microRNA in situ Hybridization for miR-211 Detection as an Ancillary Test in Melanoma Diagnosis

Sankhiros Babapoor; Michael Horwich; Rong Wu; Shauna Levinson; Manoj Gandhi; Hanspaul Makkar; Arni Kristjansson; Mary Chang; Soheil S Dadras


Mod Pathol. 2016;29(5):461-475. 

In This Article


miRNAs are highly stable in paraffin-embedded tissues up to 10 years,[18] and their expression levels can be reliably measured by qRT-PCR or sequencing. Therefore, the capability to qualitatively detect miRNA expression by light microscopy could have a significant impact in diagnostic pathology where specific probes can be used to distinguish true cancer cells from normal counter parts with atypia or resolve a differential diagnosis of a tumor from its histological mimic. For example, using multi-color miRNA in situ hybridization, higher expression of miR-205 could distinguish basal cell carcinoma from its histological mimic, Merkel cell carcinoma, and higher expression of miR-375 distinguished Merkel cell from basal cell carcinoma.[19] Moreover, miRNA in situ hybridization can provide prognostic information using a single unstained section, eg, low levels of miR-205 expression showed a significantly shorter melanoma-specific survival.[16]

Our results showed that miR-211 miRNA in situ hybridization, independent of histology, could reliably classify melanoma from nevus groups in 90% (19 of 21) of lesions with the exception of two cases, where modest levels of miR-211 was detected. In addition, applying classification and regression tree analysis with patient's age at diagnosis and miRNA in situ hybridization intensity as parameters 97% of cases were correctly classified. Although these two cases would have been underdiagnosed, the histopathology was characteristic of melanoma. There is clearly a range of measured miR-211 expression, and some overlap between the lower end of nevus and higher end of melanoma groups. In such cases, we noted that measured fluorescence was a combination of partial, residual expression of miR-211 by melanoma cells and high expression by the adjacent keratinocytes, likely giving a falsely high readout. This situation will be avoided by using chromogenic miRNA in situ hybridization where the expression of miR-211is nevus specific, ie, not detected in the epidermal or follicular keratinocytes. Finally, we do not recommend the use of miR-211 miRNA in situ hybridization alone without a complete, thorough histological examination because miRNA in situ hybridization is an ancillary test. If the histopathology is dysplastic, spitzoid or ambiguous, then a miR-211 chromogenic miRNA in situ hybridization could be helpful in devising a more personalized treatment and clinical follow-up strategy for the patient.

miRNA in situ hybridization intensity in lesions with Spitz morphology retained miR-211 expression independent of morphological classification even in spitzoid melanomas, where clinical follow-up of all 35 patients showed no recurrence at the site or metastasis in mean and median of 3 (ranging 2–5) years. There are two possible explanations for retained miR-211 expression in such cases: (1) lesions with Spitz morphology were overdiagnosed because of cytological atypia and/or (2) the follow-up of the spitzoid melanomas was not long enough. The current, published literature supports the fact that many patients with spitzoid melanocytic tumor have a favorable clinical outcome. Based on this, miRNA in situ hybridization could significantly relieve the stress of unambiguity of atypical Spitz tumors, where a simple excision with 5-mm clear peripheral margins would be curative. It is possible that our follow-up of 3 years (ranging from 2 to 4 years) may not be long enough in a 2- or a 10-year-old child (cases SM29 and SM30); however, a 3-year follow-up is more than adequate for older individuals ranging from 23 to 72 (cases SM 31–35). Interestingly, miR-211 expression did not correlate with any of the American Joint Committee on Cancer prognostic parameters (especially tumor thickness, ulceration or mitotic index) or tumoral melanosis, suggesting that miR-211 miRNA in situ hybridization could have a broader diagnostic utility in melanocytic pathology regardless of the tumor stage or melanin content.

Using a chromogenic probe with branched DNA can significantly improve specificity because of the proven, powerful design of the nucleic acid chemistry.[20] Chromogenic miRNA in situ hybridization provides additional advantages: (1) melanoma can be distinguished from nevus cells on the same tumor section; (2) miR-211 expression was specific to the nevus cells; (3) the results are readily interpreted by the pathologist using light microscopy; and (4) miRNA in situ hybridization is less expensive than DNA-based fluorescent in situ hybridization or comparative genomic hybridization and widely available to pathology laboratories. Overall, the current results combined with sequencing and qRT-PCR support miR-211 as a leading miRNA candidate for melanoma diagnosis and miRNA in situ hybridization as a uniquely uncomplicated ancillary test.