Human high molecular weight-melanoma-associated antigen: utility for detection of metastatic melanoma in sentinel lymph nodes

Abstract

Purpose: Detection of micrometastasis in melanoma-draining lymph nodes is important for staging and prognosis. Immunohistochemical staining (IHC) using S-100p-HMB-45-, and MART-1-specific antibodies is used for detecting metastases in sentinel lymph nodes (SLN). However, improvement in IHC is needed for melanoma micrometastasis detection.

Experimental design: Paraffin-embedded archival tissue (PEAT) specimens were obtained from 42 non-SLN macrometastases, 42 SLN metastases, and 16 tumor-negative SLNs of 100 melanoma patients who underwent SLN biopsy. PEAT specimens were assessed by IHC with high molecular weight-melanoma-associated antigen (HMW-MAA)-specific monoclonal antibodies (mAb) and with S-100p-, HMB-45-, and MART-1-specific antibodies. Quantitative real-time reverse-transcriptase PCR assay was used for HMW-MAA and MART-1 mRNA detection.

Results: Expression frequency and immunostaining intensity were higher for HMW-MAA than MART-1 in nodal macrometastases (P < 0.0001 and P < 0.0001, respectively) and micrometastases (P < 0.0001 and P = 0.004, respectively). All 52 (100%) macrometastases were positive with HMW-MAA-specific mAbs, whereas 43 (83%) were positive with MART-1-specific mAbs. In a comparison analysis, 23 of 23 (100%) micrometastases were HMW-MAA-positive, whereas 21 (91%) and 18 (78%) specimens were S-100p- and HMB-45-positive, respectively. Quantitative real-time reverse-transcriptase PCR analysis of 48 nodal metastases showed HMW-MAA mRNA detection in SLNs with metastases.

Conclusions: HMW-MAA is more sensitive and specific than MART-1, S-100p, and HMB-45 for IHC-based detection of SLN micrometastases. SLN PEAT-based detection specificity of melanoma micrometastases can be improved by IHC with HMW-MAA-specific mAbs.

Fig. 1

A to F, comparison of MART-1and HMW-MAA expression in SLNs by IHC. A, MART-1 (+; ×100). B, MART-1 (+; ×400). The cells immunoreactive for MART-1show red cytoplasmic staining. C, HMW-MAA (+; ×100). D, HMW-MAA (+; ×400). The cells immunoreactive for HMW-MAA show purple membrane staining. E, normal mouse IgG (−), negative control (×100). F, normal mouse IgG (−; ×400). G to L, comparative IHC between MART-1and HMW-MAA IHC in SLN macrometastasis. G, MART-1 (−; ×100). H, MART-1 (−; ×400). I, HMW-MAA (+; ×100). J, HMW-MAA (+; ×400). K, normal mouse IgG (−; ×100) L, normal mouse IgG (−; ×400). Scale bars, 100 μm.

Fig. 2

A, HMW-MAA mRNA expression in melanoma cell lines. HMW-MAA mRNA expression was designated as relative mRNA copies (absolute mRNA copies of HMW-MAA/absolute mRNA copies of GAPDH). B, HMW-MAA mRNA expression in nodal macrometastases and micrometastases. HMW-MAA mRNA expression was designated as relative mRNA copies (absolute mRNA copies of HMW-MAA/absolute mRNA copies of GAPDH). Dotted bars, mean copy numbers. The cutoff for HMW-MAA positivity was 2.95 × 10⁻², corresponding to the mean relative HMW-MAA copy number plus1SD in tumor-negative nodes. C, MART-1mRNA expression in macrometastases, micrometastases, and tumor-free nodes. MART-1mRNA expression was designated as relative mRNA copies (absolute mRNA copies of MART-1/absolute mRNA copies of GAPDH). Dotted bars, mean copy numbers.