Pediatric brainstem gangliogliomas show overexpression of neuropeptide prepronociceptin (PNOC) by microarray and immunohistochemistry

Abstract

Background: Gangliogliomas (GGs) primary to brainstem are rare, with the overwhelming majority of GGs occurring in supratentorial, especially temporal lobe, locations. A less favorable prognosis exists for brainstem GGs, despite their usually identical WHO grade I status. Few large clinical series, and limited biological information, exists on these tumors, especially gene expression.

Procedure: Seven pediatric brainstem GGs, all with classic histological features, seen at our institution since 2000 were identified. Frozen section material was available for gene expression microarray profiling from five of seven brainstem GGs and compared with that from three non-brainstem pediatric GGs.

Results: Significant upregulation of a number of genes was identified, most of which were involved in pathways of neural signaling, embryonic development, and pattern specification in pediatric brainstem GGs compared to non-brainstem. The single largest upregulated gene was a 256-fold increase in the expression of the neuropeptide prepronociceptin (PNOC); the protein product of this gene has been implicated in neuronal growth. Overexpression was validated by Western blot and by immunohistochemistry (IHC). Strong IHC expression of PNOC was seen in neoplastic neurons of 7/7 brainstem GGs, but was significantly weaker in non-brainstem GGs, and completely negative in normal pediatric autopsy brainstem controls.

Conclusions: PNOC IHC was often superior to IHC for NeuN, synaptophysin, or neurofilament for highlighting neoplastic neurons.

Figure 1

(a) All brainstem gangliogliomas were classified as WHO grade I and manifested classic histological features of GG on light microscopy, with numerous cytologically-atypical neurons, a low-grade glial component, and variable amounts of perivascular non-neoplastic lymphocytic cuffing (lower left). Hematoxylin and eosin, 400X; Patient 5 from Table I illustrated. (b) Immunohistochemical staining for PNOC protein revealed strong (scored as +++) expression in large numbers of neoplastic neurons in all 7 of the brainstem GGs; IHC for PNOC with light hematoxylin counterstain, 400X; Patient 5 from Table I illustrated. (c) Immunostaining for PNOC in brainstem gangliogliomas was predominantly cytoplasmic and was distributed throughout the cytoplasm. IHC for PNOC with light hematoxylin counterstain, 600X; Patient 7 from Table I illustrated. (d) Careful matching of serial sections taken from the same tissue block from the same case was undertaken to directly compare similar areas of the tumor in terms of quality of PNOC [see 1c], versus for NeuN, as illustrated in this image. PNOC [see 1c] highlighted the cell body, similar to NeuN [this Fig.] but showed immunostaining in a considerably larger number of the neoplastic neurons. IHC for NeuN with light hematoxylin counterstain, 600X; Patient 7 from Table I illustrated. (e) Immunostaining for synaptophysin was strong in the background of the gray-matter rich brainstem; compare this heavy IHC+ background with that for PNOC [see 1c] in the same area of the tumor. Because synaptophysin tended to identify increased synaptic density at the cell membrane/perimeter it was sometimes more difficult to confidently identify IHC+ neoplastic neurons with this immunostain than it was for PNOC. IHC for synaptophysin with light hematoxylin counterstain, 600X; Patient 7 from Table I illustrated. (f) Immunostaining for neurofilament predominantly highlighted axons in brainstem gangliogliomas and not cell bodies, as seen in this image. Indeed, unlike neurofilament immunohistochemistry that decorates axons and only occasional cell perikarya in GGs, PNOC did not appear to decorate axonal processes [see 1c for comparison]. IHC for neurofilament with light hematoxylin counterstain, 600X; Patient 7 from Table I illustrated. (g) PNOC IHC in non-brainstem gangliogliomas (GGs) was similar to that in brainstem GGs in only 1 of 5 cases, as illustrated in this image, although even in this example, the number of immunoreactive neurons appeared fewer than with brainstem GGs (scored as ++). IHC for PNOC with light hematoxylin counterstain, 400X; Patient 5 from Supplemental Table I illustrated. (h) PNOC IHC was significantly weaker and more infrequent in the remaining 2 of 5 non-brainstem GGs, as illustrated in this photomicrograph (scored as +). IHC for PNOC with light hematoxylin counterstain, 400X; Patient 10 from Table II illustrated. (i) No observable PNOC expression was found in any brainstem neurons at any level (midbrain, pons, or medulla) of control autopsy specimens; note the negative immunostaining (scored as 0) and clean background in these basis pontis neurons. IHC for PNOC with light hematoxylin counterstain, 400X.

Figure 2

Western blot analysis of Prepronociceptin (PNOC). Protein lysate (40 ug) from 5 brainstem GGs and 4 supratentorial GGs were probed with anti-PNOC antibody. Anti-A-tubulin antibody was used as an internal control for protein loading.