and stored in liquid nitrogen or fixed in Bouin's fixative and embedded in paraffin. No differences in intensity and distribution of NGFR immunoreactivity were ...
American Journal of Pathology, Vol. 139, No. 1, July 1991 Copyrigbt © American Association of Pathologists
Nerve Growth Factor Receptor Expression in Peripheral and Central Neuroectodermal Tumors, Other Pediatric Brain Tumors, and During Development of the Adrenal Gland David L. Baker,* Willemina M. Molenaar,t John Q. Trojanowski,t Audrey E. Evans,* Alonzo H. Ross,¶ Lucy B. Rorke, Roger J. Packer*, Virginia M-Y Lee,t and David Pleasurell From the Division of Anatomic Pathology, Department of
Pathology and Laboratory Medicine, University of Pennsylvania,t the Divisions of Oncology,* Pathology,3* and Neurology Research,11 Children's Hospital of Philadelphia, Pennsylvania; and the Worcester Foundation for Exerimental Biology,$ Shrewsbury, Massachusetts
Nerve growth factor (NGF) is important to the survival development, and differentiation of neurons. Its action is mediated by a specific cell surface transmembrane glycoprotein, nerve growthfactor receptor (NGFR). In this study, NGFR expression by human fetal and adult adrenal medullary tissue, peripheral nervous system (PNS) neuroectodermal tumors (neuroblastoma, ganglioneuroblastoma, ganglioneuroma), pediatric primitive neuroectodermal tumors (PNETs) of the central nervous system (CNS), and CNS gliomas was examined by an immunohistochemical technique. Sixty-nine tumors in total were probed in this manner. Nerve growth factor receptor immunoreactivity was confined to nerve fibers and clusters ofprimitive-appearing cells in the fetal adrenal, and to nerve fibers and ganglion cells of the adult adrenal medulla; adrenal chromaffin cells were negative. In PNS neuroectodermal tumors, there was NGFR expression in tumor cells of 6 of 1I neuroblastomas and 6 of 6 ganglioneuroblastomas or
ganglioneuromas. Thirteen of thirty-five CNS PNETs showed NGFR positivity. In most CNS PNETs, NGFR was restricted to scattered single or small groups of cells, but two tumors with astroglial differentiation showed much more extensive immunoreactivity. Most astrocytomas (11 of 14) and all ependymomas
(3 of 3) were intensely NGFR positive. (Am JPathol 1991, 139:115-122)
Tumors of neuroectodermal tissue are found predominantly in the pediatric age group, and include a group that has been termed 'primitive neuroectodermal tumors' (PNETs).15 This all-inclusive term, which has gained wide but controversial6,7 acceptance in the literature, has been proposed in an endeavor to classify a group of malignancies composed of small undifferentiated cells with dark oval to irregular nuclei and usually with little observable cytoplasm.1 45 They share many morphologic features, but may originate in the brain (medulloblastoma, cerebral neuroblastoma), adrenal glands and sympathetic ganglia (neuroblastoma), soft tissues (neuroepithelioma), or bone (Ewing's sarcoma). Use of the phrase, 'primitive neuroectodermal,' or 'neuroepithelial,' is based on the resemblance of the histologically undifferentiated cells of these tumors to germinal or matrix cells of the embryonic neural tube and neural crest.a In addition to such primitive cells, PNETs also may contain scattered regions or foci of neuronal, glial, or ependymal differentiation.>1 Nerve growth factor is a neurotrophic protein capable of mediating a variety of biologic responses. Its vital importance as a survival and differentiation factor in the developing peripheral nervous system (PNS) has been well established.14 The role of NGF in central nervous system (CNS) is less well documented, but several studies have indicated that it plays a role in the development of cholinSupported in part by NIH grants CA47983, CA36245, NS08075, NS18616, NS25044, and HD26979. Dr. W. M. Molenaar was a Fulbnght Scholar on sabbatical leave from the University of Gronigen in the Netherands during the course of these studies. Accepted for publication February 27, 1991. Address reprint requests to D. Pleasure, MD, Neurology Research, Children's Hospital of Philadelphia, Philadelphia, PA 19104.
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ergic pathways of the forebrain and
neurons of
the
cerebellum.1I17 The mechanism of action of NGF is not fully understood, but is known to be mediated by a specific cell surface transmembrane glycoprotein, the NGF receptor (NGFR).18 Most studies have concluded that there are two forms of the NGFR, which, although containing the same NGFR protein, differ by almost two orders of magnitude in affinity for NGF. The high-affinity NGFR is responsible for NGF internalization and biologic response by susceptible neurons or neuronal tumors.'1-22 There have been few reports of the NGFR expression in tissue from central PNETs and brain tumors, and contradictory evidence for its expression in peripheral neuroectodermal tumors.2>26 In addition, only two CNS PNETs (medulloblastomas) have been studied.26 To explore further the expression of NGFR within neuroectodermal tumors, a panel of frozen or paraffin-embedded central (PNETs, astrocytomas, ependymomas) and peripheral (neuroblastomas, ganglioneuroblastomas, ganglioneuromas) tumors were screened using immunohistochemical techniques and the NGFR monoclonal antibody, Me 20.4.27 The findings in the PNS tumors were compared with the expression of NGFR in human fetal and adult adrenal medullary cells. The NGFR analysis of 35 central PNETs, 14 astrocytomas, 3 ependymomas, and 17 peripheral neuroectodermal tumors in this report is the most extensive study of these neural tumors to date.
Bouin's-fixed, paraffin-embedded sections were compared with frozen sections from the same specimen. Most tumor tissue was collected from children operated on at the Children's Hospital of Philadelphia. Material from five cases in the neuroblastoma group was made available by Dr. R. C. Seeger from the Neuroblastoma Registry in Los Angeles. Staging of the neuroblastomas has been described by Molenaar et al.28 Fetal adrenal tissue was obtained from spontaneous and therapeutic abortions at estimated gestational ages of 6, 9, 10, 13, 15, 17, 18, 19, 20, 21, 24, 25, 32, and 34 weeks, respectively. From the four earliest ages, cross sections were made of the total fetus, but at later stages the adrenals were embedded separately. Two adrenals from adult patients, aged 18 and 53 years, respectively, were also studied.
Immunohistology All sections were probed with the anti-NGFR antibody Me 20.4,27 using the avidin-biotin complex peroxidase technique or the peroxidase-antiperoxidase technique, as previously described.23' Normal human gut was used as positive control tissue and hybridoma supernatant from unfused mouse myeloma cells was used as a negative antibody control in all cases. Slides were graded as positive for NGFR if either scattered cells or sheets of cells were strongly immunoreactive (see results for further details).
Materials and Methods Results Tissue
Fetal and Adult Normal Adrenal The samples studied and methods of slide preparation are summarized in Table 1. All tissues were snap frozen and stored in liquid nitrogen or fixed in Bouin's fixative and embedded in paraffin. No differences in intensity and distribution of NGFR immunoreactivity were noted when Table 1. Specimens Examined Tissue examined Fetal adrenals Adult adrenals Neuroblastomas
Ganglioneuroblastomas Ganglioneuromas CNS PNETs Astrocytomas Ependymomas
From the earliest fetal stage on, strong immunoreactivity observed in nerve fibers throughout the adrenal. At gestational ages of 6, 9, and 10 weeks, a well-formed adrenal medulla could not yet be distinguished, but the
was
Frozen sections
Paraffin sections* 14 2
11 2 4
23t 10
The case material and fixation of the samples are summarized in Table 1. * All paraffin sections were prepared from specimens fixed in Bouin's solution. t Includes two cases studied both in paraffin and frozen tissue sections.
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NGF Receptors in Neuroectodermal Tumors 117 AJP' uly 1991, Vol. 1239, No. 1
cortex contained immunoreactive nerve fibers and occasional positive presumptive medullary cells at gestational ages of 9 and 10 weeks. At later stages, hematoxylinand-eosin-stained sections of the adrenal medulla showed clusters of small, primitive-appearing cells and scattered larger cells with pale nuclei and ill-defined cytoplasm (for more details see reference 31). Strong NGFR immunoreactivity was observed in nerve fibers in the medulla and in small fibers surrounding the clusters of primitive-appearing cells (Figure 1A). In addition, occasional small and larger cells showed cytoplasmic NGFR immunoreactivity. In the adult, NGFR immunoreactivity was largely restricted to nerve fibers, but some ganglion cells appeared weakly positive, especially at their surfaces (Figure 1 B).
Peripheral Tumors
Nerve growth factor receptor immunoreactivity was detected in all ganglioneuromas and ganglioneuroblastomas, and in more than half of the neuroblastomas (Table 2). The examples shown in Figure 1 exemplify the intensity of NGFR immunoreactivity and the relative abundance of NGFR-positive cells in this group of tumors. The immunoreactivity in the neuroblastomas was largely restricted to stromal septae separating nodules of tumor cells (Figure 1 C). Cellular NGFR positivity was observed in six cases, and was almost exclusively in single tumor cells, with only an occasional cluster of positive cells (Figure 1 D). In contrast to the neuroblastomas, all ganglioneuroblastomas and ganglioneuromas showed strong
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Figure 1. Photomicrographs of NGFR immunoreactivitiy in the human fetal adrenal medulla at a gestational age of 24 to 25 u'eeks (A), in the adult human adrenal medulla (B), and in two different neuroblastomas (C, D). A: In thefetal human adrenal medulla, the arrowspoint to clusters offetal adrenal medullary blast cells (presumptive neuroblasts or chromaffin cell blasts) surrounded bh NGFR-positive supporting cells (probably nascent Schu'ann cells). Isolated embryonal adrenal medullary cells are also NGFR positive in this specimen. B: In the adult human adrenal medulla, onlv nerves and a rareganglion cell are NGFR positive. The arrow points to a mature adrenal medullary ganglion cell surrounded byP an NGFR-positive Schu'ann cell. C: In a neuroblastoma the arrows point to NGFR-positive cells in the septae. D: In another neuroblastoma, the arrows point to clusters ofNGFR-positive tumor cells. NGFR staining in the neuroblastomas u'as restricted to septae and/or clusters of tumor cells. The sections in A and B were obtainedffrom Bouin's-fixed, paraffin-embedded material, while those in C and D are frozen sections. All of the sections uere counterstained with hemato.xylin, and the magnification in each panel is x250.
118 Baker et al AJPJulu 1991, Vol. 139, No. 1
Table 2. NGFR Immunoreactiviy Tumor type
Number NGFR positive
Number NGFR negative
6 2 4 13
5
Neuroblastomas Ganglioneuroblastomas Ganglioneuromas CNS PNETs Astrocytomas Ependymomas
11
3
0 0
22 3 0
The results obtained with paraffin and frozen sections from the cases examined are summarized. As noted in the text, cases are scored as NGFR positive if any NGFR-positive tumor cells were detected. Figures 1 to 3 exemplify the range of NGFR labeling in these tumors. It should be emphasized that nearly all of the CNS PNETs scored as NGFR positive contained a minority of NGFR-positive tumor cells (less than 5%), as described in Results.
NGFR immunoreactivity, located in the fiber bundles of differentiated tumor cells (data not shown).
Central Tumors Although the majority of the CNS PNETs were NGFR negative, other than in incarcerated Purkinje cells, 13 of the 35 PNETs did display NGFR immunoreactivity (Table 2). In 10 of these, this was restricted to individual cells and occasional small clusters of cells (less than 5% of total tumor cells in the sections) (Figure 2C, Figure 3A, B), comparable to the findings in neuroblastomas. Representative examples of the extent of NGFR-positive tumor cells (both with respect to the intensity of staining and the abundance of labeled tumor cells) in these PNETs are shown in Figures 2 and 3. In three cases, however, the immunoreactivity was widespread and involved confluent fields of tumor cells, sometimes centered around blood vessels (Figure 2A, B; Figure 3C, D). It is of interest that two of these cases histologically showed a combination of PNET and anaplastic astrocytoma (Figures 2A, B, and 3A, B) (for more details, see reference 32). In CNS PNETs containing NGFR-positive tumor cells, immunoreactivity could be detected equally well in frozen and paraffin sections from the same specimen (compare Figure 3A, B). Eleven of fourteen astrocytomas and all three ependymomas showed extensive NGFR immunoreactivity (Table 2). The staining was characterized by a fine fibrillar pattern, corresponding to the fibrillary processes of the tumor cells. It was often concentrated around blood vessels. Nerve growth factor receptor immunoreactivity was especially strong in one case classified as a superficial cerebral astrocytoma, in which staining of both fibers and of cell bodies was observed.
Discussion In this report we describe an analysis of NGFR expression in a panel of 52 CNS malignancies (PNET, astrocy-
toma, ependymoma) and compare their expression with 17 PNS tumors of neural lineage and 14 normal fetal and two adult adrenal gland specimens. In the PNS group, NGFR immunoreactivity was demonstrated in scattered tumor cells of 6 of 11 neuroblastomas. More widespread NGFR immunoreactivity was seen in the fiber bundles and ganglion cells of the two ganglioneuroblastomas and the four ganglioneuromas. These results can be compared with the data of Perosio and Brooks,23 in which 6 of 10 neuroepitheliomas and neuroblastomas were NGFR positive, and that of Chesa et al,24 who failed to demonstrate immunoreactivity in eight neuroblastomas but detected positivity in two of two ganglioneuroblastomas and two of two ganglioneuromas. The discrepancy in neuroblastoma results between our study and that of Chesa et al24 may be related to their use of formalin rather than Bouin's as a fixative. Alternatively their collection of neuroblastomas may have been dominated by a group similar to our NGFR-negative neuroblastomas. Our immunohistologic findings with the PNS tumors are in keeping with the extensive documentation of NGFR expression in cell lines of peripheral neuroectodermal tumors, 3 and show that NGFR expression correlates positively with the degree of neuronal differentiation of such tumors. The expression of NGFR in these PNS neuroectodermal tumors can be compared with that in human fetal and adult adrenal medullary tissue. In the fetal adrenal, NGFR immunoreactivity was confined to nerve fibers and clusters of primitive-appearing cells, presumably sympathoadrenal neuroectodermal progenitor cells,35 and was present in nerve fibers and ganglion cells of the adult adrenal medulla. That NGFR-positive PNS neuroectodermal tumors arise from these fetal sympathoadrenal progenitor cells is an attractive presumption, but it is also possible that NGFR expression in these PNS tumors is a consequence of the inappropriate activation of NGFR gene transcription during transformation of NGFRnegative neural crest precursor cells. Nerve growth factor receptor expression by the PNS neuroectodermal tumors is consistent with the hypothesis of autocrine stimulation of tumor maturation.36 In fact, Per-
NGF Receptors in Neuroectodermal Tumors
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Figure 2. Photomicrographs of NGFR immu-
noreactivity in central PNETs (posterior fossa medulloblastomas) u'ith (A and B) and without (C) evidence of astrocytic differentiation.
A: In a PNET with evidence of astrocytic differentiation, the asterisks identifi' two blood vessels surrounded by NGFR-positive tumor cells. B: In another PNET uwith evidence of astroc-rtic diferentiation, the asterisks identifil two blood vessels lined by NGFR-positive tumor cells. C: In a PNET uwithout evidence of astroc-ytic differentiation, the arrows identifir clusters of NGFR-positive tumor cells; labeled cells are less frequent than in the PNETs uwith astroctic differentiation. The sections in A and B were obtainedfrom Bouin's-fixed, paraffin-embedded material, whereas that shoun in C is a frozen section. All of the sections were counterstained u'ith hematox}lin and the magnification in each panel is x250.
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osio and Brooks25 have provided direct evidence for the coexpression of growth factors and their receptors in a number of soft-tissue tumors.25 Further support for this mechanism lies in the demonstration of NGF secretion by neuroblastoma cell lines37'38and in the finding that NGF stimulates neuronal maturation in some NGFR-positive cell lines.20 To test further the hypothesis of autocrine stimulation of tumor differentiation, assessment of NGF synthesis and NGFR expression in the group of stage IV S neuroblastomas39 would be of particular interest, as
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these tumors have an exceedingly high propensity to spontaneously differentiate that might be related to this mechanism. Of the CNS PNETs we studied, 22 did not show NGFR immunoreactivity, 10 displayed minimal positivity, restricted to scattered individual cells and small cell clusters, and only in three cases was NGFR immunoreactivity widespread. Two of these three displayed histology consistent with classification as a PNET with astroglial differentiation. There is a paucity of data in the literature de-
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sections, ubile B is a fiozen section. Note that qpproximately the same levels of NGFR immunoreactivitj are evident in paraffin (A) and frozen (B) sections from the same tumor. The magnification in (A-C) is X.300, while that in D is x400.
scribing NGFR immunoreactivity in CNS PNETs. Indeed, results of NGFR immunostaining of only two medulloblastoma tumor specimens have been reported; both had variable NGFR immunoreactivity.26 Eight permanent human CNS PNET cell lines have been described,40-5 and NGFR expression has been documented in only one of these.45 Further studies will be necessary to determine whether or not those CNS PNETs that are NGFR positive represent a PNET subset derived from neural tube progenitors that are themselves NGFR positive.17 The generic classification of CNS neuroectodermal tumors as PNETs may be challenged in future by establishment of diverse patterns of proto-oncogene expression and karyotypic abnormalities, as has already occurred with PNS neuroectodermal tumors. It has been demonstrated, for example, that neuroblastoma and neuroepithelioma differ both in proto-oncogene expression (N-myc in neuroblastoma versus c-myc in neuroepithelioma) and karyotypic abnormality (1 p deletion, homolo-
gous staining regions, and double minutes in neuroblastoma versus 1 1 to 22 translocation in neuroepithelioma).647 Molecular analysis of CNS PNETs and CNS PNET lines indicates that some express amplified c-myc, whereas others do not.45'48 Cytogenetic analysis of central PNETs has shown that most have structural or numeric chromosomal abnormalities, the most consistent being an i(1 7q), present in approximately one third of tumors, sometimes in the absence of other demonstrable
abnormalities.49'50 Most of the astrocytomas (11 of 14) and all of the ependymomas (3 of 3) showed extensive NGFR immunoreactivity in the tumor cell bodies and fibrillary processes. As noted earlier, the two central PNETs with astroglial differentiation also displayed prominent NGFR immunoreactivity, consistent with our findings in pure astroglial malignancies. Once again there are few reports in the literature describing NGFR immunoreactivity in astrocytomas. Chesa et a124 and Thompson et al-" noted
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patchy immunoreactivity in only 1 of 6 and 3 of 9 gliomas, respectively, and Thomson et al51 showed positivity in 9 of 21 astrocytoma cell lines that they investigated (although 6 of the negative lines were subclones of a parent line). In conclusion, we have detected immunoreactive NGFR expression in the majority of PNS neuroectodermal tumors and CNS gliomas and in a significant proportion of CNS PNETs, particularly those with glial features. Although these NGFR-positive tumors may be responsive to the effects of NGF, recent reports by Pleasure et a122 and Azar et al34 demonstrate that mere expression of abundant NGFRs does not guarantee that NGF will induce differentiation of such tumors.
12.
13.
14.
15.
16.
Acknowledgments The authors thank Drs. Robert C. Seeger, Luis Schut, Leslie Sutton, and Arno Fried as well as Kathy Bonner, RN, for providing tissue samples. Anne O'Brien and Paul Newman are thanked for their expert technical assistance.
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aglione M, Beigel JA, Emanual B, LoPresti P, Trojanowski JO, Evans A, Roy A, Venkatakrishnan G, Chen J, Ross AH, Pleasure D: Human CNS primitive neuroectodermal tumor expressing NGF receptors: CHP707M. Ann Neurol 1990, 28:136-145 46. Thiele CJ, McKeon C, Triche TJ, Ross RP, Reynolds CP, Israel MA: Differential proto-oncogene expression characterizes histo-pathologically indistinguishable tumors of the peripheral nervous system. J Clin Invest 1987, 80:804-811 47. Whang-Peng J, Triche TJ, Knutsen T, Miser J, Kao-Shan S, Tsai S, Israel MA: Cytogenetic characterization of selected small round cell tumors of childhood. Cancer Genet Cytogenet 1986, 21:185-208 48. Wasson JC, Saylors RL l1l, Zeltzer P, Friedman HS, Bigner SH, Burger PC, Bigner DD, Look AT, Douglass EC, Brodeur GM: Oncogene amplification in pediatric brain tumors. Cancer Res 1990, 50:2987-2990 49. Bigner SM, Mark J, Freidman MS, Beigel JA, Bigner DD: Structural chromosomal abnormalities in human medulloblastomas. Cancer Genet Cytogenet 1988, 30:91-1 01 50. Biegel JA, Rorke LB, Parker RJ, Sutton LN, Schut L, Bonner K, Emmanual BS: Isochromosome 1 7q in primitive neuroectodermal tumors of the central nervous system. Genes, Chromosomes and Cancer 1989, 1:139-147 51. Thomson TM, Rettig WJ, Chesa PG, Green SM, Mena AC, Old U: Expression of human nerve growth factor receptor on cells derived from all three germ layers. Exp Cell Res 1988, 174:533-539
