31. Update-on-classification-of-melanocytic-tumors-and-the-_2022_Seminars-in-Dia.pdf

Seminars in Diagnostic Pathology 39 (2022) 248–256 Contents lists available at ScienceDirect Seminars in Diagnostic Pathology journal homepage: www.elsevier.com/locate/semdp Review article Update on classification of melanocytic tumors and the role of immunohistochemistry and molecular technique...

Seminars in Diagnostic Pathology 39 (2022) 248–256 Contents lists available at ScienceDirect Seminars in Diagnostic Pathology journal homepage: www.elsevier.com/locate/semdp Review article Update on classification of melanocytic tumors and the role of immunohistochemistry and molecular techniques Iwei Yeh a, * a Departments of Dermatology and Pathology, University of California, San Francisco, United States A B S T R A C T 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. Introduction The diagnosis of melanocytic tumors is an age old and central problem in dermatopathology. The best diagnoses are made with inte­ gration of clinical features and understanding of the pathogenesis and evolution of melanocytic tumors. It is clear that melanoma can be divided into subcategories with different clinical courses.1 These mela­ noma subtypes are caused by different oncogenic drivers and mutagenic factors. The 2018 WHO classification of skin tumors outlines nine pathways to melanoma.2,3 This pathway classification will continue to evolve with our understanding of melanocytic tumor biology. For some of these pathways of melanoma development, a melano­ cytic nevus within the pathway has been clearly identified (Table 1). The oncogenic mutations characteristic of the pathway lead to development of a melanocytic nevus in which every melanocyte harbors the initiating mutation.4,5(p200),6,7,8,9,10,11,12,13,14 Genetic work over the past decade has clearly demonstrated that there are melanocytic tumors that are intermediate in terms of their genetic progression as they have genetic alterations in addition to those in melanocytic nevi and show benign or low-grade biologic behavior.15,16 The histopathologic and genetic fea­ tures of some of these intermediate tumors, referred to as melanocyto­ mas, are now well characterized. It is likely that there are many melanocytomas still to be characterized and some may not be distin­ guishable from nevi based on histopathology alone. The melanoma pathway most encountered and best understood is pathway 1, the low-cumulative sun damage (low-CSD) pathway. The melanocytic nevi of this pathway typically harbor a BRAF V600E mu­ tation (in approximately 90% of cases) and have conventional cyto­ morphology. They commonly arise in adolescence or early adulthood. * Corresponding author. E-mail address: [email protected]. https://doi.org/10.1053/j.semdp.2022.02.001 Available online 8 February 2022 0740-2570/© 2022 Elsevier Inc. All rights reserved. We routinely encounter low-CSD melanoma within or adjacent to a precursor conventional melanocytic nevus. We are familiar with many types of melanocytomas that arise within conventional melanocytic nevi, including deep penetrating melanocytomas (previously referred to as deep penetrating nevi), BAP1-inactivated melanocytomas, and mel­ anoma in situ. Progression from melanocytic nevus to melanoma occurs when the nevus initiating oncogene is acquired before other driver mutations. Due to the increased number of melanocytes at risk of further progression within a melanocytic nevus, acquisition of an initiating oncogene is more likely to occur before other driver events are acquired. A melanoma may overgrow and obscure its precursor melanocytic nevus or stimulate an immune response that clears the nevus but not the melanoma itself. Thus, some melanomas without an observable pre­ cursor nevus may still have originated from one. However, it remains consistent with the step-wise model of melanoma progression that melanomas can arise de novo. For example, it is possible that nodular melanomas acquire an initiating oncogene after acquiring other onco­ genic alterations that do not promote proliferation, such as loss of CDKN2A or TERT promoter mutation. Alternatively, some genomic events may lead to the simultaneous acquisition of driver mutations. Examples of such catastrophic genomic events include chromothripsis and chromoplexy that result from “shattering” of chromosomes.17,18 A single catastrophic event may produce a combination of driver alter­ ations that result in melanoma. The histopathologic features used to categorize a tumor as benign or malignant vary across the pathways. For example, scatter of melano­ cytes into the upper levels of the epidermis, minimal maturation, dermal mitotic activity and enlarged epithelioid melanocytes are often present in Spitz nevi but are concerning for melanoma in the low-CSD I. Yeh Seminars in Diagnostic Pathology 39 (2022) 248–256 and malignant diagnoses are under consideration.29 When possible, we should attempt to uncouple these distinct scenarios moving forward. For a well-characterized melanocytoma with low-grade biologic behavior, a re-excision may be optional, whereas for a melanocytic tumor for which melanoma is a diagnostic consideration, complete excision is often recommended to reduce the risk of persistent melanoma. In this review, I will discuss how I integrate immunohistochemistry and molecular testing into this new diagnostic paradigm. Table 1 Pathways to melanoma. Pathway Nevus Low-grade melanocytoma High-grade melanocytoma Melanoma 1 Common or conventional nevus BRAF V600E > NRAS Q61 melanoma in situ low-CSD melanoma TERT promoter mutation CDKN2A inactivation NF1 inactivation high-CSD melanoma desmoplastic melanoma Spitz melanoma TERT promoter mutation CDKN2A inactivation 2 ? deep penetrating CTNNB1 mutation, APC inactivation BAP1inactivated PRKAR1Ainactivated ? 3 ? ? 4 CDKN2Ainactivated Spitz melanocytoma 5 Spitz nevus HRAS Q61, fusions of receptor tyrosine kinases, fusions of kinases in the MAPK pathway ? ? melanoma in situ 6 ? ? melanoma in situ 7 congenital melanocytic nevus NRAS Q61 > BRAF V600E, BRAF fusion proliferative nodule 8 blue nevus GNAQ and GNA11 mutation ? atypical proliferative nodule copy number losses and gains of entire chromosomes ? 9 uveal nevus GNAQ and GNA11 mutation ? ? melanoma in situ melanoma in situ high grade Spitz melanocytoma Assignment to the correct pathway When approaching a case, after ascertaining that I am dealing with a melanocytic proliferation, I assign the proliferation to a pathway. Three of the pathways to melanoma have well-characterized cutaneous nevi: common acquired or conventional melanocytic nevi, Spitz nevi and blue nevi. During routine daily practice, identifying a melanocytic prolifer­ ation and assigning it to a pathway may take only a moment and occur almost automatically (engaging system 1 thinking as described by Daniel Kahneman’s Thinking, Fast and Slow30). However, when I am considering the possibility of melanoma, I try to engage a more delib­ erative and logical process to pathway classification (system 2 thinking). Spitz versus conventional (low-CSD) pathway acral melanoma CCND1 amplification mucosal melanoma TERT amplification SPRED1 or NF1 inactivation melanoma arising in congenital melanocytic nevus Melanocytic tumors of Spitz and conventional lineage can be difficult to differentiate in some cases. Spitz nevi are more common in childhood but can arise in adults as well. In children, Spitz tumors may demon­ strate considerably more cellular atypia and mitotic activity and still be considered nevi or melanocytomas that can be treated conservatively with good outcomes. In adults, melanocytic tumors of the low-CSD pathway are much more prevalent than Spitz nevi and the incidence of melanoma increases significantly with age, making discrimination of Spitz nevus from a melanoma mimic particularly treacherous.31,32 Nevertheless, correct diagnosis of Spitz nevus in adults is necessary to prevent overtreatment. Many tumors classified as atypical Spitz tumors before our understanding of the genetics of Spitz tumors were actually of low-CSD lineage as they harbor BRAF V600E mutations.33,34 This may explain in part why tumors classified as atypical Spitz tumors have worse outcomes in adults than in children (as melanoma is more com­ mon in adults, more atypical Spitz tumors in adults may actually be of low-CSD lineage). When the lineage of a melanocytic proliferation is uncertain, and the determination of lineage will affect diagnosis and management, geno­ typic information can be used to guide classification. Gene panel testing (to be discussed later) can be used to test for many initiating oncogenes in parallel but is not practical in many cases due to cost and reim­ bursement issues as well as turnaround time. Immunohistochemistry can be used to assess for a limited number of alterations but is not comprehensive and the results must be considered in the context of the clinical and histopathologic findings. Mutation specific antibodies include VE1 which recognizes BRAF V600E.35 BRAF V600E is present in 80–90% of tumors of conventional lineage and is very infrequent if present in the Spitz lineage.5,9 In adults, a tumor that appears Spitz-like but harbors a BRAF V600E mutation is likely a low-CSD melanoma with a superficial spreading pattern. There are also other activating point mutations that affect BRAF V600 and other nearby residues that may predict response to BRAF inhibitors but are not detected by the VE1 antibody. Presence of BRAF V600E by VE1 immunohistochemistry argues for classification in the low-CSD pathway, though absence does not exclude low-CSD lineage. Approximately 10% of common or conventional nevi harbor NRAS mutations, and NRAS mutations are present at similar prevalence in lowCSD, high-CSD, acral, and mucosal melanomas.36–42 NRAS mutations are present in the majority giant congenital melanocytic nevi.6,10 RAS mutation specific antibodies are available, but there are several different NRAS Q61 amino acid substitutions without one being dominant. In melanoma arising in blue nevus BAP1 inactivation SF3B1, EIF1AX mutation uveal melanoma BAP1 inactivation SF3B1, EIF1AX mutation pathway.19–23 While many driver genetic alterations that contribute to melanoma development have been identified, it seems that the order in which mutations arise may differ across pathways and thus the diag­ nostic significance of a given genetic alteration may differ between pathways. A subset of genetically intermediate melanocytomas are wellcharacterized because they often arise within a melanocytic nevus and the acquisition of additional driver alterations result in a characteristic phenotypic change.24–28 While most melanocytomas have benign behavior, due to their additional oncogenic alterations, their risk of progression to melanoma is likely increased relative to their precursor melanocytic nevi though the level of this risk remains to be determined. In the past melanocytomas have been grouped with unusual melanocytic tumors with a large degree of diagnostic uncertainty where both benign 249 I. Yeh Seminars in Diagnostic Pathology 39 (2022) 248–256 gene is regulated by the promoter of the 5′ partner. The kinase gene contributes the portion that encodes its kinase domain to the fusion gene and the absence of the negative regulatory portion of the native kinase results in constitutive activity. Antibodies that target the kinase domain of the native kinase can be used to identify the presence of a kinase fusion in some cases. ALK and ROS1 are kinases are not expressed in mature melanocytes. A positive immunohistochemistry result is there­ fore highly specific for the presence of a fusion (Fig. 1). Due to the va­ riety of fusion partners and their effects on the overall level of expression addition, due to the similarity between the RAS isoforms, the antibodies cannot distinguish between mutations in NRAS and the other Ras iso­ forms, such as those in HRAS that occur in a subset of Spitz nevi. Because nevi in distinct pathways may be initiated by a spectrum of mutations, a single negative result, while providing information, is not definitive. Tumors in the Spitz pathway can be initiated by a spectrum of oncogenes, a subset of which can be identified by immunohistochem­ istry. The oncogenic kinase fusions that can initiate Spitz nevi are formed by the fusion of two genes. The expression of the resulting fusion Fig. 1. Pathway classification by immunohistochemistry. (A) low-CSD melanoma with BRAF V600E mutation left: H and E, right: immunohistochemistry for VE1 (B) Spitz nevus with ALK fusion left: H and E, right: immunohistochemistry for ALK kinase domain (C) Spitz nevus with NTRK1 fusion left: H and E, right: immuno­ histochemistry for NTRK1 kinase domain. 250 I. Yeh Seminars in Diagnostic Pathology 39 (2022) 248–256 of the fusion protein and its localization, several different expression patterns can be seen including diffuse cytoplasmic and dot-like expres­ sion. NTRK fusions (NTRK1 and NTRK3 are most common in Spitz tu­ mors, though NTRK2 has also been observed) typically lead to strong uniform expression of the NTRK kinase domain. Because NTRK isoforms are expressed in melanocytes, a positive result is more subject to inter­ pretation and less specific. Strong uniform expression of NTRK by me­ lanocytes is indicative of a fusion whereas moderate or patchy staining should be considered equivocal. In some cases, particularly when guiding targeted therapy, a confirmatory test is recommended. For other kinases, immunohistochemistry does not appear helpful in predicting the presence of a kinase fusion (BRAF, RAF1, MAP3K8). Because of the large spectrum of Spitz initiating mutations, it can be helpful to recog­ nize histopathologic features associated with different kinase fusions to guide select application of immunohistochemistry.43–45,46(p3),47,48(p3) BAP1-inactivated melanocytomas of the low-CSD pathway often mimic Spitz tumors. They may arise within a conventional melanocytic nevus, and identification of the precursor melanocytic nevus can be a helpful clue to the correct pathway. Most BAP1-inactivated melanocy­ tomas arise within the low-CSD pathway and a significant majority harbor BRAF V600E mutation. BAP1-inactivated melanocytomas are typically intradermal and comprised of melanocytes with well-defined nuclear membranes, abundant amphophilic glassy cytoplasm, eccen­ tric nuclei and pleomorphism of cell and nuclear size and are often inflamed (Fig. 2). Before these tumors were recognized as melanocyto­ mas and the pathway classification was established, many were classi­ fied as atypical Spitz tumors, or halo Spitz nevi.49,22,25 Immunohistochemistry can be helpful to demonstrate BAP1 inactiva­ tion, which typically occurs due to a truncating mutation of one allele and loss of the other wild-type allele (with loss of all or part of chromosome 3 on which BAP1 resides). BAP1 is a nuclear protein, and keratinocytes and lymphocytes serve as internal positive controls. Loss of nuclear expression of BAP1 within melanocytes is quite specific for BAP1-inactivation, however, inactivating missense mutations may occur that disrupt nuclear localization of BAP1 resulting in cytoplasmic expression of BAP1, or inactivate the function of the protein with retained nuclear expression of BAP1.50(p1),51 Thus, expression of BAP1 may be retained in BAP1-inactivated tumors. As compared to Spitz nevi, BAP1-inactivated melanocytomas typically have a lower mitotic index and elevated proliferative activity (>2 per mm2) is concerning for melanoma. People with germline inactivating mutations of BAP1 are at increased risk for developing BAP1-inactivated melanocytomas and BAP1-related malignancies including uveal and cutaneous melanoma, mesothelioma and renal cell carcinoma.52 I typically include a statement about referral for discussion of genetic testing and highlight the need for increased suspicion in children and patients with multiple BAP1-i­ nactivated tumors or a personal or family history of related cancers. Conventional versus blue nevus pathway While typical blue nevi are easy to distinguish from tumors in the low-CSD or conventional pathway, there are some melanocytomas within the low-CSD pathway that may mimic tumors of the blue pathway. Deep penetrating melanocytomas (previously referred to as deep penetrating nevus) harbor mutations that activate the beta-catenin pathway. These are most often point mutations in beta-catenin, although bi-allelic loss of function mutations in APC have been observed as an alternative mechanism. Deep penetrating melanocyto­ mas are composed of pigmented melanocytes with moderate to Fig. 2. BAP1-inactivation in the low-CSD pathway. (A) low power view of BAP1-inactivated melanocytoma (B) high power view showing characteristic epithelioid melanocytes (C) BAP1 immunohistochemistry demonstrates nuclear staining in keratinocytes and lymphocytes but absence of nuclear staining in the epithelioid melanocytes. 251 I. Yeh Seminars in Diagnostic Pathology 39 (2022) 248–256 abundant amounts of pigmented and vacuolated cytoplasm arrayed as small aggregates and slightly elongated nests with scattered mela­ nophages in the surrounding dermis (Fig. 3). They lack maturation with depth, and melanocytes stay uniform in size and dispersion. Due to their features, they may mimic blue nevi or melanoma. Mutational activation of the beta-catenin pathway is not exclusive to melanocytomas, it is present in about 10% of melanomas. Activation of the beta-catenin pathway can be assessed by immunohistochemistry for beta-catenin or LEF1 (the binding partner of beta-catenin in the nucleus) .53,54 Expres­ sion of both is strong and uniform in tumors with beta-catenin activation Fig. 3. Deep penetrating melanocytoma. (A) low power view of a deep penetrating melanocytoma and associated precursor nevus (left: H and E, right: immuno­ histochemistry for beta-catenin) (B) medium power view left: Melanocytes with deep penetrating cytomorphology are present in the deep dermis below a precursor conventional nevus. right: The deep penetrating melanocytes show strong uniform expression of beta-catenin. The precursor nevus shows patchy expression with a slight gradient pattern with less staining in the deeper portion. (C) high power view left: The deep penetrating melanocytes have abundant vacuolated pigmented cytoplasm and there are scattered melanophages. right: The deep penetrating melanocytes show strong uniform cytoplasmic expression of beta-catenin. This case demonstrates strong nuclear staining, though this is not a constant feature. 252 I. Yeh Seminars in Diagnostic Pathology 39 (2022) 248–256 and shows a gradient pattern in conventional nevi. Beta-catenin shows strong cytoplasmic and variable nuclear staining in deep penetrating melanocytoma (Fig. 3). Blue nevi typically show uniform membranous expression of beta-catenin. Another melanocytoma has been previously referred to as epithelioid blue nevus (of Carney complex) and is a subset of pigmented epithelioid melanocytoma.55–57,28 I refer to this tumor as PRKAR1A-inactivated melanocytoma. It is most common in the low-CSD pathway and typically harbors a BRAF V600E mutation. While these tumors are pigmented and epithelioid, they are genetically distinct from the blue nevus pathway which is characterized by a mutation that activates Gαq pathway rather than a BRAF V600E mutation. The melanocytes of PRKAR1A-inactivated melanocytoma typically have larger vesicular nuclei with large faintly eosinophilic nucleoli and are distributed as single cells more than nests (Fig. 4). These tumors are less common than deep penetrating melanocytomas (most likely because they require two hits to a tumor suppressor rather than a single oncogenic alteration) and can be con­ cerning for melanoma. Loss of PRKAR1A expression by immunohisto­ chemistry can be helpful in identifying these tumors. Patients with Carney complex due to germline inactivation of PRKAR1A have a higher risk of developing these and other characteristic tumors such as myx­ omas and psammomatous melanotic schwannoma.56 Tumors of melanocytic differentiation not within the current pathway classification Clear cell sarcomas demonstrate melanocytic differentiation though they are typically amelanotic without epidermal involvement. These tumors can be confused with Spitz tumors, cellular blue nevi, and other sarcomas. They are characterized by EWSR1 fusions, typically involving ATF1 and less commonly CREB1.58 Melanocytic tumors with CTRC1-TRIM11 fusions have similar clinical and histopathologic fea­ tures.59,60 Benign precursors of these tumors have not been identified. Assessment of tumor suppressors We already discussed the evaluation of the tumor suppressors BAP1 and PRKAR1A in the context of melanocytomas of the low-CSD pathway. Notably, BAP1 is a tumor suppressor that is often lost in uveal melanoma or melanoma arising within blue nevus and loss of BAP1 in these mel­ anoma pathways is highly concerning for melanoma as a low-grade tumor with loss of BAP1 in these pathways has not been identified. This is an example of how classifying a tumor into the correct pathway is a critical foundational component of the diagnostic paradigm. Another important tumor suppressor in melanoma is CDKN2A which encodes p16 and p14. About half of melanomas across all pathways demonstrate inactivation of this tumor suppressor and germline inacti­ vating mutations predispose to melanoma. While loss of p16 often cor­ responds with malignant transformation in the low-CSD pathway, it does not necessarily indicate malignant transformation in the Spitz pathway.15,61 CDKN2A-inactivated Spitz melanocytoma, defined as a Spitz tumor with isolated inactivationof CDKN2A without other addi­ tional secondary driver alterations likely account for most atypical Spitz tumors with inactivation of CDKN2A. These tumors typically have benign biologic behavior and early studies suggest that TERT promoter alterations or other telomere maintenance mechanisms are required for malignant transformation.16 p16 can be evaluated by immunohisto­ chemistry, but its interpretation can be challenging. This tumor sup­ pressor is not expressed at significant levels in normal melanocytes, unlike BAP1 or PRKAR1A. Rather, its expression is induced by onco­ genic signaling, and it is a mediator of oncogene induced senescence. Some nevi express little p16 and this may reflect lack of induction of p16 rather than inactivation of CDKN2A. Thus, lack of p16 expression should be interpreted in the context of the histopathology. Genetic inactivation of CDKN2A often occurs by homozygous deletion but can also occur due to a truncating mutation in one allele followed by loss of heterozygosity, often due to loss of part of all of chromosome 9. However, many mela­ nocytic tumors show single copy loss of chromosome 9 without inacti­ vation of CDKN2A (p16 expression is retained). Because of these various methods of CDKN2A inactivation, homozygous deletion by fluorescence in situ hybridization is specific but not entirely sensitive for genetic inactivation of CDKN2A. p16 expression can be either cytoplasmic or nuclear and often occurs in mosaic fashion, with some melanocytes expressing p16 with adjacent melanocytes completely lacking expres­ sion (Fig. 5). Other tumor suppressors that are inactivated in melanoma include NF1 and SPRED138(p1),39(p1),42(p1). Immunohistochemistry can be used to assess their status, but the expression of these tumor suppressors across different types of melanocytic nevi, melanocytomas, and melanomas remains to be further characterized. Fig. 4. PRKAR1A-inactivated melanocytoma. (A) low power view (B) high power view On the left there are nests of melanocytes with moderate amounts of cytoplasm, representing a precursor melanocytic nevus. On the right, scat­ tered melanocytes have large nuclei and occasionally prominent nucleoli. There are many melanophages in the surrounding dermis. (C) PRKAR1A immuno­ histochemistry shows cytoplasmic expression in the conventional nevus. The large melanocytes on the right lack PRKAR1A expression. 253 I. Yeh Seminars in Diagnostic Pathology 39 (2022) 248–256 Fig. 5. Assessment of p16 by immunohistochemistry. (A) Common nevus with low to moderate expression of p16. Note the gradient pattern with less p16 expression at the base of the melanocytic nevus. (B) Common nevus with moderate to high expression of p16. Most melanocytes but not all express p16. (C) Biphenotypic melanocytic tumor, with large pigmented melanocytes in the superficial component and small melanocytes below. (D) p16 immunohistochemistry shows strong expression in the small melanocytes and lack of expression in the larger melanocytes, indicating genetic progression with CDKN2A-inactivation. Molecular assessment of melanocytic tumors Nevi and melanocytomas can demonstrate losses or gains of limited portions of the genome. Spitz nevi with HRAS mutations often have gain of 11p (on which HRAS resides). Spitz nevi initiated by kinase fusions often display copy number alterations flanking one or both fusion partners. Amplification of the fusion gene may also occur. If a tumor in the Spitz pathway is a diagnostic consideration, copy number transitions that reflect breakpoints within Spitz nevus associated kinase genes can indicate a kinase fusion. Atypical proliferative nodules that arise within giant congenital nevi mimic melanoma histopathologically but have benign behavior and are often show copy number losses or gains of whole chromosomes. With aCGH or other genome wide assessments, one can also observe evidence of chromothripsis or chromoplexy in both benign/low-grade tumors and melanomas. Dual-colored FISH probes can also be used to detect rearrangements. Depending on the design, a specific assay can test for splitting of a specific gene or fusion of two specific partners. FISH assays can result in false negatives (a small inversion can result in a fusion gene without a detectable difference in probe distribution) or false positives (a struc­ tural rearrangement affects the target gene but does not result in oncogene formation, for example, the kinase portion of the gene may be fused to an intergenic region that does not result in a fusion transcript). Immunohistochemistry can be used to help assign melanocytic tu­ mors to a specific pathway as well as classify their progression status. However, immunohistochemistry directed towards specific oncogenes or tumor suppressors should be used in combination with histopatho­ logic features since broad assessment of drivers is not practical by immunohistochemistry and many driver alterations do not cause detectable or reproducible alterations in protein expression levels. Molecular techniques can provide additional information and can be divided into assessment of genomic breakpoints, genomic copy number status, genetic mutations, and expression signatures. Copy number status Melanomas often demonstrate losses and gains of genomic mate­ rial.62,63 These losses and gains can be classified by their size and amplitude. The methods used for copy number assessment include fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH). Some gene panel tests also provide copy number information. Each method of copy number assessment has its own sensitivity and specificity for detecting specific copy number aberrations. FISH is typically used to interrogate 4–6 genomic positions at a time. While the areas of the genome that can be assessed are limited, FISH can be used for sparsely cellular and small samples. aCGH has varying degrees of resolution depending on the platform. With lower resolution, small copy number aberrations may not be detected. Traditional four probe FISH for melanoma assesses copy number status and balance of 6p and 6q as well as the CCND1 locus on 11q. Of note, the genomic copy number changes typically observed in melanoma arising in blue nevus are quite different that those that are typical of the other melanoma pathways and traditional melanoma FISH should not be applied to tumors for which melanoma arising in blue nevus is a consideration. Gene panel testing Massively parallel short read sequencing technology has been adopted for gene panel or exome sequencing for risk stratification, diagnosis, and treatment selection. Cell free DNA sequencing is used for prenatal diagnosis as well as cancer detection and monitoring. Gene panel testing can be applied to the diagnosis of melanocytic tumors, but the testing platform and the interpretation of results is not yet systematized or algorithmic. Gene panel tests are typically designed for broad application to several tumor types and vary from test to test, so understanding the information that can be provided is critical. Depending on the design of the assay, DNA sequencing can provide 254 Seminars in Diagnostic Pathology 39 (2022) 248–256 I. Yeh information on non-coding sequences, such as TERT promoter mutations that are common in melanoma as well as detect structural rearrange­ ments that give rise to fusion genes. DNA sequencing is generally less sensitive for fusion detection as compared to RNA sequencing, and some tests incorporate analysis of both DNA and RNA. Some gene panel tests also provide copy number information, of varying accuracy and reso­ lution depending on the platform and the analysis algorithm. For the moment, interpretation of gene panel testing requires integration of clinical and histopathologic findings and an understanding of the test characteristics. 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Gene expression profiling Commercial tests based on gene expression profiling are available for both diagnosis and prognosis of melanocytic tumors. For diagnosis, some tests are based on material from tape stripping the surface of the pigmented lesion, without requiring biopsy. These tests, similar to aCGH and gene panel sequencing take as input a mixture of tumor cells and adjacent normal or inflammatory cells. The sensitivity of the tests are typically above 90% but less than 95% with varying specificities. It re­ mains to be seen how best to integrate these tests into clinical practice in combination with expert clinical examination and dermoscopy or expert histologic assessment with immunohistochemistry and other ancillary molecular tests. Expression of PRAME (preferentially expressed antigen in melanoma) is assessed in most of the gene expression tests and expression is associated with melanoma is also a biomarker for other cancers. PRAME expression can be assessed in melanocytic tumors by immunohistochemistry and is more commonly expressed in melanoma than melanocytic nevi.64,65 The fraction of melanocytic nevi and mela­ nomas that express PRAME vary across the WHO defined pathways and thus PRAME expression should be interpreted in the context of mela­ noma subtype. Conclusion It is an exciting time given the many scientific advances in our un­ derstanding of melanocytic tumor development and progression and technological advances that enable multidimensional molecular testing of small archival specimens. At the moment, a complete diagnostic al­ gorithm that incorporates clinical and histopathologic features, the subtypes of melanoma and their pathways to progression, and genotypic and expression data has not been developed. 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