Enhanced Detection of Spitzoid Melanomas Using Fluorescence In Situ Hybridization With 9p21 as an Adjunctive Probe

Abstract

The use of molecular diagnostic methods such as fluorescence in situ hybridization (FISH) for challenging melanocytic neoplasms is becoming more widespread. In light of the diagnostic difficulty they pose, spitzoid melanocytic neoplasms constitute an area of greatest potential utilization. In this study we wished to evaluate the sensitivity of the currently used melanoma FISH probe assay in a group of unambiguous spitzoid melanomas. On the basis of comparative genomic hybridization data, copy number losses at chromosome 9 have long been recognized as a frequent event in melanoma. In this study we wished to evaluate the efficacy of 9p21 as a potential FISH target and then evaluate the added benefit of reflexing the standard melanoma FISH assay, with FISH targeting 9p21 and the centromere of chromosome 9 (Cep9). Cep9 was included as a control to exclude inadequate hybridization or truncation as a source of homozygous deletions. We first studied a training set of 85 melanomas and 58 nevi with FISH targeting 9p21 and Cep9. As per previous methodology, 30 cells were enumerated. In the training set, the nevi had a mean number of cells with homozygous 9p21 loss of 0.97, with a standard deviation of 1.26. The melanomas had a mean of 7.1 and a standard deviation of 6.76. This difference was significant (P=2×10(-12)). On the basis of the training set, we identified a cutoff of 10 homozygous deletions to distinguish between melanoma and nevi. In a subsequent validation set consisting of 76 melanomas and 88 nevi, we found this cutoff to have a sensitivity of 33% and a specificity of 100%. Finally, in our group of 43 unequivocal spitzoid melanomas, standard FISH against chromosomes 6 and 11 was 70% sensitive. Homozygous 9p21 loss was present in 11 of 27 (41%) cases tested. By combining the standard melanoma FISH assay with the 9p21 FISH assay, a combined sensitivity of 85% was found. Among these 27 cases tested with 9p21, there were 7 cases that were negative by the standard melanoma FISH assay but that were positive by 9p21, suggesting that the 9p21 assay may be highly complementary to the standard melanoma FISH assay. Hence, in this study, we validated the efficacy of 9p21/Cep9 as a diagnostic FISH assay in melanoma, and demonstrated its complementary effect to the standard FISH assay. 9p21 may be particularly helpful in lesions with spitzoid morphology.

An independent validation of a gene expression signature to differentiate malignant melanoma from benign melanocytic nevi

“…The sensitivity and specificity of the FISH method varies dramatically among existing reports with the lesion subtype, the probe set used, the number of observers, and the cutoff thresholds used. Various authors have reported a FISH sensitivity ranging from 43% to 94% and a specificity ranging from 60% to 98% 24, 25, 26, 27, 28. The original 4‐probe FISH assay targeting 6p25 (RREB1), 6q23 (MYB), Cep6 (centromere 6), and 11q13 (CCND1) was reported to discriminate between histologically unequivocal melanomas and benign nevi with a sensitivity of 86.7% and a specificity of 95.4% 24.…”

“…The original 4‐probe FISH assay targeting 6p25 (RREB1), 6q23 (MYB), Cep6 (centromere 6), and 11q13 (CCND1) was reported to discriminate between histologically unequivocal melanomas and benign nevi with a sensitivity of 86.7% and a specificity of 95.4% 24. However, the sensitivity was subsequently found to be only 70% for melanomas with a Spitzoid morphology 25, 26, 29, 30, 31, 32…”

A diagnostic algorithm for atypical spitzoid tumors: guidelines for immunohistochemical and molecular assessment

“…It turns out that heterozygous losses of 9p21 were enumerated in both melanomas and nevi in the early stage of probe selection data, and as a result, the 9p21 probe failed to demonstrate a statistically significant difference between melanoma and nevi. 34 Current 5-probe FISH assay for diagnosis of melanoma. In 2012, after testing three training sets using 322 tumors, including 152 melanomas and 170 nevi, the initial FISH panel has been redefined to the currently used 5-probe set of 6p25, 9p21, CEN9, and 11q13, and myelocytomatosis oncogene (MYC, 8q24) (Figure 4b).…”

“…In 2012, after testing three training sets using 322 tumors, including 152 melanomas and 170 nevi, the initial FISH panel has been redefined to the currently used 5-probe set of 6p25, 9p21, CEN9, and 11q13, and myelocytomatosis oncogene (MYC, 8q24) (Figure 4b). 34 This probe set was validated on 51 melanomas and 51 nevi, and displayed improved discriminatory power with a sensitivity of 94% and specificity of 98% and significantly improved sensitivity of spitzoid melanoma detection to 94% (Table 3A and Figure 4b). 32 Table 3A summarizes the chronological improvement in sensitivity and specificity of the 4-and 5-probe FISH assay for the diagnosis of melanomas and demonstrates relatively consistent results between American and European groups even when different cutoff criteria were applied.…”

“…32 Table 3A summarizes the chronological improvement in sensitivity and specificity of the 4-and 5-probe FISH assay for the diagnosis of melanomas and demonstrates relatively consistent results between American and European groups even when different cutoff criteria were applied. 31,32,[34][35][36][37][38] 39 The FISH-based risk assessment for atypical spitzoid tumors applied to this algorithm was derived from a retrospective data set of Gerami et al 40 showing a striking difference in clinical risk according to the subtype of the isolated chromosomal aberrations in atypical spitzoid tumors. However, this data set is very limited and additional studies with a larger number of cases and longerterm follow-up are warranted to further substantiate its significance.…”