Serum polysialylated neural cell adhesion molecule in childhood neuroblastoma.

Neuroblastoma cells express the polysialylated form of the neural cell adhesion molecule (NCAM), which normally becomes restricted to a few neural tissues after embryogenesis. In this study, we investigated serum levels of polysialylated NCAM in 14 children with different grades and stages of neuroblastoma using an immunoluminescence assay, and compared the results to 269 healthy control subjects. Simultaneously, the polysialylated NCAM content of the tumours was determined by immunohistochemistry. Serum levels were dramatically elevated (more than sixfold) in children with advanced stages and fatal courses of disease, whereas children with differentiated tumour types and limited disease had low or normal levels. Serum concentrations correlated with the polysialylated NCAM content of the tumours, and they decreased during successful therapy. We therefore suggest polysialylated NCAM to be a useful marker monitoring childhood neuroblastoma.

Polysialylated NCAM forms are transiently expressed in many tissues during embryogenesis and become restricted to areas of permanent neural plasticity in the adult brain (Rougon. 1993). Some poorly differentiated tumours such as neuroblastoma. smallcell lung cancer (Moolenaar et al. 1990) and multiple myeloma (van Camp et al. 1990) re-express polysialylated NCAM forms. 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 tumour cells (Yang et al. 1992). NCAM isoforms occur not only membrane bound. but also as soluble molecules detectable in the serum. Recently. it has been shown that the serum levels of polysialylated NCAM are elevated in adult patients with small-cell lung cancer (Jaques et al. 1993) and multiple myeloma (Kaiser et al. 1994). high levels being associated with a poor prognosis (Ledermann et al. 1994: Smith et al, 1996.
In this study. we investigated children with different histological grades and stages of neuroblastoma and compared their serum levels with those of 269 healthy control subjects. We present evidence that serum polysialylated NCAM is a useful prognostic marker for childhood neuroblastoma.

Patients
Serum was collected from 14 children with different grades and stages of neuroblastoma (Table 1). Simultaneously. histological specimens were obtained for immunohistochemistry from all patients but two (nos 1 and 11). Staging and histological grading were applied according to the international criteria set by Brodeur et al (1988) and Hughes et al (1974). N-mwc gene amplification was analysed by Southern blotting (performed at the Department of Paediatrics. Univ ersity of Gie,en. Germany. by H Christiansen. MD. and F. Lampert. MD: data shown with permission) in all tumours but one (no. 1). The control group consisted of 269 children with an age range from 1 day to 16 years affected by other unrelated disorders. The project was approved by local ethics and scientific committees.

Immunoluminescence assay
Serum samples were thawed and assayed for soluble polysialylated NCAM by a chemiluminescent immunoassay developed by the Research Laboratories of Behring Diagnostics. Marburg.
Germany (Takamatsu et al. 1994). This assay system uses two monoclonal antibodies (MAbs): the polysialic acid-specific antibody 735 (Frosch et al. 1985) for capture and the anti-NCAM antibody BW SCLC-1 for detection of soluble poly sialylated NCAM.
MAb 735 specifically recognizes to a high degree a-2.8-linked sialic acid polymers with oligosaccharide segments > 8 (Hvyrinen et al. 1989). whereas MAb BW SCLC-1 specifically recognizes an epitope on the third immunoglobulin-like domain of human NCAM (M Eckhardt and R Gerardv Schahn. unpublished data). An aliquot of 200 gl of Tris-buffered incubation medium containing 0.5%c Tween 20 (pH 7.0) and 20 gl of sample or standard was filled into tubes coated with MAb 735 and incubated for 1 h at room temperature. After washin. 200 gl of MAb BW SCLC-1 conjugated to an acridinium N-acylsulfonamide label was added to each well. After incubation for 1 h at room temperature. the reaction was terminated bv an additional washing cycle. The chemiluminescence activitv was determined by using the BeriLux Analyser 250 (Behringwerke. Marburg. Germany). The results were expressed as kU 1-1 as previously descnrbed (Takamatsu et al. 1994). All measures were performed at least twice for internal control.  Fgure 4 Follow-up polysaytaed NCAM levels during therapy: serum concentrabons decreased in patients with high serum levels initially and remained low in patents with normal levels. Note variable scale was no correlation with age or sex of the children. In this study. all values above 60 kU 1were designated as pathological.
The data of the neuroblastoma patients are summarized in Table   1: two infants (nos 1 and 2) with stage 4 disease and one child (no. 3) with stage 3 disease had excessively high (1377.0kU 1-1) or high serum levels (301.3, 280.6 kU 1-1 respectively). These children died of rapid tumour progression despite intensive chemotherapy. Patient no. 4 with stage 3 disease and an initial serum level of 264.9 kU 1-1 is currently still receiving chemotherapy. One infant with stage 4S disease (no. 5) had an initial serum level of 255.4 kU 1-1. Another child (no. 6) with a large stage 1 neuroblastoma of histological grade 3 (tumour weight 200 g. the tumour was limited to one adrenal gland) had a serum level of 221.7 kU 1-1. Two infants with small adrenal stage 1 neuroblastoma (nos 9 and 10. tumour weight 9.5 g and 4.5 respectively) had serum levels within normal ranges. Both children with more differentiated tumour types but extensive disease (nos 7 and 8) had moderately raised levels (128.5. 90.2 kU 1-' respectively). All children with ganglioneuroblastoma stage (nos 11-14) had normal serum levels (20.4. 15.0, 38.8. 30.7 kU 1-' respectively) at diagnosis. N-mvc gene amplification (25to 40-fold) was found in the patients with the highest serum levels (nos 2-4). whereas no amplification of the gene was found in the other tumours (Table 1). Immunohistochemistry revealed high expression of polysialylated NCAM in the tumour specimens of patients nos 2-6 and no. 9 (Table 1 and Figure 2). whereas the specimens of the other patients had considerably smaller amounts of polysialylated NCAM (Table 1 and Figure 3).
As shown in Figure 4. eight children were available for follow-up studies. In patients with high levels initially the serum concentrations

DISCUSSION
In adult patients, serum polysialylated NCAM levels above 20 kU 1-' have been shown to be pathological (Jaques et al, 1993;Kaiser et al, 1994). We found levels of up to 62.9 kU 1-l in healthy children in whom the levels are on average higher (mean 26.8 kU 1-') than in adults. As the values cover a wide range (from 4.4 to 62.9 kU 1-1) we could not establish an age-dependent decrease in serum polysialylated NCAM. This is in contrast to previous findings in cerebrospinal fluid when a decrease was observed during the first year of life (Weisgerber et al, 1990). The mechanism by which NCAM forms appear in the serum is not known. The fact that there seems to be a relationship between the serum levels and the estimated amount of polysialylated NCAM-positive cells may indicate a constant release of this molecule from the cell surface. PSA-NCAM is expressed also on natural killer cells and a subset of T lymphocytes (van Riet et al. 1991). Theoretically. reactive shifts within these populations may be of relevance to the PSA-NCAM serum level. However, the serum levels of PSA-NCAM are low initially and during the course of most other malignant and benign childhood diseases. Moreover, we found a strong correlation between the levels of PSA-NCAM and the number of viable cells in cultures of neuroblastoma cells, indicating that the molecule in fact derives from the tumour cells (unpublished observations).
In a recent study (Figarella-Branger et al, 1996), polysialylated NCAM levels in cerebrospinal fluid were shown to correlate with clinical stage and outcome of patients with medulloblastoma. Our data show that polysialylated NCAM is a useful marker in the far more easily accessible serum of children suffering from neuroblastoma. A reliable and widely accepted prognostic factor in childhood neuroblastoma is amplification of the N-mvc gene in the tumour (Brodeur et al, 1984). In our study. N-mc gene amplification was found in the three children with the highest serum levels of polysialylated NCAM, two of whom died subsequently of progressive disease. In contrast, amplification was not present in the tumours of the patients with moderately raised or normal serum levels. However, two children with non-amplifying tumours had high (>200 kU 1-') serum levels, one with stage 4S disease and one with a large stage 1 adrenal tumour. Obviously, serum polysialylated NCAM did not distinguish these children from the others with a poor prognosis. As a consequence, it seems possible to speculate that, rather than indicating a defined tumour stage, serum polysialylated NCAM levels reflect the total amount of polysialylated NCAM-positive tumour cells, which in turn represent the viable tumour mass.
Polysialylation of NCAM has previously been shown to modulate the adhesive functions of tumour cells and to enhance their invasive and metastatic potential (Yang et al, 1992). Therefore, it is conceivable that high serum levels of polysialylated NCAM indicate more malignant tumour types. In our study, significantly lower serum levels were found in children with differentiated tumour types compared with undifferentiated tumours. In differentiated ganglioneuroblastomas, pathological levels occurred only in children with advanced stages of disease. Thus, serum polysialylated NCAM levels are influenced by both the tumour mass and histological differentiation. Serum levels decreased during successful therapy and remained low in patients during remission. This indicates that soluble polysialylated NCAM may be a useful marker for maintaining therapy in neuroblastoma patients.