Molecular classification of melanomas and nevi using gene expression microarray signatures and formalin-fixed and paraffin-embedded tissue

Abstract

Melanoma may be difficult to identify histologically and relatively high rates of misdiagnosis leads to many malpractice claims. Currently separation of melanomas from nevi is based primarily on light microscopic interpretation of hematoxylin and eosin stained sections with limited assistance from immunohistology. To increase the accuracy of discrimination of benign and malignant melanocytic lesions we identified DNA microarray-derived gene expression profiles of different melanocytic lesions and evaluated the performance of these gene signatures as molecular diagnostic tools in the molecular classification and separation of melanomas and nevi. Melanocyte-derived cells were isolated by laser capture microdissection from 165 formalin-fixed and paraffin-embedded melanocytic nevi and melanoma tissue sections. RNA was isolated, amplified, labeled, and hybridized to a custom DNA microarray. In all 120 samples were used to identify differentially expressed genes and generate a gene expression classifier capable of distinguishing between melanomas and nevi. These classifiers were tested by the leave-one-out method and in a blinded study. RT-PCR verified the results. Unsupervised hierarchical clustering identified two distinct lesional groups that closely correlated with the histopathologically identified melanomas and nevi. Analysis of gene expression levels identified 36 significant differentially expressed genes. In comparison with nevi, melanomas expressed higher levels of genes promoting signal transduction, transcription, and cell growth. In contrast, expression of L1CAM (homolog) was reduced in melanomas relative to nevi. Genes differentially expressed in melanomas and nevi, on the basis of molecular signal, sub classified a group of unknown melanocytic lesions as melanomas or nevi and had high concordance rates with histopathology. Gene signatures established using DNA microarray gene expression profiling can distinguish melanomas from nevi, indicating the feasibility of using molecular classification as a supplement to standard histology. Our successful use of a standard formalin-fixed and paraffin-embedded tissue further supports the practicability of combining molecular diagnostic testing with histopathology in evaluation of difficult melanocytic lesions.

Differential gene expression profiles of neurothekeomas and nerve sheath myxomas by microarray analysis

“…These advances include newer formalin-fixed paraffin-embedded tissue extraction protocols, cDNA-mediated annealing, selection, extension and ligation, the addition of random hexamer priming to oligo(dT) priming and newly developed array platforms with redesigned probes. [30][31][32][33][34]57,[67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][82] Although sensitivity is low, high specificity and positive predictive value suggest that transcript detection is reliable from formalin-fixed paraffin-embedded tissue. 33 Although RNA isolation techniques from formalin-fixed paraffin-embedded tissues have improved, problems remain, largely because of the greater fragmented nature of RNA in formalin-fixed paraffin-embedded tissue.…”

“…Recent biotechnological advances have optimized conditions to improve the quality and quantity of RNA extracted from formalin-fixed paraffin-embedded tissues. [30][31][32] These now permit molecular analysis, including microarray-based whole-genome-wide testing, of clinically small lesions from archival tissues.…”

Streamlining Molecular Pathobiology of Malignant Melanoma

“…They also scrutinize DNA mutations and polymorphism [56,57]. Indeed, mutations altering proteins as a result of amino acid substitution differ from silent mutations (polymorphism) affecting a given amino acid.…”

The role of osteopontin expression in melanoma progression

“…A total of 93 FFPE melanomas were included in the tissue microarray analysis (TMA) [37]. The clinicopathological data of the primary melanomas are summarised in Table 1.…”