Diagnostic Utility of Neural Stem and Progenitor Cell Markers Nestin and SOX2 in Distinguishing Nodal Melanocytic Nevi From Metastatic Melanomas

Pei-Ling Chen; Wei-Shen Chen; Jianping Li; Anne C Lind; Dongsi Lu


Mod Pathol. 2013;26(1):44-53. 

In This Article

Materials and Methods

Study Population and Case Selection

This study was approved by the Institutional Review Board of Washington University in St Louis. The Department of Pathology database was searched for cases diagnosed with nodal melanocytic nevi from 1993 to 2011, and a matching number of metastatic melanomas to the lymph nodes were randomly selected for this study. Archival formalin-fixed, paraffin-embedded tissue blocks and slides were retrieved from our surgical pathology files. All H&E-stained slides were independently reviewed by three study pathologists (DL, AL and PLC). A total of 41 surgical specimens were included in this study: 23 lymph nodes with metastatic melanomas (16 sentinel, 7 non-sentinel), 17 lymph nodes with nodal melanocytic nevi (8 sentinel, 9 non-sentinel; 5 with history of melanoma) and 1 lymph node with 'subcapsular/intranodal melanocytic rest.' Patient demographics (age, gender, primary diagnosis, specimen type and location) are detailed in Table 1 .


Results of each immunostain were independently reviewed by three study pathologists (AL, DL and PLC). Immunostain for nestin (Milipore, 1:100, no. MAB5326) was performed on a Ventana Benchmark XT automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA) according to standard protocols after heat-induced antigen retrieval. Immunoreactivity for cytoplasmic nestin was detected using the Ultra View Universal Alkaline Phosphatase Red Detection Kit to avoid difficulty in interpreting nestin staining in cells with pigmented cytoplasm. Appropriate positive and negative controls were included for each run of immunostaining. Nestin is expressed in vascular endothelial cells, so lymph node blood vessels served as internal positive controls. Primary antibodies were omitted as negative controls. The expression of nestin antibodies was qualitatively scored as 0 (no staining), 1+ (weak), 2+ (moderate) and 3+ (strong).

Immunostaining for goat anti-SOX2 antibodies was performed manually. Briefly, the unstained slides were blocked using 1% BSA (bovine serum antigen) and subsequently incubated with the primary antibody (Neuromics, 1:200, no. GT15098) for 35 min. Secondary antibody (rabbit/mouse/goat, DAKO; no. K0679) was applied and incubated for 30 min, and DAB+ chromogen was incubated for 2 min according to the manufacture's protocol. Between each step, Tris-buffer (0.5% Tween 20, pH 7.4) was used for washing. Faint, blush-like cytoplasmic SOX2 was detected in lymph node germinal centers and was used as internal positive control. Primary antibodies were omitted as negative controls. For nodal melanocytic nevi, SOX2 immunostaining was applied to 16 cases, as the melanocytic focus of interest was lost in one case upon serial sectioning of the block.


The statistical association between each antibody and the melanocytic lesions was analyzed using Fisher's exact test. All reported P-values were based on a two-sided hypothesis, and a P-value <0.05 was considered statistically significant.