Immunohistochemical analysis of the Bcl-2 oncoprotein in human neuroblastomas. Comparisons with tumor cell differentiation and N-Myc protein

Abstract

Background: The bcl-2 gene encodes a 26-kilodalton integral membrane oncoprotein that is noteworthy for its function as a blocker of programmed cell death, and ability to render cells resistant to killing by chemotherapeutic drugs and x-irradiation.

Experimental design: To determine the in vivo patterns of bcl-2 expression in neuroblastomas (NBs), tumor specimens derived from 17 children with NB or ganglioneuromas were immunohistochemically evaluated using formalin-fixed, paraffin-embedded material and antibodies specific for the Bcl-2 and N-Myc proteins, or various markers typically used to assess the differentiation status of these tumors, including neuron-specific enolase, S-100 protein, beta 2-microglobulin (beta 2M) and the intermediate filament proteins, vimentin, and neurofilament light- and medium-chains.

Results: Using two-color immunohistochemical methods, Bcl-2 protein was found exclusively in tumor cells that did not contain N-Myc, an oncoprotein previously associated with poor prognosis in NB. Levels of Bcl-2 immunostaining were heterogeneous in the undifferentiated small, round cells typically seen in tumors with aggressive histology, and ranged from essentially undetectable, to strong in intensity. Somewhat higher levels of Bcl-2 immunostaining were found in the slightly larger, more differentiated neuroblastic cells that had more generous cytoplasm in these neoplasms. In contrast to the heterogeneous levels of Bcl-2 seen in undifferentiated NBs, Bcl-2 immunoreactivity was uniformly present at high levels in tumor cells that were more differentiated and judged to be similar to immature ganglion cells at an intermediate stage of neuronal differentiation, based on morphologic characteristics and immunophenotyping with antibodies specific for various differentiation markers. The still more differentiated ganglionic cells seen in ganglioneuroblastomas and ganglioneuromas exhibited much less intense immunoreactivity with anti-Bcl-2 antibodies, suggesting down-regulation of bcl-2 during final terminal differentiation. In contrast to cells with neuronal features, the stromal Schwann cells were uniformly negative for Bcl-2 protein in all histologic grades of tumors. Thus, bcl-2 expression in NBs was limited to cells of the neuronal lineage and tended to be highest in tumor cells with the characteristics of ganglionic cell precursors. The expression of bcl-2 in the normal fetal and postnatal adrenal medulla and sympathetic ganglia paralleled that seen in NBs, consistent with previous suggestions that NB reflects a block in the normal differentiation process.

Conclusions: Taken together, these findings indicate that the levels of the Bcl-2 protein are developmentally regulated in normal and neoplastic cells of the sympathetic branch of the autonomic nervous system, and suggest that this oncoprotein may be useful as a differentiation marker for subclassification of NBs for prognostic purposes and for investigations of normal neuronal maturation. Furthermore, strong Bcl-2 immunoreactivity was detected in islets of residual tumor cells in 6 of 7 specimens obtained from 5 patients after therapy, suggesting that this oncoprotein may be cytoprotective for NB cells in vivo.