Melanoma Arising in Association With Blue Nevus

A Clinical and Pathologic Study of 24 Cases and Comprehensive Review of the Literature

Sanam Loghavi; Jonathan L Curry; Carlos A Torres-Cabala; Doina Ivan; Keyur P Patel; Meenakshi Mehrotra; Roland Bassett; Victor G Prieto; Michael T Tetzlaff

Disclosures

Mod Pathol. 2014;27(11):1468-1478. 

In This Article

Materials and Methods

Patient Specimens

With approval from the Institutional review Board at the University of Texas, MD Anderson Cancer Center, the files of the Department of Pathology at University of Texas, MD Anderson Cancer Center were searched for cases of melanoma described as arising in association with a common or cellular blue nevus. Twenty-four cases of primary lesions in patients without prior history of invasive melanoma were identified from the past 9 years. In each case, either hematoxylin and eosin-stained slides or whole slide scanned images were reviewed to confirm the diagnosis and record the histopathologic features including tumor thickness, tumor cytology, number of mitotic figures, type of associated benign nevus, and the presence of an intraepidermal component, ulceration, regression, lymphovascular invasion, perineural invasion, satellitosis, and tumor necrosis. The criteria to distinguish between melanoma and an associated nevus component included distinctly biphenotypic foci with increased cellularity, prominent nuclear pleomorphism and nucleoli, increased mitotic figures, and the presence of coagulative tumor necrosis.[6] 'Tumor thickness' was defined as the standard Breslow thickness when the lesion abutted or came in close proximity to the epidermis or the largest tumor dimension available for a given lesion (when the lesion grew as a nodule at a location distant from the epidermis and/or there was no epidermis available for evaluation in the specimen). Only the malignant component was measured when assessing maximum tumor thickness. Using this definition, clinical attributes and follow-up information for each case were obtained from electronic medical records, when available.

Mutation Analysis

BRAF mutation analysis was performed using targeted next generation sequencing or pyrosequencing as previously described.[55,56] GNAQ mutation analysis was performed using DNA extracted from unstained formalin fixed, paraffin-embedded tissue sections. We tested for mutations in exon 5, codon 209 of the GNAQ gene using Sanger sequencing. Polymerase chain reaction amplification analyses of six samples were performed in duplicate. Polymerase chain reaction was performed in a 96-well plate with a 50 μl volume including 100 ng DNA, 10 μM of M13 tagged GNAQ Exon 5 forward and reverse primers, 25 μM MgCl2, 5 μl 10 × GoTaq buffer, 5 μl 10 mM dNTP mix, and 0.3 μl of 5 units/μl of GoTaq enzyme. The reaction mix was subjected to initial denaturation at 95°C for 10 minutes, followed by 40 cycles of amplification consisting of denaturation at 95 °C for 30 seconds, annealing at 55 °C for 30 seconds, and extension at 72 °C for 30 seconds followed by final extension at 72 °C for 7 min and hold at 4 °C. To facilitate sequencing of amplicons, the forward and reverse primers were tagged with M13 universal sequences: M13 forward, 5′-TGTAAAACGACGGCCAGT-3′ or M13 reverse, 5′-CAGGAAACAGCTATGACC-3′. PCR products were purified using AMPure magnetic beads (Agentcourt, Danvers, MA) according to the manufacturer's protocol. A quantity of 4 μl of purified amplicons was used for Sanger sequencing using 3130 DNA Analyzer (Applied Biosystems, Carlsbad, CA) for detection of mutation in codon 209 of GNAQ Exon 5. The resulting data were analyzed by SeqScape software versions 2.5 and/or 2.7 (Applied Biosystems).

Statistical Analysis

Four outcomes were interrogated: overall survival, recurrence-free survival, time to local recurrence, and time to distant recurrence. Cox proportional hazards regression models were fit to model the association between each survival parameter and clinical and tumor covariates of interest. No adjustment was made for the multiplicity of testing.

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