Abstract and Introduction
Some melanocytic tumors can be histologically ambiguous causing diagnostic difficulty, which could lead to overtreatment of benign lesions with an unwarranted psychological distress or undertreatment of malignant cancers. Previously, we demonstrated that significantly decreased miR-211 expression in melanomas compared with melanocytic nevi could accurately discriminate malignant from benign tumors. Herein we show microRNA in situ hybridization for fluorescent detection of miR-211, suitable for paraffin-embedded tissues in 109 primary melanocytic tumors. miR-211 expression was significantly lower in melanomas vs nevi (P<0.0001), and receiver operating characteristic curve (area under the curve=0.862) accurately discriminated melanomas from nevi with 90% sensitivity and 86.2% specificity. Qualitatively, all dysplastic nevi expressed miR-211 at high (86%) and low (14%) levels, independent of the degree of nuclear atypia. All 35 melanocytic tumors with Spitz morphology expressed miR-211 independent of morphological classification, where clinical follow-up of these patients showed no recurrence at the site or metastasis in mean and median of 3 (ranging 2–5) years. Moreover, a decision tree learning analysis selected age and miR-211 miRNA in situ hybridization as the predictive variables for benign or malignant outcome in 88 patients correctly classified 92% (81 out of 88) of cases as proven by receiver operating characteristic curve (area under the curve=0.9029). These results support miR-211 as a leading miRNA candidate for melanoma diagnosis and miRNA in situ hybridization as a uniquely uncomplicated ancillary test.
The incidence and mortality of melanoma have continually increased over the past decades in the United States. It is estimated that 76 250 individuals (44 250 men and 32 000 women) will be diagnosed with and 9180 men and women will die of melanoma of the skin in 2012. Increasing incidence is coupled by diagnostic discrepancies whereby a small but significant number of clinically suspicious pigmented lesions may show ambiguous histopathology making the classification between benign (common nevus) and malignant (melanoma) melanocytic tumors difficult and the clinical behavior unpredictable. Notwithstanding the distinct sets of genetic alterations demonstrated in melanoma by array-based comparative genomic hybridization and by DNA-based fluorescence in situ hybridization, the changes in non-coding RNA (part of the genome that does not encode genes) during melanoma progression and how they could be exploited in diagnostic pathology remain uncharacterized.
microRNAs (miRNAs) are endogenous ~22 nucleotide non-coding small RNAs, negative regulators of gene expression, whose levels are significantly changed in a growing number cancers, eg, breast (ductal carcinoma in situ and invasive), colorectal carcinoma, malignant melanoma and papillary thyroid carcinoma. The changes in specific miRNAs have been shown to be associated with cancer diagnosis or outcome, which can be readily detected by quantitative real-time reverse transcriptase PCR (qRT-PCR) in formalin-fixed paraffin-embedded specimens. In fact, it has been shown that specific miRNAs function as tumor suppressors and oncogenes in human malignancies;[6–8] however, their tissue-specific expression patterns remain understudied by miRNA in situ hybridization in diagnostic material.
Measuring the levels of specific miRNAs by array-based profiling strategies can classify human cancers. Using next-generation sequencing of miRNA transcriptome and validated by qRT-PCR in biopsies of common nevi, thick primary (>4.0 mm) and metastatic melanomas, we defined a set of top 40 miRNAs, which correctly classified melanomas from nevi in archived specimens. Among the top miRNAs, miR-211 expression was significantly decreased in melanomas compared with melanocytic nevi, which was confirmed in an independent validation cohort of 101 specimens by qRT-PCR. Moreover, the receiver operating characteristic curves for miR-211 expression accurately discriminated invasive melanoma (area under the receiver operating characteristic curve (AUC)=0.933), melanoma in situ (AUC=0.933) and dysplastic (atypical) nevi (AUC=0.951) from common nevi. Corroborating our findings in clinical specimens, others and we have shown that miR-211 functions as a potent tumor suppressor in vitro influencing gene pathways involved in cell invasion.[11–13] Notably, miR-211 is located within the non-coding sequence (intron 6) of Melastatin-1 (MLSN-1) gene, which is expressed at high levels in melanocytic nevi but significantly reduced in metastatic human melanomas with variable expression in primary lesions. Loss of MLSN-1 mRNA is predictive of significantly reduced disease-free survival in a study of 150 melanoma patients. These results strongly suggest that miR-211 might be associated with a malignant behavior in melanoma.
As we had previously demonstrated that significantly decreased miR-211 expression could discriminate malignant from benign in 129 histologically unambiguous melanocytic tumors, we investigated whether miRNA in situ hybridization could be applied to aid in the diagnosis of ambiguous lesions. We confirmed that miR-211 expression was decreased in melanomas compared with nevi by fluorescent and chromogenic miRNA in situ hybridization. Moreover, our present data support the diagnostic utility of miR-211 in dysplastic nevi and melanocytic tumors with Spitz morphology.
Mod Pathol. 2016;29(5):461-475. © 2016 Nature Publishing Group