Significant Reduction of miR-211 Expression in Melanomas Compared With Common and Dysplastic Nevi
The study encompassed melanocytic tumors with a wide histopathological range from unequivocal common (n=32) and dysplastic (n=21) melanocytic nevi and primary cutaneous melanoma (n=21) to more challenging lesions with Spitz morphology (Table 1). These cases included Spitz nevi (n=11), atypical Spitz tumors (n=17) and spitzoid melanomas (n=7). To examine the diagnostic utility of miR-211 in a total of 109 cases, we optimized an established methodology for miRNA in situ hybridization in paraffin-embedded specimen. The fluorescent intensity of miR-211 expression was measured for all lesions (Table 2), except for dysplastic nevi (Table 3). Given the small size of dysplastic nevi, mostly consisting of small, shave biopsies, miR-211 expression was scored in a semiquantitative manner, as fluorescent intensity could not be reproducibly measured with confidence using 3–5 overlapping fields.
The expression of miR-211 was consistently high and readily detected by fluorescent microscopy in all common and dysplastic nevi (Table 1 and Table 2). miR-211 was diffusely and strongly expressed in the dermal nevus nests and the overlying epidermal keratinocytes; the signal was detected in the nuclei and cytoplasm (Figure 1a). In contrast, miR-211 was absent to dramatically decreased in 19 of the 21 melanomas (90%) except for 2 cases that showed focal but modest expression of miR-211. miR-211 was consistently reduced to absent from the epidermal melanoma cells compared with the neighboring keratinocytes (Figure 1b). Combined immunofluorescent for MART-1 and miRNA in situ hybridization for miR-211 co-localized expression to intradermal nevus cells (results not shown). Although there was a range for miRNA in situ hybridization intensity, the mean and median values were significantly lower for melanomas compared with nevi, using two different statistical methods, showing consistent results (P<0.0001, Figure 1c). Receiver operating characteristic curve for miR-211 based on miRNA in situ hybridization intensity accurately discriminated melanomas from nevi (AUC=0.853, Figure 1d). Based on the receiver operating characteristic curve, the minimum distance from ideal of 0.17 was calculated, with sensitivity of 90%, specificity of 86.2% and a false-positive rate of 0.138.
Intradermal nevus nests (arrows) and overlying epidermal keratinocytes diffusely and strongly express miR-211 (cytoplasm, red; and nuclei, blue) detected by fluorescent miRNA in situ hybridization (a). In contrast, the pagetoid, epidermal melanoma cells (circles and arrows) are negative for miR-211 (cytoplasm, black; and nuclei, blue) in a melanoma in situ (b). Measured fluorescent intensity showed a significant decrease in miR-211 expression in primary cutaneous melanomas compared with common nevi (P-value<0.0001) (c). The fluorescent intensity was expressed in boxplots: box starts from first quartile and ends at third quartile; cross represents mean; line represents median; the ends of whiskers represent the minimum and maximum of the data that are not outliers; and black dots are outliers. Receiver operating characteristic curve showed an accurate discrimination between diagnostic primary cutaneous melanoma and common nevi groups (area under the curve=0.853) (d). Original magnifications: (a–b), × 200.
To ensure the specificity of miR-211 signal, we used a scrambled miRNA (miRNA with non-specific sequence) as negative control, which showed no background or non-specific hybridization (Figures 2a and b, insets). In contrast, diffusely strong and specific signal for miR-211 was detected in the nuclei and cytoplasm of intradermal nevus cells and overlying keratinocytes (Figure 2a). This signal was lost in the intraepidermal pagetoid melanoma cells, compared with the adjacent keratinocytes, and in the dermal melanoma cells compared with the adjacent intradermal nevus cells (Figure 2b). To ensure the integrity of small RNAs (including miRNAs) in archived paraffin material, we used U6 (a non-coding small nuclear RNA) as positive control (results not shown), virtually all the nuclei of epidermal keratinocytes and nevus cells highly expressed U6, indicating that small RNAs are intact in archived specimen. We observed a similar pattern of U6 expression in melanoma specimens (results not shown).
Coalescing intradermal nevus nests diffusely expressed miR-211 (cytoplasm, red; and nuclei, blue) (a) compared with melanoma in situ (arrowheads) and invasive dermal (circles) tumor cells are negative for miR-211 (cytoplasm, black; and nuclei, blue) (b). The insets show negative control (miRNA scramble), devoid of any background or non-specific signal (cytoplasm, black; and nuclei, blue). Serial sections (hematoxylin and eosin) are shown for the two lesions in panels (a) and (b), respectively (c and d). Melanin did not interfere with the miR-211 hybridization or signal detection (d). Dysplastic nevi diffusely expressed miR-211 both in the junctional and dermal components (e). Serial section is shown (case no. 20) from the scalp of a 26-year-old woman (f); the inset shows high magnification of the junctional melanocytic atypia. Original magnifications: (a–d), × 200; and (e) and (f), × 100.
Similar to common nevi, the great majority of dysplastic lesions (18 of 21, 86%) expressed miR-211 at high levels; the remainder (3 of 21, 14%) expressed it at lower but detectable levels (Table 3). miR-211 expression was robust and diffuse in both the epidermal and dermal components of dysplastic nevi regardless of the degree of cytological atypia (Figures 2e and f). To demonstrate the reproducibility of miR-211 miRNA in situ hybridization, additional nevi were qualitatively compared with melanomas (Figure 3). Low-power examination showed a diffusely strong expression of miR-211 in the nuclei and cytoplasm of nevus cells (Figure 3a), absent from blood vessels (results not shown). A variable but detectable expression of miR-211 was demonstrated in the keratinocytes of epidermis and follicular epithelium (results not shown). In contrast, miR-211 expression was markedly reduced to absent in melanoma cells. In fact, the intraepidermal cells typically exhibited complete loss of miR-211 expression and left a negative imprint compared with the adjacent keratinocytes. However, in some cases the intradermal melanoma cells still expressed miR-211 (Figure 3b). Examining the relationship between miRNA in situ hybridization intensity and melanoma clinicopathological and American Joint Committee on Cancer parameters, such as Breslow's thickness, ulceration or mitotic index, revealed no significant association (Table 4). In addition, miRNA in situ hybridization intensity did not correlate with gender, age or solar elastosis, indicating that miRNA in situ hybridization could be used in a wide array of melanocytic tumors.
A reproducible, evenly diffuse expression of miR-211 (red) is detected in intradermal nevus nests (a) compared with a striking partial loss of miR-211 (cytoplasm, black; and nuclei, blue) in the situ (arrowhead) and invasive melanoma cells (arrow) (b). Original magnifications: (a–b), × 100.
Chromogenic miRNA In Situ Hybridization Distinguished Invasive Melanoma From Intradermal Nevus Cells in the Same Section
We used fluorescent miR-211 miRNA in situ hybridization for its sensitivity in detection and quantification for all 109 tumors. After demonstrating the feasibility of miR-211 detection by fluorescent miRNA in situ hybridization, we sought to establish a chromogenic probe so that it could be readily and conveniently detected by light microscopy. Using the well-established branched DNA probe chemistry, we detected diffuse expression of miR-211 in intradermal nevus nests (Figures 4a and b). In a case of melanoma associated with common intradermal nevus, miR-211 was robustly expressed in the banal-appearing nevus nests (Figures 4c and d). However, a separate section on the same slide showed histologically unambiguous melanoma where miR-211 signal is nearly completely lost (Figures 4e and f). For positive control, we used let-7a, a most abundantly expressed miRNA, which was diffusely expressed in the nevus cells, epidermal and follicular keratinocytes (results not shown). In contrast, in melanoma cells the expression of let-7a albeit being decreased, it was still detected by light microscopy. For negative control, we used a probe sequence to DapB gene, which was nearly absent from all the cases tested (results not shown).
Chromogenic miRNA in situ hybridization using branched DNA probe also confirmed diffuse miR-211 expression in the nevus nests (red cytoplasmic grains) (a and b). Similarly, the nevic component (c and d) in an invasive melanoma strongly expressed miR-211. In contrast, miR-211 expression was entirely lost in the melanoma area (e and f) of the same lesion. Original magnifications: (a, c) and (e), × 100; and (b), (d) and (f), × 600.
Melanocytic Tumors With Spitz Morphology Expressed miR-211
Clinically, the Spitz nevus group was characterized by young age (mean age of 5.7 years) well defined and orange-to-pink papule or macule. The mean age of the patients diagnosed with atypical Spitz tumor and spitzoid melanoma was significantly higher than that of Spitz nevus (22.6 and 32.9 vs 5.7 years, respectively; Table 1). The median for miR-211 miRNA in situ hybridization intensity was near or above the melanoma threshold (47.5) in all 35 tumors with Spitz morphology (Table 2). Representative sections of Spitz nevus, atypical Spitz tumor and spitzoid melanoma showed diffuse expression of miR-211 (Figure 5). For example, a 2.5-mm melanoma (T3a) diagnosed on the right calf of a 2-year-old girl (case no. 30) diffusely expressed miR-211 (Figures 5e and f). She has had no recurrence or metastasis as determined by a negative PET scan after 4 years. Moreover, a 37-year-old man (case no. 32) diagnosed with spitzoid melanoma expressing miR-211 in the melanoma situ (Tis) with dermal nevus on the right ear has had no recurrence or metastasis after 2 years. A Kruskal–Wallis test for miR-211 expression confirmed no statistical difference between any of the diagnostic groups (n=35, Figure 5g). All atypical Spitz and spitzoid melanoma lesions were completely excised, and patients were followed for local recurrence by repigmentation in or near the scar, lymphadenopathy by palpation and PET scan as clinically indicated. The miR-211 expression results and follow-up results for each individual case are reported (Table 5). All 35 patients were followed up from 2 to 5 years with mean of 2.5, 3.4 and 3.0 years for Spitz nevus, atypical Spitz and SM lesions, respectively (Table 6); none of the lesions has recurred or metastasized to date. Moreover, miRNA in situ hybridization intensity did not correlate with gender, age, solar elastosis, Breslow's thickness, ulceration or mitotic index, regression or tumor-infiltrating lymphocytes response (results not shown).
All melanocytic tumors with Spitz morphology expressed miR-211. Representative serial sections (hematoxylin and eosin) followed by fluorescent miRNA in situ hybridization are shown for Spitz nevus (a and b), atypical Spitz tumor (c and d) and spitzoid melanoma (e and f). The SM shown in panel (f) (case no. 30) expressing miR-211 was diagnosed as 2.5 mm melanoma (T3a) on the right calf of a 2-year-old girl; she has had no recurrence or metastasis after 4 years. Kruskal–Wallis test confirmed no statistical difference in miR-211 expression between the diagnostic groups for all lesions tested (g). Original magnification: (a–f), × 100.
miR-211 as Potential Marker of Clinical Behavior in Melanocytic Tumor
We applied classification and regression tree analysis as a predictive model to investigate which predictors could best classify the melanocytic tumors in our study. We used all lesions with available outcome results (n=88); dysplastic nevi (n=21) were excluded because of their semiquantitative miR-211 expression results. To obtain the best predictive model, a number of predictors such as sex, age, anatomic location, American Joint Committee on Cancer stage and miR-211 miRNA in situ hybridization intensity were explored. The classification and regression tree software selected age (younger vs >48.5 years) and miRNA in situ hybridization (≤48.8, malignant; and >48.8, benign) intensity as the predictor variables for malignancy (Figure 6). The threshold for miRNA in situ hybridization intensity (≤48.8, malignant; and >48.8, benign) is determined from classification and regression tree modeling analysis. This analysis accurately classified the great majority (96.5%) of the patients aged <48.5 years as benign while 3.5% as malignant in node 1. The remainder of 31 patients aged >48.5 years could be accurately classified by miRNA in situ hybridization intensity threshold of 48.8 as benign or malignant in node 2. In node 3, only two lesions with high miRNA in situ hybridization intensity of 74.9 were misclassified as malignant. Overall, the receiver operating characteristic curve accurately discriminated between benign and malignant outcomes (AUC=0.9029).
Using classification and regression tree analysis, the decision tree diagram is predicted for melanocytic tumors (n=88) according to benign vs malignant outcome. The age and miRNA in situ hybridization intensity shaped the specific tree as shown (area under the curve=0.9029). In each node, the gray color represents benign and black represents malignant, highlighting the percentage of patients for each classification. The total number of patients is shown as 'N'.
Mod Pathol. 2016;29(5):461-475. © 2016 Nature Publishing Group