Materials and Methods
Study Population and Clinical Samples
Formalin-fixed paraffin-embedded sections from common melanocytic nevi, dysplastic nevi, primary cutaneous melanomas, Spitz nevi, atypical Spitz tumors and spitzoid melanomas were obtained from the archives of Department of Dermatology at University of Connecticut Health Center between 2009 and 2013 (Table 1). In addition, we purchased melanoma tissue microarrays, T385a and T388 from US Biomax (Rockville, MD, USA) consisting of 24 tissue cores in duplicates or triplicates. For the melanomas, 11 cases were full sections and 10 were from tissue microarrays. For each specimen, a hematoxylin–eosin-stained section was used for histopathological evaluation, diagnostic confirmation, pathological staging and imaging. In total, we examined 109 individual lesions for the study (Table 1).
The inclusion criteria for diagnostic groups were as follows. The common melanocytic nevi were either junctional or combined with proper maturation of melanocytes (cells become smaller and more spindled) with increasing dermal depth. The primary cutaneous melanomas were predominately superficial spreading or nodular subtypes. The Spitz nevi were characterized by well-defined, symmetric silhouette, melanocyte maturation by increasing dermal depth, epithelioid and spindled cells and lack of any dermal mitoses as determined by several additional deeper sections. Atypical Spitz tumor group was characterized by asymmetric silhouette, focal pagetoid scatter, focal lack of proper maturation or a single dermal mitotic figure. In addition, the cytology showed epithelioid and spindled cells with slight atypia and conspicuous nucleoli. The spitzoid melanoma were predominately invasive (two cases were in situ) exhibiting asymmetric silhouette, pagetoid scatter, lack of proper maturation, epithelioid and spindled cells and/or dermal mitotic figure(s). The cytopathology showed significant atypia with vesicular chromatin and macronucleoli. Lesions with Spitz morphology were additionally reviewed in a consensus conference seen at least by three pathologists and sent to an expert dermatopathologist for a second opinion. For the study, at least two board-certified pathologists/deramtopathologists confirmed all rendered diagnoses. The institutional review board of the University of Connecticut Health Center approved this protocol.
Fluorescent miRNA in Situ Hybridization
Formalin-fixed paraffin-embedded sections were cut at 5-μm thick, mounted on positively charged slides (Fisher Scientific, Pittsburgh, PA, USA). miRNA in situ hybridization was performed as described with slight modifications. Briefly, tissue sections were baked at 60 °C for 20 min and deparaffinized in three exchanges of xylene and rehydrated in an ethanol gradient. Then the slides were treated with 20 μg/ml proteinase K (Ambion, USA) for 10 min at 37 °C and fixed in 4% paraformaldehyde (Thermo Scientific, Rockford, IL, USA). Then, slides were rinsed in 0.13 M 1-methylimidazole (Sigma-Aldrich, St Louis, MO, USA) followed by an additional fixation with 0.16 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Sigma-Aldrich). Endogenous peroxidases were blocked in 1% hydrogen peroxide (Sigma-Aldrich). Slides were prehybridized at 50 °C in hybridization buffer followed by hybridization in 80 nM double Digoxigenin-labeled LNA-modified probe corresponding to mature miR-211 (sequence: 5′-AGGCGAAGGATGACAAAGGGAA-3′), a scrambled probe (sequence: 5′-GTGTAACACGTCTATACGCCCA-3′) and 5′ Digoxigenin-labeled U6 probe (sequence: 5′-CACGAATTTGCGTGTCATCCTT-3′) (Exiqon, Copenhagen, Denmark) for 1 h at 50 °C. Slides were washed thrice in 2 × SSC, with the first wash at hybridization temperature and subsequent washes at room temperature, blocked in 0.5% Roche blocking reagent (Roche Diagnostics, Indianapolis, IN, USA) and then incubated in 1:100 Anti- Digoxigenin-HRP antibody (Novus Biologicals, CO, USA) for 1 h at room temperature. Slides were then washed twice in 0.1% Tween-20 PBS (PBS-T) and once in PBS, then incubated in 1:50 TSA Cyanine 3 reagent (Perkin Elmer, Norwalk, CT, USA) for 30 min and washed again as above. Nuclei were counterstained with DRAQ5 (Cell Signaling Technology, MA, USA) and then the slides were mounted using Prolong Gold (Invitrogen, CA, USA) mounting medium.
Fluorescent Microscopy and Measurement of Fluorescent Intensity
Images were acquired with a Zeiss LSM ConfoCor3 confocal microscope (Carl Zeiss, Germany) and EVOS FL Auto Cell Imaging System (Advanced Microscopy Group, Life Technologies, NY, USA), under constant exposure time and gain for all specimens, including positive (U6) and negative (scramble miRNA) controls. We captured × 20 images in multiple regions, consisting of 3–5 fields per lesion. Then using multi-position imaging and MetaMorph microscopy automation and image analysis software (Sunnyvale, CA, USA), we measured the signal intensity per × 20 image fields. The results were averaged and reported as miRNA in situ hybridization intensity for each case. The mean, median, minimum, maximum and interquartile range were calculated (Table 2 and Table 4). The fluorescent expression of miR-211 was examined in the normal skin, which was expressed in the nuclei and cytoplasm of most epidermal keratinocytes. A semiquantitative, four-point ordinal reactivity score was established for dysplastic nevi: '0' reflected the lack of miR-211 and was the most common pattern in invasive and in situ melanomas in <10% of tumor cells. Weakly positive expression was designated as '1' if expression was detected in 10–30%; moderately positive '2' in 31–60% and strongly positive '3' in >61% of tumor cells. Only staining of tumor cells was scored in comparison to adjacent keratinocytes (internal positive control). Two investigators scored the slides independently. Scores for multiple cores from one case were averaged, and final miR-211 scores were categorized into a three-level grouping of Negative, Low (>0 and ≤1.4) or High (>1.5).
Chromogenic miRNA In Situ Hybridization
Two melanocytic nevi and two primary cutaneous melanomas with unambiguous histopathology were selected. Bacillus subtilis dihdrodipicolinate reductase (DapB) gene and let-7a, a highly abundant human miRNA, were used as negative and positive controls, respectively. Custom designed probes for miR-211, DapB and let7a were synthesized. Hybridization was performed using a modified protocol of the ViewRNA in situ hybridization tissue protocol (Affymetrix, Santa Clara, CA, USA). Thirty formalin-fixed paraffin-embedded sections were cut at 5-μm thickness for each case (two additional nevi and melanomas) and mounted on positively charged SurgiPath X-tra slides (Leica Biosystems, Buffalo Grove, IL, USA). Tissue sections were baked at 60 °C for 60 min, deparaffinized using xylene followed by ethanol dehydration and air-dried. Slides were rehydrated in 1 × PBS followed by protease digestion (1:150 dilution, Affymetrix) for 30 min at 37 °C and fixed in 4% formaldehyde for 5 min at RT. Slides were rinsed in 1 × PBS followed by hybridization with 12.5 nm final concentration of miRNA in situ hybridization probe for 2 h at 40 °C. Signal amplification was achieved using the branched DNA technology with a series of sequential hybridization and washes. Briefly, posthybridized slides were hybridized with the preamplifier mix for 25 min at 40 °C, followed by amplifier mix for 15 min at 40 °C, 1:1000 dilution of Alkaline-Phosphatase (AP)-labeled probe for 15 min at 40 °C and AP enhancer for 5 min at RT. The chromogenic reaction was carried out incubating Fast Red Substrate for 30 min at 40 °C. Slides were postfixed with 4% formaldehyde for 5 min at RT, counterstained with Gill's Hematoxylin and mounted using Advantage (Innovex Biosciences, Richmond, CA, USA) mounting media. Overall, the measured red dots (miRNA signal) for DapB was hardly detected (1 dot/10 cells) and for let-7a abundantly present (>10 dots/cell) in the cases tested.
The statistical analyses were performed using SAS version 9.3. Difference between two diagnostic groups was assessed using two independent sample t-test or non-parametric alternative (Mann–Whitney test). Difference between more than two diagnostic groups was assessed using Kruskal–Wallis Test. A P-value <0.05 was considered statistically significant in analysis. To evaluate how miR-211 expression distinguishes melanoma from nevus, we used different cutoff values of miRNA in situ hybridization intensity to calculate sensitivity and specificity and then generated the receiver operator characteristic curve by plotting sensitivity vs (1−specificity). From the receiver operator characteristic curve, AUC and the distance from ideal were calculated using previously described. The decision tree analysis to discriminate benign vs malignant outcome employed Classification and Regression Tree software (Salford Systems, San Diego, CA, USA). Using 10-fold cross-validation, the predictors included miRNA in situ hybridization intensity, sex, age and location.
Mod Pathol. 2016;29(5):461-475. © 2016 Nature Publishing Group