Immunoreactivity of a monoclonal antibody against human placental alkaline phosphatase

To the Editor: Immunohistochemistry is a valuable and essential diagnostic adjunct of current tumor pathology. Unexpected results, however, potentially due to cross-reactivity in the immunostaining, often create some confusion or challenges in the interpretation of the findings. We encountered such a case in which a commercially available and widely distributed monoclonal antibody against human placental alkaline phosphatase (PLAP, clone 8A9, DakoCytomation, Glostrup, Denmark) was applied to the immunohistochemical evaluation of a testicular tumor. PLAP is one of three tissue-specific alkaline phosphatase isoenzymes that catalyze the hydrolysis of phosphomonoesters with the release of inorganic phosphate, and its active form consists of dimerized subunits of a 65 kDa membrane-bound glycoprotein. PLAP is normally synthesized by placental syncytiotrophoblasts and released into the maternal circulation after the 12th week of pregnancy. In addition to germ cell tumors, a variety of non-germ cell tumors, including gastrointestinal, gynecological, hematological, pulmonary, breast and urological tumors, may also rarely express PLAP. The precise biological function of PLAP remains unclear, although it has been suggested that it is involved in the transfer of maternal IgG to the fetus or cell division in normal and transformed cells. In our immunohistochemistry study using the monoclonal anti-PLAP antibody, the antigen was expressed in many tumor cells of embryonal carcinoma that arose in the left testis of a 14-year-old boy. The immunoreactivity was seen predominantly in a membranous pattern. Surprisingly, nonneoplastic smooth muscle cells including those of the blood vessels and the ductus deferens were also stained unequivocally for PLAP in a diffuse cytoplasmic manner (Fig. 1). No information regarding the expression of PLAP in smooth muscle cells was available from the manufacturer. Therefore, the immunohistochemistry of PLAP was extended using a panel of muscular tissues and tumors to confirm the finding, and positive immunoreactivity was also found in both smooth and skeletal muscle cells at various anatomical sites as well as in myogenic tumors such as leiomyoma, leiomyosarcoma and rhabdomyosarcoma. The finding, however, was not reproducible using a polyclonal antibody against PLAP (A0268) purchased from the same supplier, thus suggesting the potential for an artifact due to cross-reactivity of the monoclonal antibody used. This possibility was addressed using a modified azocoupling enzyme histochemistry technique to evaluate alkaline phosphatase activity in the liver, myometrium and leiomyoma. No positive reactivity was found in snap-frozen tissue specimens of the myometrium and leiomyoma, which were immersed in an alkaline solution (pH 8.2) containing Fast Red and a-naphthyl AS-MX phosphate (DakoCytomation), whereas that of the liver, as a positive control, was intensely stained. Furthermore, reverse transcription– polymerase chain reaction (RT-PCR) using a specific primer set (5′-CAGCCGCCAAGAACCTCATC, 5′-AAGCGGTCC ATGGCCAGGAAG) aligned respectively to exons 2 and 3 of the PLAP gene failed to detect its transcript in the myometrium or leiomyoma. Immunoelectronmicroscopy using the monoclonal primary antibody (clone 8A9) and 10 nm gold particles conjugated with anti-mouse immunoglobulins (DakoCytomation) showed that the immunogold particles were localized on intracytoplasmic fine filaments of myometrial smooth muscle cells (Fig. 2). Recently, Goldsmith et al. described almost identical immunohistochemistry using the same anti-PLAP antibody and assumed that normal muscle cells and tumors with myogenic differentiation indeed express PLAP, showing abnormal subcellular localization. Their assumption appeared to be supported by similar immunoreactivity in selected myogenic tumors using another monoclonal anti-PLAP antibody (clone PL8-F6, Biogenex, San Ramon, CA USA) and by a