Main

RMS, the most frequent soft tissue tumor, accounts for about 5% of all pediatric malignancies(1). On the basis of morphologic appearance and clinical features, four major subtypes are differentiated: embryonal, alveolar, botryoid, and pleomorphic RMS. Because cases of botryoid RMS are classified as embryonal, and because pleomorphic RMS are very rare in children, only two basic types-embryonal and alveolar RMS-were considered in this study. Alveolar RMS displays a more aggressive behavior and has a poorer prognosis compared with embryonal RMS(2). Histologically, RMS resemble characteristics seen during the embryonic development of skeletal muscle.

NCAM is known to be involved in skeletal myogenesis and regeneration after muscle denervation or injury(3, 4). Therefore, it is likely that rhabdomyosarcoma cells, in contrast to normal muscle tissue, express high amounts of NCAM. This NCAM has been shown to be the highly sialylated form(5), described before to be reexpressed in tumors such as Wilms' tumor, neuroblastoma, and small cell lung carcinoma(6, 7). Using a MAb highly specific for polysialic acid(8), we investigated tumor specimens of nine patients with RMS of different histologic types and clinical stages and determined their amount of PSA-NCAM. In seven patients, serum levels were tested to evaluate PSA-NCAM as a diagnostic and prognostic marker in childhood RMS.

METHODS

Patients. The clinical data of the patients investigated are summarized in Table 1. The tumor specimens and/or serum samples were obtained from 11 children during routine measures, and further processed as described below. Staging and histologic classification were applied according to criteria set by the International Rhabdomyosarcoma Study Group(9). The project was approved by the local ethical and scientific committees.

Table 1 Clinical data, immunohistochemistry, and serum PSA-NCAM levels of the children investigated
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Immunohistochemistry. Cryostat sections (5 μm) of snap frozen tumor specimens were further processed for immunohistochemistry using the APAAP technique according to Cordell(10). Briefly, the slides were fixed in ice-cold acetone for 10 min and air-dried. After preincubation with normal rabbit serum the polysialic acid-specific MAb 735(8) was added for 1 h at room temperature. After washing, the slides were incubated with rabbit anti-mouse immunoglobulin and the alkaline phosphate anti-alkaline phosphate complex twice for 1 h each. Color development was obtained with naphthol AS-biphosphate and New Fuchsin. Positive reaction of the counterstained cells resulted in bright red staining of the cytoplasm. Controls included the omission of primary and/or secondary antibodies, and preincubation of the sections with bacteriophage endoneuraminidase for 2 h at 37°C, which has previously been shown to specifically recognize and degrade polysialic acid(11).

Immunoluminescence assay. Serum samples were thawed and assayed for soluble PSA-NCAM by a chemiluminescent immunoassay developed by the Research Laboratories of Behring Diagnostics, Marburg, Germany(12). In this assay system, the polysialic acid-specific MAb 735(8) is used as a capture antibody. Tris-buffered incubation medium, 200 μL, containing 0.5% Tween 20 (pH 7.0) and 20 μL of sample or standard, was put into tubes coated with MAb 735 and incubated for 1 h at room temperature. After washing, 200 μL of the anti-NCAM MAb BW SCLC-1 conjugated to an acridinium N-acylsulfonamide label were added to each well. After incubation for 1 h at room temperature, the reaction was terminated by an additional washing cycle. The chemiluminescence activity was determined by using the BeriLux Analyzer 250 (Behringwerke AG, Marburg, Germany). The results were expressed as kU/L as previously described(12). All measures were done at least in duplicate for internal control.

RESULTS

As shown in Table 1, all RMS specimens but one showed reactivity with MAb 735, regardless of alveolar or embryonal nature. Patients with extensive disease (nos. 1-6) and the patient with pulmonary metastasis(no. 11) showed strong staining of tumor cells, whereas patients with limited disease (nos. 7-9) had a distinctly lesser amount, or almost no staining. After preincubation with endoneuraminidase, previously positive cells were negative for staining with MAb 735, but remained positive with the NCAM-specific MAb UJ13A(13).

In the serum, the highest PSA-NCAM levels were found in children with stage IV disease (566.1 and 332.1 kU/L), whereas children with stage III and stage II disease had clearly lower serum levels (169.9, 113.4, 106.7, and 78.2 kU/L). One patient with a stage I paratesticular RMS had normal serum levels at diagnosis (25.2 kU/L).

Four children were available for follow-up studies. As shown in Figure 1, the serum concentrations of PSA-NCAM decreased during successful therapy. After reaching normal levels, values remained normal during follow-up. After chemotherapeutic treatment, previously positive tumors became completely negative for PSA-NCAM in immunohistochemical study, as shown in Figure 2.

Figure 1
figure 1

Follow-up serum levels of PSA-NCAM in four patients with RMS. Bar indicates threshold value at 60 kU/L.

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Figure 2
figure 2

Embryonal RMS of the biliary system (patient no. 3).(A) Immunohistochemical staining with MAb 735, showing PSA-NCAM expression on RMS cells in a liver biopsy before chemotherapy. (B) Immunohistochemical staining of the tumor after chemotherapy, showing almost no reactivity for MAb 735.

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DISCUSSION

In contrast to other tumors, especially carcinomas in adults, RMS usually respond well to chemotherapeutic agents and radiation. As a consequence, it is not imperative to completely resect an RMS primarily, endangering vital structures. Rather, chemotherapy will reduce the tumor's size and enhance the chance of complete resection at a later operation. Therefore, particularly for these tumors, specific markers are desirable that reflect the viable tumor mass and allow monitoring the response of the tumor cells to chemotherapy or radiation. However, reliable diagnostic and prognostic markers currently are not available for childhood RMS.

RMS cells are known to express the highly sialylated form of the NCAM(5). PSA-NCAM forms are transiently expressed in many tissues during embryogenesis and become restricted to areas of permanent neural plasticity in the adult brain(14). The polysialic acid moiety has been shown to modulate the adhesive functions of NCAM and other adhesion molecules and to be relevant for an enhanced invasive and metastatic potential of tumor cells(15). High levels of PSA-NCAM have been previously shown to be associated with a poor prognosis in adult patients with small cell lung cancer(16). Likewise in our study, the expression of PSA-NCAM was high in advanced stages (stages III or IV) and the pulmonary metastasis, although the lower stages (stages I and II) had considerably less PSA-NCAM. Chemotherapy dramatically removed all PSA-NCAM-positive cells, but expression was high in the pulmonary metastasis that occurred 3 y after treatment of the primary tumor. Because PSA-NCAM was present in both tumor types, embryonal and alveolar, it obviously does not differentiate between them.

NCAM isoforms occur not only membrane-bound but also as soluble molecules detectable in serum, cerebrospinal fluid(17), and amniotic fluid(18). Recently, it has been shown that serum PSA-NCAM is elevated in adult patients with small cell lung cancer(19) and multiple myeloma(20) proving it as a reliable serum marker for these tumors. The mechanism by which NCAM forms appear in the serum is not known. However, the fact that there seems to be a relationship between the serum levels and the estimated amount of PSA-NCAM-positive tumor cells may indicate that there is a constant release of this molecule from the cell surface. This would imply that serum levels of PSA-NCAM, rather than indicating a defined tumor stage or prognosis, reflect the total amount of positive tumor cells, which in turn represent the viable tumor mass. Serum levels decreased during successful therapy and remained low in patients during remission. This indicates that soluble PSA-NCAM may be a useful marker for monitoring therapy in RMS patients.