Amiierican Journal/ of Pathology, Vo.I 146, Ao. 3. tlIarcb 1995 (opyhn,ght ©D Amnericai .Societ'Jfor Investigative Pathology Expression of c-ErbB2 in Human Neuroblastoma Tissues, Adrenal Medulla Adjacent to Tumor, and Developing Mouse Neural Crest Cells Junko Goji,* Hajime Nakamura,* Hiroshi Ito,t proto-oncogene1 that is similar to the epidermal Osamu Mabuchi,t Kimio Hashimoto,§ and growth factor receptor. c-erbB2lneu was first identi- Kimihiko Sano* fied as a transforming gene of ethylnitrosourea- From the Departments of Pediatrics* anzd Pathology,t Kobe induced rat neuroblastoma.2 Sequencing data have University School ofMedicine, and Departments of demonstrated that oncogenic p185neu differs from HematologyIOncologyt and Pathology,5 Kobe ChildrenS c-Neu by a single point mutation within the transmem- Hospital of Hvogo Prefectnre, Kobe, Japan brane region of the glycoprotein. This mutation results in the substitution of a glutamic acid residue for a valine residue at amino acid position 664 of the rat neu We have examined the expression of c-ErbB2 in gene product. In humans, the gene product is called primary neuroblastoma tissues, mouse neural ErbB2 (also NGL and HER2)3 and its amplification crest-derived tissues, and human adrenal gland and overexpression correlate with a poor prognosis in adjacent to neuroblastoma tissue and of age- breast, ovarian, gastric, and endometrial cancers.47 matched controls. c-ErbB2 expression was ob- The ligands for c-ErbB2/Neu protein were purified served in approximately 60% of cases analyzed, from transformed rat fibroblast and a human breast and there were two stainingpatterns; one showed tumor cell line and designated as neu differentiation focal and cytoplasmic and the other showed dif- factor (NDF)8 and heregulin,9 respectively. These pro- fuse and membrane staining patterns. The ex- teins stimulate p185neu autophosphorylation and in- pression ofc-ErbB2 in neuroblastoma tissues was duce differentiation of mammary tumor cells to milk- confirmed by reverse transcription polymerase producing, growth-arrested cells.10 Independently, chain reaction and Western blot analysis. Diffuse the cDNA for the glial growth factor (GGF), which and membrane staining ofc-ErbB2 was weUl cor- stimulates DNA synthesis and cell division in cultured related with high urinary catecholamine secre- Schwann cells, has been cloned, and it was revealed tion. In mouse tissues, cytoplasmic expression of that NDF and GGF are encoded by alternatively c-ErbB2 was observed in immature peripheral spliced transcripts of the same NDFIGGF gene.11 neurons and adrenomedullary cells. In mature Moreover, the acetylcholine receptor-inducing activ- neurons, the immunoreactivity was confined to ity (ARIA), which is present in motor neurons and their theplasma membrane. These results suggest that synaptic terminals at the neuromuscular junction and the expression of c-ErbB2 in neuroblastoma re- upregulates acetylcholine receptor, is found to be a flects the phenotype of developing pertiperal product of the NDF/GGFgene. 12 Alternative splicing neurons. Postnatal human and mouse ad- of the NDFIGGFIARIA mRNA generates at least 14 renomedullary ceUs lacked c-ErbB2 immunoreac- different transcripts that encode putative membrane- tivity, although apparently normal adrenomedul- attached, intracellular, and secreted signaling pro- lary ceUls adjacent to neuroblastoma tissues teins. These results indicate that c-ErbB2/Neu protein showed strong cytoplasmic expression of mediates differentiation and/or proliferation of secre- c-ErbB2. It is not known whether the phenotypic conversion of adjacent adrenal medullary cells had occurred before or after tumor progression Supported in part by a grant for cancer research from the Hyogo at present. (AmJPathol 1995, 146:660-672) Total Health Association (1993). Accepted for publication November 22, 1994. Address reprint requests to Dr. Kimihiko Sano, Department of c-ErbB2/Neu protein is a 185-kd transmembrane re- Pediatrics, Kobe University School of Medicine, 7-5-1 Kusunoki- ceptor tyrosine kinase encoded by the c-erbB2lneu cho, Chuo-ku, Kobe 650, Japan. (Fax 81-78-371-6239) 660 c-ErbB2 in Neuroblastoma and Adrenal Gland 661 AJP March 1995, Vol. 146, No. 3 tory epithelial cells, Schwann cells, and muscle cells the tumor had undergone phenotypic conversion to through interaction with different ligands arising from an immature phenotype. a single gene. The localization of the NDF mRNA in mouse em- bryo was examined by in situ hybridization.13 In E14.5 Materials and Methods mouse embryo, the highest expression was observed in the neuroepithelium lining the lateral ventricles of Tumor Specimens and Histology the brain, the ventral horn of the spinal cord, and in- Tumor specimens were obtained from patients re- testinal as well as dorsal root ganglia. Other tissues ferred to the Hyogo Prefectural Children's Hospital expressing the NDFmRNA were adrenal cortex, liver, between 1986 and 1993. In total, 62 specimens were and skin. The ARIA mRNA in E7 chick embryo is lo- analyzed. Patients were staged according to the calized in the ventral horn of the spinal cord, and a Evans staging system.16 All specimens were ana- faint signal was observed in the dorsal root ganglia.12 lyzed before treatment, and histology was deter- The expression of the GGFmRNA was examined by mined as described by Beckwith and Martin.17 The in situ hybridization.11 In mouse embryo from El 1 to definition of this classification was as follows: grade El 5, the GGFmRNA is highly expressed in the ventral 1, predominantly differentiated, >50% differentiating horn of the spinal cord and dorsal root ganglia. Sym- elements; grade 2, predominantly differentiated, 5 to pathetic ganglia, cranial sensory ganglia, trigeminal 50% differentiating elements; grade 3, slightly differ- ganglia, and other primary motor neurons also ex- entiated, <5% differentiating elements; and grade 4, pressed the GGF mRNA. Although the function of no recognizable neurogenesis. N-myc gene amplifi- NDF in developing neural tissue is not known, Shah cation was determined by Southern blot analysis. et al14 showed that GGF suppresses neuronal differ- entiation of rat neural crest stem cells while promoting or allowing Schwannian differentiation. Immunohistochemical Study The level of the c-erbB2lneu mRNA is highest in Tumor tissues from patients were processed for rou- E14 rat embryo.15 Immunohistochemical analysis has tine light microscopic analysis. Mouse embryos re- revealed that p185neu is expressed in a variety of tis- moved from the uterus were fixed in 4% paraformal- sues including nervous system, connective tissue, dehyde at 4 C and rinsed with phosphate-buffered and secretory epithelium in the E14 embryo.15 In the saline (PBS) containing 10, 15, and 20% sucrose by peripheral nervous system, p185neu is expressed in turns at 4 C. The specimens were embedded in OCT the neural perikarya of the developing dorsal root compound, frozen in dry ice-acetone, and kept at -80 ganglia. These results suggest that c-ErbB2/Neu and C until analysis. The primary antibodies used in this its ligands may be involved in proliferation and/or dif- study were as follows. Rabbit polyclonal antibody for ferentiation of immature peripheral neurons by an au- c-ErbB2 (A485) was obtained from Dako Japan tocrine or paracrine pathway. These results prompted (Kyoto, Japan). This antibody was raised against 21 us to examine the expression of c-ErbB2/Neu protein amino acids of the carboxy terminus of human in human neuroblastoma tissues, as this childhood c-ErbB2 protein and was diluted 1:10,000 for frozen tumor is derived from immature peripheral neural tis- sections, 1:1,000 for paraffin sections, and 1:100 for sue and possesses various phenotypes common to electron microscopic immunohistochemical study. those found in normal developmental processes. We We have also used another rabbit polyclonal antibody have examined 62 primary neuroblastoma tissues for c-ErbB2 protein (NCO01, Novo Castra, Newcastle and found that approximately 60% of cases ex- upon Tyne UK), which recognizes the same region as pressed c-ErbB2 protein. Diffuse expression of Dako's antibody, and obtained similar results. Rabbit c-ErbB2 protein in tumor cell membrane was asso- polyclonal antibody for N-Myc protein was obtained ciated with high urinary catecholamine secretion. Fur- from MBL (Nagoya, Japan). This antibody was raised thermore, the apparently normal adrenomedullary against a polypeptide corresponding to amino acids cells adjacent to tumor tissue showed strong c-ErbB2 169 to 253 of exon 2 and does not react with c-Myc immunoreactivity whereas adrenomedullary cells in protein. This antibody was diluted 1:40 for frozen sec- age-matched controls did not show significant ex- tions. Rabbit polyclonal antibody for dopamine pression. These results suggest that c-ErbB2 expres- (AB122S) was obtained from Chemicon (Temecula, sion is related to the differentiation state of neuroblas- CA). To stain dopamine in mouse tissues, sections toma cells and that adrenomedullary cells adjacent to were treated as follows. Embryos removed from the 662 Goji et al AJP March 1995, Vol. 146, No. 3 c-ErbB2 in Neuroblastoma and Adrenal Gland 663 AJP March 1995, Vol. 146, No. 3 uterus were sliced into 3-mm-thick sections and pre- supernatants were then boiled in sodium dodecyl sul- washed in 1 % sodium meta-bisulfite in 50 mmol/L ca- fate and 2-mercaptoethanol for 3 minutes and loaded codylate buffer at pH 6.5 to prevent oxidation of do- onto a 7.5% sodium dodecyl sulfate polyacrylamide pamine and fixed in glutaraldehyde solution (5% gel. After electrophoresis was completed, the pro- glutaraldehyde and 1% sodium meta-bisulfite in 50 teins on the gel were transferred to a polyvinlidene mmol/L cacodylate buffer at pH 6.5). Sections were difluoride-nitrocellulose membrane (Immobilon-P, then washed three times with 50 mmol/L Tris-HCI, pH Millipore, Bedford, MA) with a semi-dry blotting de- 7.5, containing 1% sodium meta-bisulfite and 10, 15, vice (ATTO, Kyoto, Japan). The membrane was and 20% sucrose by turns at 4 C for 2 hours each and blocked in BlockAce (Dainippon Seiyaku, Osaka, frozen in dry ice-acetone.