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.

BRAF Mutated and Morphologically Spitzoid Tumors, a Subgroup of Melanocytic Neoplasms Difficult to Distinguish From True Spitz Neoplasms.

Drivers of Spitz neoplasms include activating point mutations in HRAS and Spitz-associated genomic fusions. It has become evident that some BRAF -mutated melanocytic neoplasms can morphologically mimic Spitz tumors (STs). These have been termed BRAF mutated and morphologically spitzoid (BAMS). In this study, 17 experts from the International Melanoma Pathology Study Group assessed 54 cases which included 40 BAMS and 14 true STs. The participants reviewed the cases blinded to the genomic data and selected among several diagnostic options, including BAMS, ST, melanoma, and other. A total of 38% of all diagnostic selections in the BAMS cases were for BAMS, whereas 32% were for ST. In 22 of the BAMS cases, the favored diagnosis was BAMS, whereas in 17 of the BAMS cases, the favored diagnosis was ST. Among the 20 cases in the total group of 54 with the highest number of votes for ST, half were BAMS. Of BAMS, 75% had a number of votes for ST that was within the SD of votes for ST seen among true ST cases. There was poor interobserver agreement for the precise diagnosis of the BAMS (kappa = 0.16) but good agreement that these cases were not melanoma (kappa = 0.7). BAMS nevi/tumors can closely mimic Spitz neoplasms. Expert melanoma pathologists in this study favored a diagnosis of ST in nearly half of the BAMS cases. There are BAMS cases that even experts cannot morphologically distinguish from true Spitz neoplasms.

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.

Next-generation sequencing improves agreement and accuracy in the diagnosis of Spitz and spitzoid melanocytic lesions.

BACKGROUND: Spitzoid melanocytic neoplasms can be challenging to diagnose on histopathology alone. Next-generation sequencing (NGS) offers promise as a valuable aid in the diagnosis. Recently, one study reported increased inter-rater agreement in the diagnosis of spitzoid melanocytic neoplasms among 20 expert melanoma pathologists after incorporating NGS data. We hypothesized that NGS would carry a similar utility in a broader group of dermatopathologists and general pathologists. METHODS: Sixty-three participants of a live online (www.Dermpedia.org) CME course rendered a diagnosis on 70 cases composed of melanocytic neoplasms with spitzoid features. In Survey 1, cases included H&E slides and demographic information only, while Survey 2 included NGS data. RESULTS: With NGS information, inter-rater agreement significantly improved from "fair" to "almost perfect" and from "fair" to "substantial" for categorizing lesions as Spitz versus non-Spitz and conventional melanoma versus not, respectively. There was also an increase in diagnostic accuracy, evidenced by improved recognition of three metastatic tumors as being conventional melanomas. CONCLUSION: The study supports the adoption of NGS as a valuable diagnostic adjunct for both expert and broader dermatopathologists in their assessments of spitzoid neoplasms.

Next-Generation Sequencing Reveals a New Class of Melanocytic Neoplasms With Hybrid Genomic Features of PEM Including Protein Kinase R 1 Alpha Gene Inactivation and Spitz Tumor-Defining Protein Kinase Fusions.

BACKGROUND: Pigmented epithelioid melanocytoma (PEM) is a subtype of melanocytic tumor with frequent involvement of the sentinel lymph node but rare distant metastasis. Rendering a diagnosis and prognosis based on histology can be challenging. Recent genomic studies identified 2 molecular variants of PEM. One variant is characterized by the activation of the mitogen-activated protein kinase pathway and inactivation of the PRKAR1a gene. The other is associated with genomic fusions involving the protein kinase C ( PRKC ) gene family. OBJECTIVE: We investigated the molecular and clinicopathologic features of previously unreported PEM cases to improve tumor classification and report new classes of PEM. METHODS: Next-generation sequencing and histomorphologic assessment was performed on 13 PEM cases. RESULTS: We identified 2 novel PEM classes. Three cases harbored PRKAR1a inactivation and genomic fusions ( ALK , NTRK , and MAP3K8 ). These tumors had overlapping histologic features with pigmented Spitz neoplasms. Three cases had genomic fusions involving PRKCB . These cases had overlapping features with PRKCA fusions but, in 2 cases, had a notable spindle cell component. LIMITATIONS: The overall sample size and amount of clinical follow-up is limited, leaving some uncertainty regarding the expected clinical course of these novel cases. CONCLUSIONS: PRKAR1a-inactivated/Spitz fusion-associated PEMs and PRKCB fusion-associated PEMs represent 2 new molecular classes of PEM.

Impact of Next-generation Sequencing on Interobserver Agreement and Diagnosis of Spitzoid Neoplasms.

Atypical Spitzoid melanocytic tumors are diagnostically challenging. Many studies have suggested various genomic markers to improve classification and prognostication. We aimed to assess whether next-generation sequencing studies using the Tempus xO assay assessing mutations in 1711 cancer-related genes and performing whole transcriptome mRNA sequencing for structural alterations could improve diagnostic agreement and accuracy in assessing neoplasms with Spitzoid histologic features. Twenty expert pathologists were asked to review 70 consultation level cases with Spitzoid features, once with limited clinical information and again with additional genomic information. There was an improvement in overall agreement with additional genomic information. Most significantly, there was increase in agreement of the diagnosis of conventional melanoma from moderate (κ=0.470, SE=0.0105) to substantial (κ=0.645, SE=0.0143) as measured by an average Cohen κ. Clinical follow-up was available in all 70 cases which substantiated that the improved agreement was clinically significant. Among 3 patients with distant metastatic disease, there was a highly significant increase in diagnostic recognition of the cases as conventional melanoma with genomics (P<0.005). In one case, none of 20 pathologists recognized a tumor with BRAF and TERT promoter mutations associated with fatal outcome as a conventional melanoma when only limited clinical information was provided, whereas 60% of pathologists correctly diagnosed this case when genomic information was also available. There was also a significant improvement in agreement of which lesions should be classified in the Spitz category/WHO Pathway from an average Cohen κ of 0.360 (SE=0.00921) to 0.607 (SE=0.0232) with genomics.

Histological features and outcome of inverted type-A melanocytic nevi.

The presence of enlarged epithelioid/spindled nests located deep in the reticular dermis of a biphasic melanocytic neoplasm can mimic melanoma arising in a pre-existing nevus, causing over-interpretation of malignancy. We aimed to define the clinicopathologic significance of epithelioid/spindled nests in melanocytic nevi. Retrospectively using clinical and histologic information, we characterized 121 patients with a single lesion showing epithelioid/spindled melanocytes in the reticular dermis or subcutaneous fat, surrounded by melanophages, sometimes blending in with the adnexa. The majority of nevi occurred in women in the ages of 10 to 39 years, where the most frequent presentation was a changing mole. While 78% of the lesions displayed an anatomic (Clark's) level of IV-V, there was no ulceration, significant regression or inflammation. Up to 2 mitoses were found in only 12% of the cases, not correlating with the severity of cytological atypia. No recurrence or metastasis occurred during 45.5 months (mean) of clinical follow up in 26 patients. Notwithstanding the deep dermal extension, these findings suggest a benign histopathology and clinical outcome. Having compared the overlapping histopathology and clinical features between deep penetrating/clonal nevus and combined nevus, we posit that "inverted type-A nevus" might be considered a variant of the two.

Blue Nevi and Related Tumors.

The major entities related to blue nevus are common blue nevus, cellular blue nevus, atypical blue nevus, and malignant blue nevus. These lesions share presence of dermal pigmented dendritic melanocytes derived from embryonal precursors to melanocytes, Schwann cells, and glial cells migrating to the skin from the ventral neural crest. Genetically, blue nevi harbor mutations in G-protein-coupled receptor subunits GNAQ and GNA11. Progression to malignant blue nevus is associated with additional mutations and partial gains and losses of chromosomal material. This article discusses recent advances in pathology of blue nevi with emphasis on differential diagnosis and molecular pathology.

Genomic Analysis of Pigmented Epithelioid Melanocytomas Reveals Recurrent Alterations in PRKAR1A, and PRKCA Genes.

Pigmented epithelioid melanocytoma (PEM) is a rare cutaneous melanocytic tumor first described as epithelioid blue nevus in patients with the Carney Complex (CC). PEM was among the first established examples of an intermediate class of melanocytic tumors, including atypical Spitz tumors, with frequent metastasis to lymph nodes but only rare extranodal spread. Sporadic and CC-associated PEM are essentially histologically indistinguishable. A subset of PEM shows loss of cytoplasmic expression of the protein kinase A regulatory subunit alpha (PRKAR1A), a tumor suppressor gene mutated in 70% of families with CC. However, molecular studies of such tumors have been limited. Therefore, we used next-generation sequencing to assess 480 cancer-related genes and performed PrkaR1α immunohistochemistry on 13 cases morphologically consistent with PEM. Six cases demonstrated loss of PrkaR1α expression by immunohistochemistry. Three cases were "combined" PEM arising in association with a common nevus. These lesions harbored PRKAR1A genetic alterations in addition to BRAF mutations. Three "pure" PEM, not associated with a common nevus, showed no evidence of PRKAR1A genetic alterations despite loss of PrkaR1α expression. Two of these PEM demonstrated MAP2K1 in frame deletions. PrkaR1α protein expression was preserved in 7 cases. Two of these lesions revealed fusions of the gene encoding the protein kinase C alpha isoform (PRKCA) to 2 distinct partners (ATP2B4-PRKCA and RNF13-PRKCA). Two lesions may represent misdiagnosed "blue nevus with epithelioid features" as they demonstrated GNAQ hotspot mutations. A conceivable explanation, but one we do not favor is that rare PEM are caused by GNAQ mutations. No genetic aberrations were detected in 3 lesions. None of our 13 cases demonstrated TERT alterations or significant chromosomal copy number changes. These results further validate the concept of PEM as a distinctive intermediate/borderline melanocytic tumor, and also illustrate its molecular heterogeneity.

Metastatic atypical fibroxanthoma: a series of 11 cases including with minimal and no subcutaneous involvement.

Atypical fibroxanthoma (AFX) is a dermal mesenchymal neoplasm arising in sun-damaged skin, primarily of the head and neck region of older men. Conservative excision cures most. However, varying degrees of subcutaneous involvement can lead to a more aggressive course and rare metastases. Thus, AFX involving the subcutis are termed pleomorphic dermal sarcomas or other monikers by some to recognize the more threatening natural history. We reviewed cases of "metastatic AFX" from our institution and from the files of a consultative dermatopathology practice. Nine of 152 patients with AFX were identified at a single institution (2000-2011). Two additional patients were identified from the files of a consultative practice. Clinical, radiological, and pathological features were reviewed and cases with histologically verified metastases identified. Median age was 67 (range, 45-91) years, all male, and involving the head and neck region. Two cases had no documented involvement of the subcutis, and 2 cases had only superficial subcutis involvement. Median time to metastases was 13 (range, 8-49) months. Three patients developed solitary regional lymph node metastases while 8 had widespread metastases. Five patients developed local recurrence within 8 months, and all 5 developed widespread metastasis. With median follow-up of 26 (range, 10-145) months, 6 died of disease (median, 19 months; range, 10-35 months), 4 were alive and well, and 1 was alive with disease. AFX has very rare metastatic potential, even those without or with minimal subcutis involvement, and can lead to mortality. Most metastasis and local recurrence occurred within 1 year of presentation. Solitary regional metastases were associated with better outcomes than those with multiple distant metastases. Patients with repeated local recurrences portended more aggressive disease including development of distant metastases.

Soluble adenylyl cyclase antibody (R21) as a diagnostic adjunct in the evaluation of lentigo maligna margins during slow Mohs surgery.

Margin-controlled staged excision (slow Mohs) has emerged as a preferred method for the treatment of lentigo maligna (LM). The interpretation of margins for LM is one of the most challenging tasks faced by a dermatopathologist. R21 is a mouse monoclonal antibody against soluble adenylyl cyclase (sAC), overexpressed in the nuclei of LM but not in native melanocytes. The objective of this study was to validate the use of sAC immunohistochemistry in histological assessment of slow Mohs surgery margins for LM. Seventeen randomly selected cases of patients who underwent slow Mohs surgery for LM at Lahey Clinic, Burlington, MA, were studied. Ninety-nine margins were stained with R21 and microphthalmia transcription factor antibodies and reevaluated blindly by 2 observers. Sixteen of 17 lesions expressed sAC. In all cases, observers agreed on interpretation of R21 stains. In 85 (86%) margins, there was concordance between routine sections and R21 stains. In 14 margins (14%), the results were discrepant. In 2 margins, R21 identified foci of LM missed on routine sections. In 8 margins, atypical melanocytes, interpreted as positive in routine sections, were negative for R21 questioning the accuracy of the original interpretation. Microphthalmia transcription factor stained nuclei of melanocytes in all margins. We found significant correlation between assessment of margins by sAC immunohistochemistry and routine histology. Evaluation of sAC expression using R21 antibody is a useful diagnostic adjunct in the evaluation of margins of LM during slow Mohs surgery.

Expression of soluble adenylyl cyclase in lentigo maligna: use of immunohistochemistry with anti-soluble adenylyl cyclase antibody (R21) in diagnosis of lentigo maligna and assessment of margins.

CONTEXT: Soluble adenylyl cyclase (sAC) is an enzyme that generates cyclic adenosine monophosphate, a signaling molecule involved in regulating melanocyte functions. R21, a mouse monoclonal antibody against sAC, shows a striking pan-nuclear staining in lentigo maligna, indicating possible utility for diagnosis and margin assessment. OBJECTIVE: To evaluate R21 in the diagnosis and evaluation of margins in lentigo maligna. DESIGN: Thirty one re-excision specimens for lentigo maligna were evaluated for R21 expression using previously published protocol. In addition, 153 cases including 41 lentigo malignas, 30 non-lentigo maligna-type melanomas, 38 lentigos, and 44 nevi were evaluated using a modified stringent protocol to eliminate all nonmelanocyte staining. RESULTS: The sensitivity of nuclear staining with R21 in lentigo maligna was 87.8%. Nuclear expression of sAC was observed in 40% of other melanomas and 2.3% of benign nevi. R21 did not stain nuclei of resting melanocytes but was observed in 28.9% of melanocytic hyperplasias. These cases were easily distinguished from lentigo maligna in routine sections. R21 staining facilitated extent of the lesion in resection margins. In cases examined under the less stringent conditions, interpretation was facilitated by comparing R21 and Mart1/Melan A staining. Greater than 9 pan-nuclear staining melanocytes within one high-power field along with a pan-nuclear sAC/Melan A ratio greater than 0.5 was consistent with a positive margin whereas 5 or less pan-nuclear staining melanocytes along with a sAC/Melan A ratio of less than 0.3 constituted a negative margin. CONCLUSION: R21 is a useful diagnostic adjunct in the diagnosis and evaluation of margins in re-excision specimens in lentigo maligna.

Correlation between histologic assessment and fluorescence in situ hybridization using MelanoSITE in evaluation of histologically ambiguous melanocytic lesions.

CONTEXT: The 4-probe, multicolor, fluorescence in situ hybridization (FISH) panel targeting chromosomes 6 and 11 has shown promising sensitivity and specificity in distinguishing between benign nevi and malignant melanoma. Only a few studies have assessed the potential utility of FISH in classification of histologically ambiguous melanocytic lesions. In the United States, this assay is exclusively licensed to NeoGenomics Laboratories (Irvine, California), which provides the technical component and has developed an innovative service (MelanoSITE) allowing pathologists to interpret FISH results using a dedicated Web portal. Thus far, use of MelanoSITE as a diagnostic adjunct in the diagnosis of melanocytic lesions has not, to our knowledge, been reported in the literature. OBJECTIVE: To analyze 1.5 years of experience with the MelanoSITE melanoma FISH assay in the evaluation of histologically ambiguous lesions in the context of second opinion and routine dermatopathology practice. DESIGN: A prospective histologic/FISH correlation study of 140 cases. RESULTS: Twenty-seven percent of abnormal FISH results were false-positive results because of tetraploidy. After correcting for known false-positive results, all lesions considered atypical nevi showed normal FISH signals. Abnormal FISH signals were reported in 30% of lesions considered histologically borderline and in 48% of lesions in which a diagnosis of melanoma was favored. CONCLUSIONS: Four-probe, multicolor FISH results for melanoma correlate with the microscopic assessments of histologically ambiguous lesions. Pathologists using MelanoSITE must be aware of the high rate of false-positive results from tetraploidy.

Syringotropic melanoma: a variant of melanoma with prominent involvement of eccrine apparatus and risk of deep dermal invasion.

We report 7 cases which can be regarded as a syringotropic melanoma-a unique presentation of melanoma defined as melanoma spreading within the eccrine apparatus into the reticular dermis and/or subcutaneous tissue deeper than any (if present) associated invasive melanoma. Six patients were females, and 1 was a male. Their ages ranged from 32 to 85 years old (average 63). The lesions showed a wide site distribution, occurring on the extremities (4), trunk (2), and head and neck (1). Five melanomas were superficial spreading type; 1 was acral lentiginous type; 1 was unclassified. Four lesions (57%) invaded from within eccrine apparatus at a depth and anatomical level greater than that of an adjacent conventional invasive melanoma arising from the surface epidermis. In 1 lesion, which presented clinically as a pigmented macule, deep dermal syringocentric invasive tumor was the only site of invasion and tumorigenic growth. Thus, this variant of melanoma carries a significant risk of syringocentric deep dermal invasion, which may be unsuspected clinically but must be detected on histological examination to provide the most accurate prognosis and staging information.

Update on fluorescence in situ hybridization in melanoma: state of the art.

CONTEXT: Recent advances in understanding the molecular basis of melanoma have resulted in development of fluorescence in situ hybridization (FISH) protocols designed to detect genetic abnormalities discriminating melanoma from nevi. The most extensively studied is a 4-probe multicolor FISH probe panel targeting chromosomes 6 and 11. Validation studies showed promising sensitivity and specificity for distinguishing benign nevi and malignant melanoma by FISH. Recent studies show that a melanoma FISH assay has great potential for becoming an important diagnostic adjunct in classification of melanocytic lesions and in diagnosis of melanoma. OBJECTIVE: To present a comprehensive review of the science and practical aspects of FISH in melanoma for pathologists considering the use of melanoma FISH in their practice. DATA SOURCES: Review of the literature and personal experience of the authors. CONCLUSIONS: Judicious use of a 4-probe multicolor melanoma FISH procedure can enhance accuracy for diagnosis of melanoma and improve classification of melanocytic proliferations.

Subcutaneous thrombotic vasculopathy syndrome: an ominous condition reminiscent of calciphylaxis: calciphylaxis sine calcifications?

Ischemic skin necrosis can be a cause of severe morbidity and mortality. It can be due to a number of systemic conditions such as (1) thrombotic vasculopathy syndromes, (2) calciphylaxis, (3) septic or cholesterol emboli, and (4) cutaneous vasculitis. We present 3 patients with a clinicopathological syndrome consisting of ischemic skin necrosis associated with histological pattern of subcutaneous thrombotic vasculopathy-extensive microvascular thrombosis confined to small vessels and capillaries of the subcutaneous tissue. All 3 patients were obese and had severe pre-existing medical conditions. Skin biopsies showed intravascular thrombosis involving small arterioles and capillaries of the subcutaneous tissue. Distribution of vascular involvement by thrombotic process was similar to that observed in calciphylaxis, but calcifications were not observed. Two patients died within 3 months of diagnosis. One patient died 2 years after the presentation. Review of 15 biopsies of calciphylaxis revealed areas of subcutaneous thrombotic vasculopathy in 11 cases (73%). Our study shows that subcutaneous thrombotic vasculopathy syndrome is a potentially lethal condition showing overlapping features between thrombotic vasculopathy syndromes and calciphylaxis. Clinicopathological analysis suggests that it may be a rare variant of calciphylaxis sine calcifications or an early prodromal stage of calciphylaxis. This conclusion is in keeping with increasing appreciation of importance of thrombosis and vascular injury in calciphylaxis.