ hybridisation (FISH) or RNAseq were re-evaluated for identification of notable smooth muscle hyperplasia. Fifty-one of these slides had already been studied in a previous publication from our group. RESULTS: The hyperplasia ranged from hypertrophic arrector pili muscles to extensive horizontal bundles of disorganised fibres constantly associated and limited within a biphasic dermal melanocytic component. At least one arrector pili muscle was always visible within the tumour, with occasionally direct extension of the hyperplastic fibres from the main muscle body. These muscle fibres were devoid of a PKC-fusion signal by FISH. PKC molecules are involved in the regulation of smooth muscle function, offering an explanatory framework. CONCLUSIONS: These data suggest incorporating smooth muscle hyperplasia as a diagnostic morphological feature of PKC-fused blue melanocytic tumours.

The Impact of Next-generation Sequencing on Interobserver Agreement and Diagnostic Accuracy of Desmoplastic Melanocytic Neoplasms.

Next-generation sequencing (NGS) is increasingly being utilized as an ancillary tool for diagnostically challenging melanocytic neoplasms. It is incumbent upon the pathology community to perform studies assessing the benefits and limitations of these tools in specific diagnostic scenarios. One of the most challenging diagnostic scenarios faced by skin pathologists involves accurate diagnosis of desmoplastic melanocytic neoplasms (DMNs). In this study, 20 expert melanoma pathologists rendered a diagnosis on 47 DMNs based on hematoxylin and eosin sections with demographic information. After submitting their diagnosis, the experts were given the same cases, but this time with comprehensive genomic sequencing results, and asked to render a diagnosis again. Identification of desmoplastic melanoma (DM) improved by 7%, and this difference was statistically significant ( P <0.05). In addition, among the 15 melanoma cases, in the pregenomic assessment, only 12 were favored to be DM by the experts, while after genomics, this improved to 14 of the cases being favored to be DM. In fact, some cases resulting in metastatic disease had a substantial increase in the number of experts recognizing them as DM after genomics. The impact of the genomic findings was less dramatic among benign and intermediate-grade desmoplastic tumors (BIDTs). Interobserver agreement also improved, with the Fleiss multirater Kappa being 0.36 before genomics to 0.4 after genomics. NGS has the potential to improve diagnostic accuracy in the assessment of desmoplastic melanocytic tumors. The degree of improvement will be most substantial among pathologists with some background and experience in bioinformatics and melanoma genetics.

Amplification of Mutant NRAS in Melanocytic Tumors With Features of Spitz Tumors.

NRAS activating mutations are prevalent in melanocytic neoplasia, occurring in a subset of common acquired melanocytic nevi and ∼30% of cutaneous melanomas. In this study, we described a cohort of 7 distinctive melanocytic tumors characterized by activating point mutations in codon 61 of NRAS with amplification of the mutant NRAS allele and shared clinicopathologic features. These tumors occurred predominantly in younger patients, with a median age of 20 years (range, 6-56 years). They presented as papules on the helix of the ear (4 cases) or extremities (3 cases). Microscopically, the tumors were cellular, relatively well-circumscribed, compound, or intradermal proliferations. The tumor cells often extended into the deep reticular dermis and involved the superficial subcutaneous fat in some cases. The melanocytes were epithelioid to spindled with moderate amounts of cytoplasm and conspicuous nucleoli. They were arranged in short plexiform fascicles, nests, and cords. Some cases had occasional pleomorphic and multinucleated melanocytes. Rare dermal mitotic figures were present in all cases. The dermis contained thick collagen bundles and minimal solar elastosis. Follow-up data were available for 5 patients, with a median period of 4.2 years (range, 1-9 years), during which no recurrences or metastases were reported. Our series highlights a clinicopathologically and molecularly distinctive subset of NRAS-mutated tumors with amplification of the mutant NRAS allele.

Distinct senescence mechanisms restrain progression of dysplastic nevi.

Telomerase reverse transcriptase (TERT) promoter mutations (TPMs) are frequently found in different cancer types, including ∼70% of sun-exposed skin melanomas. In melanoma, TPMs are among the earliest mutations and can be present during the transition from nevus to melanoma. However, the specific factors that contribute to the selection of TPMs in certain nevi subsets are not well understood. To investigate this, we analyzed a group of dysplastic nevi (DN) by sequencing genes commonly mutated in melanocytic neoplasms. We examined the relationship between the identified mutations, patient age, telomere length, histological features, and the expression of p16. Our findings reveal that TPMs are more prevalent in DN from older patients and are associated with shorter telomeres. Importantly, these TPMs were not found in nevi with BRAF V600E mutations. Conversely, DN with BRAF V600E mutations were observed in younger patients, had longer telomeres and a higher proportion of p16-positive cells. This suggests that these nevi arrest growth independently of telomere shortening through a mechanism known as oncogene-induced senescence (OIS). These characteristics extend to melanoma-sequencing datasets, where melanomas with BRAF V600E mutations were more likely to have a CDKN2A inactivation, overriding OIS. In contrast, melanomas without BRAF V600E mutations showed a higher frequency of TPMs. Our data imply that TPMs are selected to bypass replicative senescence (RS) in cells that were not arrested by OIS. Overall, our results indicate that a subset of melanocytic neoplasms face constraints from RS, while others encounter OIS and RS. The order in which these barriers are overcome during progression to melanoma depends on the mutational context.

Mosaic BRAF Fusions Are a Recurrent Cause of Congenital Melanocytic Nevi Targetable by MAPK Pathway Inhibition.

Among children with multiple congenital melanocytic nevi, 25% have no established genetic cause, of whom many develop a hyperproliferative and severely pruritic phenotype resistant to treatment. Gene fusions have been reported in individual cases of congenital melanocytic nevi. We studied 169 patients with congenital melanocytic nevi in this study, 38 of whom were double wild type for pathogenic NRAS/BRAF variants. Nineteen of these 38 patients had sufficient tissue to undergo RNA sequencing, which revealed mosaic BRAF fusions in 11 of 19 patients and mosaic RAF1 fusions in 1 of 19. Recurrently, fusions involved the loss of the 5´ regulatory domain of BRAF or RAF1 but preserved the kinase domain. We validated all cases and detected the fusions in two separate nevi in 5 of 12 patients, confirming clonality. The absence of the fusion in blood in 8 of 12 patients indicated mosaicism. Primary culture of BRAF-fusion nevus cells from 3 of 12 patients demonstrated highly increased MAPK activation, despite only mildly increased BRAF expression, suggesting additional mechanisms of kinase activation. Trametinib quenched MAPK hyperactivation in vitro, and treatment of two patients caused rapid improvement in bulk tissue, improving bodily movement and reducing inflammation and severe pruritus. These findings offer a genetic diagnosis to an additional group of patients and trametinib as a treatment option for the severe associated phenotypes.

Melanoma in infants, caused by a gene fusion involving the anaplastic lymphoma kinase (ALK).

We describe the first cases of pediatric melanoma with ALK fusion gene arising within giant congenital melanocytic nevi. Two newborn boys presented with large pigmented nodular plaques and numerous smaller satellite nevi. Additional expansile nodules developed within both nevi and invasive melanomas were diagnosed before 10 months of age in both boys. Oncogenic driver mutations in NRAS and BRAF were absent in both cases. Instead, oncogenic ZEB2::ALK fusion genes were identified in both the nevus and melanoma developing within the nevus. In both cases, tumors were noted by ultrasound in utero, demonstrated significant nodularity at birth, and progressed to melanoma in the first year of life suggesting that congenital nevi with ALK fusion genes may behave more aggressively than those with other mutations. As ALK kinase inhibitors are effective against a range of tumors with similar ALK fusion kinases, identifying ALK fusion genes in congenital melanocytic nevi may provide an opportunity for targeted therapy.

STmut: a framework for visualizing somatic alterations in spatial transcriptomics data of cancer.

Spatial transcriptomic technologies, such as the Visium platform, measure gene expression in different regions of tissues. Here, we describe new software, STmut, to visualize somatic point mutations, allelic imbalance, and copy number alterations in Visium data. STmut is tested on fresh-frozen Visium data, formalin-fixed paraffin-embedded (FFPE) Visium data, and tumors with and without matching DNA sequencing data. Copy number is inferred on all conditions, but the chemistry of the FFPE platform does not permit analyses of single nucleotide variants. Taken together, we propose solutions to add the genetic dimension to spatial transcriptomic data and describe the limitations of different datatypes.

Tracing cancer evolution and heterogeneity using Hi-C.

Chromosomal rearrangements can initiate and drive cancer progression, yet it has been challenging to evaluate their impact, especially in genetically heterogeneous solid cancers. To address this problem we developed HiDENSEC, a new computational framework for analyzing chromatin conformation capture in heterogeneous samples that can infer somatic copy number alterations, characterize large-scale chromosomal rearrangements, and estimate cancer cell fractions. After validating HiDENSEC with in silico and in vitro controls, we used it to characterize chromosome-scale evolution during melanoma progression in formalin-fixed tumor samples from three patients. The resulting comprehensive annotation of the genomic events includes copy number neutral translocations that disrupt tumor suppressor genes such as NF1, whole chromosome arm exchanges that result in loss of CDKN2A, and whole-arm copy-number neutral loss of homozygosity involving PTEN. These findings show that large-scale chromosomal rearrangements occur throughout cancer evolution and that characterizing these events yields insights into drivers of melanoma progression.

Proceedings from the Melanoma Research Foundation Mucosal Melanoma Meeting (December 16, 2022, New York, USA).

Mucosal melanoma remains a rare cancer with high mortality and a paucity of therapeutic options. This is due in significant part to its low incidence leading to limited patient access to expert care and downstream clinical/basic science data for research interrogation. Clinical challenges such as delayed and at times inaccurate diagnoses, and lack of consensus tumor staging have added to the suboptimal outcomes for these patients. Clinical trials, while promising, have been difficult to activate and accrue. While individual institutions and investigators have attempted to seek solutions to such problems, international, national, and local partnership may provide the keys to more efficient and innovative paths forward. Furthermore, a mucosal melanoma coalition would provide a potential network for patients and caregivers to seek expert opinion and advice. The Melanoma Research Foundation Mucosal Melanoma Meeting (December 16, 2022, New York, USA) highlighted the current clinical challenges faced by patients, providers, and scientists, identified current and future clinical trial investigations in this rare disease space, and aimed to increase national and international collaboration among the mucosal melanoma community in an effort to improve patient outcomes. The included proceedings highlight the clinical challenges of mucosal melanoma, global clinical trial experience, basic science advances in mucosal melanoma, and future directions, including the creation of shared rare tumor registries and enhanced collaborations.

The genetic evolution of acral melanoma.

Acral melanoma is an aggressive type of melanoma with unknown origins, arising on the sole, palm, or nail apparatus. It is the most common type of melanoma in individuals with dark skin and is notoriously challenging to treat. Our study examined exome sequencing data from 139 tissue samples, spanning different progression stages, collected from 37 patients. We found that 78.4% of the melanomas displayed one or more clustered copy number transitions with focal amplifications, recurring predominantly on chromosomes 5, 11, 12, and 22. These genomic "hailstorms" were typically shared across all progression stages within individual patients. Genetic alterations known to activate TERT also arose early. By contrast, mutations in the MAP-kinase pathway appeared later during progression, often leading to different tumor areas harboring non-overlapping driver mutations. We conclude that the evolutionary trajectories of acral melanomas substantially diverge from those of melanomas on sun-exposed skin, where MAP-kinase pathway activation initiates the neoplastic cascade followed by immortalization later. The punctuated formation of hailstorms, paired with early TERT activation, suggests a unique mutational mechanism underlying the origins of acral melanoma. Our findings highlight an essential role for telomerase, likely in re-stabilizing tumor genomes after hailstorms have initiated the tumors. The marked genetic heterogeneity, in particular of MAP-kinase pathway drivers, may partly explain the limited success of targeted and other therapies in treating this melanoma subtype.

Histologic and Genetic Features of 51 Melanocytic Neoplasms With Protein Kinase C Fusion Genes.

Fusion genes involving homologs of protein kinase C (PKC) have been identified in a variety of tumors. We report the clinical and histologic presentation of 51 cutaneous melanocytic neoplasms with a PKC fusion gene (involving PRKCA in 35 cases, PRKCB in 15 cases, and PRKCG in a single case). Most tumors were in young adults (median age, 29.5 years; range, 1-73 years) but some presented in newborns. Histologically, 42 tumors were classified as benign, presenting predominantly as biphasic dermal proliferation (88%) with nests of small melanocytes surrounded by fibrosis with haphazardly arranged spindled and dendritic melanocytes, resembling those reported as "combined blue nevi." Most tumors (60%) were heavily pigmented and in 15%, hyperpigmented epithelioid melanocytes were present at the dermoepidermal junction. Two lesions were paucicellular and showed marked sclerosis. Three tumors, including 2 proliferating nodules, were considered intermediate grade. Six tumors had sheets of atypical melanocytes infiltrating the dermis and were classified as melanomas. Two of the melanomas displayed loss of BAP1 nuclear expression. The median follow-up time was 12 months, with 1 patient alive with metastatic disease and 1 dying of their melanoma. These results suggest that melanocytic tumors with PKC fusion genes have characteristic histopathologic features, which are more similar to blue nevi than to pigmented epithelioid melanocytomas. As is the case with GNA-mutated blue nevi, they can progress to melanomas via BAP1 inactivation and metastasize.

Distinct senescence mechanisms restrain progression of dysplastic nevi.

TERT promoter mutations (TPMs) are frequently found in different cancer types, including approximately 70% of sun-exposed skin melanomas. In melanoma, TPMs are among the earliest mutations and can be present during the transition from nevus to melanoma. However, the specific factors that contribute to the selection of TPMs in certain nevi subsets are not well understood. To investigate this, we analyzed a group of dysplastic nevi (DN) by sequencing genes commonly mutated in melanocytic neoplasms. We examined the relationship between the identified mutations, patient age, telomere length, histological features, and the expression of p16. Our findings reveal that TPMs are more prevalent in DN from older patients and are associated with shorter telomeres. Importantly, these TPMs were not found in nevi with BRAF V600E mutations. Conversely, DN with BRAF V600E mutations were observed in younger patients, had longer telomeres, and a higher proportion of p16-positive cells. This suggests that these nevi arrest growth independently of telomere shortening through a mechanism known as oncogene-induced senescence (OIS). These characteristics extend to melanoma sequencing data sets, where melanomas with BRAF V600E mutations were more likely to have CDKN2A inactivation, overriding OIS. In contrast, melanomas without BRAF V600E mutations showed a higher frequency of TPMs. Our data imply that TPMs are selected to bypass replicative senescence (RS) in cells that were not arrested by OIS. Overall, our results indicate that a subset of melanocytic neoplasms face constraints from RS, while others encounter OIS and RS. The order in which these barriers are overcome during progression to melanoma depends on the mutational context.

Spectrum of Melanocytic Tumors Harboring BRAF Gene Fusions: 58 Cases With Histomorphologic and Genetic Correlations.

We report a series of 58 melanocytic tumors that harbor an activating fusion of BRAF, a component of the mitogen-activated protein kinase (MAPK) signaling cascade. Cases were diagnosed as melanocytic nevus (n = 12, 21%), diagnostically ambiguous favor benign (n = 22, 38%), and diagnostically ambiguous concerning for melanoma (n = 12, 21%) or melanoma (n = 12, 21%). Three main histopathologic patterns were observed. The first pattern (buckshot fibrosis) was characterized by large, epithelioid melanocytes arrayed as single cells or "buckshot" within marked stromal desmoplasia. The second pattern (cords in whorled fibrosis) demonstrated polypoid growth with a whorled arrangement of cords and single melanocytes within desmoplasia. The third pattern (spindle-cell fascicles) showed fascicular growth of spindled melanocytes. Cytomorphologic features characteristic of Spitz nevi were observed in most cases (n = 50, 86%). Most of the cases (n = 54, or 93%) showed stromal desmoplasia. Histomorphology alone was not sufficient in distinguishing benign from malignant melanocytic tumors with BRAF fusion gene because the only histopathologic features more commonly associated with a diagnosis of malignancy included dermal mitoses (P = .046) and transepidermal elimination of melanocytes (P = .013). BRAF fusion kinases are targetable by kinase inhibitors and, thus, should be considered as relevant genetic alterations in the molecular workup of melanomas. Recognizing the 3 main histopathologic patterns of melanocytic tumors with BRAF fusion gene will aid in directing ancillary testing.

Melanocytic naevi, melanocytomas and emerging concepts.

With the elucidation of the genetics of melanocytic tumours, new concepts have emerged. An important one is the identification of 'intermediate' melanocytic tumours, those with genetic progression events beyond those of melanocytic naevi but that are not fully malignant. Thus, melanocytic tumours exist on a genetic spectrum that likely corresponds to biological behaviour. There are multiple pathways to melanoma development with different initiating events and characteristic benign melanocytic neoplasms and the precise placement of tumours on these pathways remains to be established and the corresponding risks of progression quantified. In this review, I discuss the classification of melanocytic naevi based on clinical, histopathological and genetic features, as well as the concept of melanocytomas with discussion of specific recognisable subtypes.

Revision of the Melanocytic Pathology Assessment Tool and Hierarchy for Diagnosis Classification Schema for Melanocytic Lesions: A Consensus Statement.

Importance: A standardized pathology classification system for melanocytic lesions is needed to aid both pathologists and clinicians in cataloging currently existing diverse terminologies and in the diagnosis and treatment of patients. The Melanocytic Pathology Assessment Tool and Hierarchy for Diagnosis (MPATH-Dx) has been developed for this purpose. Objective: To revise the MPATH-Dx version 1.0 classification tool, using feedback from dermatopathologists participating in the National Institutes of Health-funded Reducing Errors in Melanocytic Interpretations (REMI) Study and from members of the International Melanoma Pathology Study Group (IMPSG). Evidence Review: Practicing dermatopathologists recruited from 40 US states participated in the 2-year REMI study and provided feedback on the MPATH-Dx version 1.0 tool. Independently, member dermatopathologists participating in an IMPSG workshop dedicated to the MPATH-Dx schema provided additional input for refining the MPATH-Dx tool. A reference panel of 3 dermatopathologists, the original authors of the MPATH-Dx version 1.0 tool, integrated all feedback into an updated and refined MPATH-Dx version 2.0. Findings: The new MPATH-Dx version 2.0 schema simplifies the original 5-class hierarchy into 4 classes to improve diagnostic concordance and to provide more explicit guidance in the treatment of patients. This new version also has clearly defined histopathological criteria for classification of classes I and II lesions; has specific provisions for the most frequently encountered low-cumulative sun damage pathway of melanoma progression, as well as other, less common World Health Organization pathways to melanoma; provides guidance for classifying intermediate class II tumors vs melanoma; and recognizes a subset of pT1a melanomas with very low risk and possible eventual reclassification as neoplasms lacking criteria for melanoma. Conclusions and Relevance: The implementation of the newly revised MPATH-Dx version 2.0 schema into clinical practice is anticipated to provide a robust tool and adjunct for standardized diagnostic reporting of melanocytic lesions and management of patients to the benefit of both health care practitioners and patients.

Interobserver agreement in the histopathological classification of desmoplastic melanomas.

Desmoplastic melanoma is a subtype of melanoma characterised by amelanotic fusiform melanocytes dispersed in a collagenous stroma. Cell-poor and fibrous stroma-rich 'pure' variants have been distinguished from 'mixed' variants with areas of higher cell density and/or less desmoplastic stroma. This distinction is relevant because patients whose tumours display a pure phenotype have a lower risk for regional lymph node metastasis and distant recurrence. However, little is known about interobserver agreement among pathologists in the subclassification of desmoplastic melanoma. To address this issue, we conducted a study in which eleven dermatopathologists independently evaluated whole slide scanned images of excisions from 30 desmoplastic melanomas. The participating pathologists were asked to classify the tumours as pure or mixed. They were also asked to record the presence or absence of neurotropism and angiotropism. We found substantial interobserver agreement between the 11 dermatopathologists in the classification of tumours as pure versus mixed desmoplastic melanoma (kappa=0.64; p<0.0001). There was fair agreement between the 11 dermatopathologists in the evaluation of presence versus absence of neurotropism (kappa=0.26; p<0.0001), and slight agreement in the assessment of angiotropism (kappa=0.13; p<0.0001). The level of concordance in the subclassification of desmoplastic melanomas is encouraging for the acceptance of this prognostic parameter in the real-world practice of melanoma pathology.

Integrated genomic analyses of acral and mucosal melanomas nominate novel driver genes.

BACKGROUND: Acral and mucosal melanomas are aggressive subtypes of melanoma, which have a significantly lower burden of somatic mutations than cutaneous melanomas, but more frequent copy number variations, focused gene amplifications, and structural alterations. The landscapes of their genomic alterations remain to be fully characterized. METHODS: We compiled sequencing data of 240 human acral and mucosal melanoma samples from 11 previously published studies and applied a uniform pipeline to call tumor cell content, ploidy, somatic and germline mutations, as well as CNVs, LOH, and SVs. We identified genes that are significantly mutated or recurrently affected by CNVs and implicated in oncogenesis. We further examined the difference in the frequency of recurrent pathogenic alterations between the two melanoma subtypes, correlation between pathogenic alterations, and their association with clinical features. RESULTS: We nominated PTPRJ, mutated and homozygously deleted in 3.8% (9/240) and 0.8% (2/240) of samples, respectively, as a probable tumor suppressor gene, and FER and SKP2, amplified in 3.8% and 11.7% of samples, respectively, as probable oncogenes. We further identified a long tail of infrequent pathogenic alterations, involving genes such as CIC and LZTR1. Pathogenic germline mutations were observed on MITF, PTEN, ATM, and PRKN. We found BRAF V600E mutations in acral melanomas with fewer structural variations, suggesting that they are distinct and related to cutaneous melanomas. Amplifications of PAK1 and GAB2 were more commonly observed in acral melanomas, whereas SF3B1 R625 codon mutations were unique to mucosal melanomas (12.9%). Amplifications at 11q13-14 were frequently accompanied by fusion to a region on chromosome 6q12, revealing a recurrent novel structural rearrangement whose role remains to be elucidated. CONCLUSIONS: Our meta-analysis expands the catalog of driver mutations in acral and mucosal melanomas, sheds new light on their pathogenesis and broadens the catalog of therapeutic targets for these difficult-to-treat cancers.

Ferrous iron-activatable drug conjugate achieves potent MAPK blockade in KRAS-driven tumors.

KRAS mutations drive a quarter of cancer mortality, and most are undruggable. Several inhibitors of the MAPK pathway are FDA approved but poorly tolerated at the doses needed to adequately extinguish RAS/RAF/MAPK signaling in the tumor cell. We found that oncogenic KRAS signaling induced ferrous iron (Fe2+) accumulation early in and throughout mutant KRAS-mediated transformation. We converted an FDA-approved MEK inhibitor into a ferrous iron-activatable drug conjugate (FeADC) and achieved potent MAPK blockade in tumor cells while sparing normal tissues. This innovation allowed sustainable, effective treatment of tumor-bearing animals, with tumor-selective drug activation, producing superior systemic tolerability. Ferrous iron accumulation is an exploitable feature of KRAS transformation, and FeADCs hold promise for improving the treatment of KRAS-driven solid tumors.

Update on classification of melanocytic tumors and the role of immunohistochemistry and molecular techniques.

The nine pathways to melanoma outlined in the most recent WHO classification of skin tumors attempt to capture the typical evolution of different subtypes of melanoma as governed by cell of origin, mutagenic factors and oncogenic drivers. This pathway-based classification will continue to be refined as we learn more about melanocytic tumor biology. I review the practical application of immunohistochemistry and molecular techniques in the diagnosis of melanocytic tumors in the context of this classification.