Detection of carbonic anhydrase 9–expressing tumor cells in the lymph nodes of vulvar carcinoma patients by RT‐PCR

Abstract

Regional lymph node status is an important prognostic factor for vulvar cancer. The goal of our study was to elaborate a reliable test for detecting micrometastases, undetectable by traditional methods, in the lymph nodes of vulvar squamous carcinoma patients. For this purpose, carbonic anhydrase-9 (CA9) was investigated as a cancer-related marker by RT-PCR. Firstly, primary carcinoma specimens were examined for CA9 expression by immunohistochemistry with M75 monoclonal antibody. All 19 tissues exhibited a variable degree of staining, which was mostly confined to the plasma membranes of tumor cells. Correspondingly, all primary tumor specimens and the control A-431 vulvar cancer cell line gave a positive signal in the nested RT-PCR assay designed to detect CA9-expressing cells with a high sensitivity. Analysis of 77 lymph node specimens from 20 patients revealed a full correlation between RT-PCR results and standard hematoxylin-eosin staining in 75% of samples, whereas 25% of specimens were negative by the standard method and positive for CA9 mRNA, accounting for 28% of all histologically negative lymph nodes. There were no false-negatives with RT-PCR. A positive inguinal lymph node with a negative sentinel node was observed in the same groin only once in 38 specimens. Our findings clearly indicate potential value of CA9 as a molecular marker for the assessment of regional lymph node status in vulvar cancer patients and support a possible utility of our RT-PCR assay in the detection of micrometastases.