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 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 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 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 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 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 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. Then, 6-p sections were Japan) for 1 hour at room temperature and then in- incubated with the antibody for dopamine at 4 C over- cubated with the antibody for c-ErbB2 diluted in Tris- night. Antigen-antibody complexes were detected buffered saline (20 mmol/L Tris-HCI, pH 7.5, and 137 with an avidin-biotin-peroxidase technique (Histofine mmol/L sodium chloride) with 0.1% Tween 20 at ABC kit, Nichirei, Tokyo, Japan). Peroxidase activity 1:1000 for 1 hour at room temperature. After three was detected with 3,3'-diaminobenzidine. washes with Tris-buffered saline with 0. 1% Tween 20, the membrane was incubated with horseradish Electron Microscopic peroxidase-conjugated anti-rabbit immunoglobulin (MBL) at a 1:5000 dilution for 1 hour at room tem- Immunohistochemical Study perature. After three washes with Tris-buffered saline Sections (6 p) were obtained by cryostat, blocked with 0.1 % Tween 20, peroxidase activity was de- with 10% goat serum at room temperature for 10 min- tected with ECL detection reagent (Amersham, Buck- utes, and incubated with the antibody for c-ErbB2 at inghamshire, UK). 4 C overnight. After five washes with PBS containing 10% sucrose at 4 C, sections were incubated with Reverse Transcriptase-Polymerase Chain goat anti-rabbit immunoglobulin (MBL) diluted 1:400 at 4 C overnight. After five washes with PBS contain- Reaction (RT-PCR) ing 10% sucrose, sections were fixed in PBS con- Total RNA was extracted with 4 mol/L guanidinium taining 0.5% glutaraldehyde and incubated with 50 thiocyanate and purified by ultracentrifugation mmol/L Tris-HCI, pH 7.5, containing 1% dimethylsul- through a 5.7 mol/L CsCI cushion.18 The amount of foxide and 3,3'-diaminobenzidine at room tempera- total RNA was determined by absorbance at 260 nm, ture for 30 minutes. Sections were then stained with and 2 pg of total RNA and 1 pg of random hexamer 50 mmol/L Tris-HCI, pH 7.5, containing 0.2 mg/ml were heated at 65 C for 5 minutes and then chilled on 3,3'-diaminobendizine and 0.05% H202 at room tem- ice. cDNA synthesis was carried out in a solution con- perature for 10 minutes. After sections were washed taining 200 U of Moloney murine leukemia virus re- in PBS, they were postfixed in 2% osmium in PBS, verse transcriptase, 50 mmol/L Tris-HCI at pH 8.3, 74 dehydrated, and embedded. Ultrathin sections were mmol/L KCI, 3 mmol/L MgCI2, 1 mmol/L dithiothreitol, obtained with an Ultratome (Reichert-Jung, Vienna, and 20 U of placenta ribonuclease inhibitor at 37 C for Austria) and stained with lead citrate. 60 minutes in a final volume of 20 pi. Reverse tran- scribed RNA was subjected to PCR amplification with Western Blot Analysis pairs of oligonucleotide primers derived from the cDNA sequence of the human erbB2 cDNA.3 The se- Frozen tumor specimens were homogenized in five quences of the primers were as follows: forward volumes of 20 mmol/L Tris-HCI, pH 7.5, containing 5 primer, 5' CTGACCAGTGTGTGGCCTGT (corre- mmol/L EDTA, 10 pmol/L 4-amidinophenylmethane- sponding to 1916 to 1935 nt) and reverse primer, 5' sulfonyl fluoride, and 1% Triton X-100 with a Polytron ATCGCTCCGCTAGGTGTCAG (corresponding to homogenizer. After leaving the homogenates on ice 2291 to 2272 nt). The expected size of the amplified for 30 minutes, the samples were centrifuged at product was 376 bp. The cDNA for glyceraldehyde- 10,000 x g for 30 minutes at 4 C. The aliquots of the 3-phosphate dehydrogenase was also amplified

Figure 1. Imnunuoperoxidalse staining of c-ErbB2 in primary nteutroblastomca tissues. Tumor specimnens were assessedfor c-ErbB2 expressionl with a rabbit polyclonal antibody for c-ErbB2. a: Focal-type staininig (histological gracle 3); an intensive immuntjoreactivity was observed in cytoplasm of tutnior cells. b: Diffuise-t)pe staintitng of metastatic lymph niode (histological grade 2): immnoreactivity was observed in the plasma membrane of tumor cells (arrowheads) and cellularprocesses. The remaining lymphocytes (asternsk) uere negative. C: D/fuse-tvpe staining ofgantglionzeuroblas- toma tissute (histological grade 1); strotng inmunoreactiiity was obseri'ed in the plasma mnembrane of tuimor cells (arroirheads) and celltular pro- cesses (star). d: Aegative-tlpe stainiing ofganglionieluroblastoma (histological grade 1). Magnification, X 400. 664 Goji et al AJP Mllarch 1995, Vol. 146, NVo. 3

A focal

RT + + + + - + - + - +

M Li Li LJLJ LJLI

z diffuse u

- + - + -+ + -+ - +

L i L4Li Li LJiLJ M

"A & 4 " z B *W 4 Z D3 >; X4 CA ;: :4 -@ v4 X4 s d = d uS= X = Y X FX0g Figure 2. RT-PCR of primaory nieroblastonma tissue(s. A: Amnplif'icationi of'c-erbfB2 cDVA Total RAA extractedfrom neutroblastonma tissues was subjected to RT-PG-R in the presence (+) or ab- seice (-) of rev'erse trauscrlptase (RT) as de- scribed in Materials and Methods. Huiman pla- cenital genomic DNA (100 ng) was also amplfijed fbr the sanme coniditioni. Thce stainting pattern of c-ErbB2 is indicated ahoie and ini- tials of the patientts are indicated helo'. M in- dicates 4X174/1-incII digest DNA size marker. B: Aniplification of'glvceraldehi'de-3-phosphate dehbdrogenase cDVA.

to verify an integrity of RNA. The primers for (NH4)2SO4, 33.3 mmol/L MgCI2, 0.85 mg/ml bovine glyceraldehyde-3-phosphate dehydrogenase were serum albumin, 50 mmol/L 2-mercaptoethanol, 335 forward primer, 5' TGGTATCGTGGAAGGACTCAT- mmol/L Tris-HCI at pH 8.8, 34 pmol/L EDTA, and 2.5 GAC (corresponding to 537 to 560 nt) and reverse U of Taq DNA polymerase (Perkin-Elmer Cetus, Nor- primer, 5' ATGCCAGTGAGCTTCCCGTTCAGC (cor- walk, CT) in a final volume of 50 pl. Amplification was responding to 725 to 702 nt).19 These primers were achieved by performing 25 cycles, each cycle con- designed not to amplify genomic DNA.20 Two micro- sisting of denaturation at 94 C for 1 minute followed liters of a cDNA template was used in the PCR. The by 1 minute of annealing at 60 C and 1 minute of PCR mixture contained 50 pmol each of the oligo- extension at 72 C in an automated Perkin-Elmer Cetus nucleotide primer, 0.2 mmol/L dNTPs, 83 mmol/L DNA thermal cycler. The oligonucleotide primers c-ErbB2 in Neuroblastoma and Adrenal Gland 665 AJP March 1995, Vol. 146, No. 3 were synthesized on an automated DNA synthesizer brane domain was amplified. As shown in Figure 2A, (Model 391, Applied Biosystems). The PCR product the c-erbB2 gene transcript was detected in focal and from patient KK was treated with T4 DNA polymerase diffuse-type tumors to various extents but not in a and then ligated with Smal-cut pGEM7Zf(+). The negative-type tumor. This negative-type tumor speci- DNA sequence was determined by a dideoxy chain men did not contain adjacent adrenal medulla. The termination method with fluorescence-tagged prim- amplified region contains at least one intron in the ers and Taq DNA polymerase according to the pro- genomic sequence, and amplification of genomic tocol recommended by the manufacturer and ana- DNA yielded a larger product. Sequence analysis of lyzed on an automated DNA sequencer (Model 370A, the amplified product from patient KK revealed that Applied Biosystems). this is derived from the c-erbB2 gene and there was no abnormality in this region. The integrity of RNA was confirmed by amplification of glyceraldehyde-3- Statistical Analysis phosphate dehydrogenase cDNA (Figure 2B). Fisher's protected least significant difference method Tumor specimens from four patients (two were was used to assess the significance of the staining diffuse type and two were focal type) were examined pattern of c-ErbB2 with stage of disease and urinary by Western blot analysis with the same antibody as concentration of vanillylmandelic acid (VMA) and used in the immunohistochemical study. This anti- (HVA), with StatView software body detected a single polypeptide with an apparent (Abacus Concepts, Berkeley, CA). molecular weight of 185,000 in all samples examined as shown in Figure 3. These results suggest that cy- toplasmic staining of c-ErbB2 in these tissues is not Results due to nonspecific reaction of the antibody with other proteins or intracellular degradation of the c-ErbB2 Expression of c-ErbB2 in Neuroblastoma protein. Tumor Tissue We have examined the expression of c-ErbB2/Neu Relationship between Immunohistological protein in 62 cases of neuroblastoma by an immu- Types and Clinical Findings nohistochemical technique. The specimens were ei- ther resected tumor upon primary surgery or biopsied Table shows clinical profiles of the patients analyzed metastatic lymph node before chemotherapy. These in this study. Of 15 patients showing a focal-type tumors were divided into three groups depending on staining pattern, 14 (93%) were at a favorable stage the staining pattern with anti-c-ErbB2 antibody. Typi- cal examples of the staining are shown in Figure 1. difmse focal Fifteen samples showed a focal staining pattern as shown in Figure 1 a. This group of tumors showed fo- W; w cal positivity with the antibody in which 20 to 80% of M.W. 03 :e the area was positive in the cytoplasm and cellular (kDa) processes. Twenty-one samples showed a diffuse 194 - 185 c-ErbB2 staining pattern in which all of the area showed posi- tive staining in cellular processes and plasma mem- 116 - brane as shown in Figure lb. Figure 1c represents a ganglioneuroblastoma tissue of diffuse type, which 85 - shows prominent expression of c-ErbB2 in the plasma membrane and cellular processes. A faint signal was also observed in the cytoplasm. Twenty-six samples 49 - were negative for c-ErbB2 expression. Figure ld shows a ganglioneuroblastoma tissue of negative type. There was no correlation between c-ErbB2 staining pattern and histological grade. To verify the expression of the c-erbB2 gene in Figure 3. Western blot analysis of neuroblastoma tissuesfor c-ErbB2 protein. Proteins ( 100 ,ug) extractedfrom tumor tissues ofthepatients these tumor tissues, we have performed RT-PCR as indicated above were subjected to sodium dodecyl sulfate poly- analysis. Analyzable RNA was obtained from 19 acrylamide gel electrophoresis, transferred to a nitrocellulose mem- brane, and incubated with a rabbitpolyclonal anti-c-ErbB2 antibody samples, and a region of cDNA coding the transmem- as described in Materials and Methods. 666 Goji et al AJP March 1995, Vol. 146, No. 3

Table 1. Clinical Profiles Number of patients Age of Stage N-myc Histologicalgrade Urinalysis onset gene (pg/mg ) Tumor type (months) + II IlIl + IV IVs amplification Dead 1 + 2 3 + 4 HVA VMA Focal (n = 15) 14.5 ± 3.9 13 1 1 1 1 11 4 37.6 ± 6.1 27.3 ± 2.1 Diffuse (n = 21) 16.6 + 4.6 5 12 4 1 2 12 9 253.9 ±- 34.5* 303.8 + 55.0* Negative (n = 26) 19.1 ± 3.9 1 1 14 1 5 4 12 14 50.6 ±- 6.5 38.5 + 4.6 Values are represented as mean + standard error. *P < 0.0001 for the diffuse group compared with focal and negative groups.

(stage 1, 11, or IVs). On the other hand, the number of was a marked difference in urinary VMA and HVA the patients at the favorable stage in the diffuse type secretion among the three groups. The concentration and negative type were 9 (43%) and 12 (54%), re- of these catecholamine metabolites in the diffuse spectively. The number of patients with N-myc gene group was significantly higher than those in the focal amplification was high in the negative type. There and negative groups (P < 0.0001). This difference

I

Figure 4. Immunioperoxidase staining ofN-Myc (a), dopamine (b), and c-ErbB2 (c and d) in adrenal gland of mouse embryo at E13 (a to c) and E16 (d). a: N-Myc immunoreactivity was stronger in extra-adrenal chromoblasts (large arrow) than in intra-adrenal chromoblasts. Ovary (0) was negative. Magnification, X 150. b: Strong immunoreactivity ofdopamine was observed in intra-adrenal chromoblasts whereas extra-adrenal chro- moblasts (large arrow) uere negative. Magnification, X 150. c: c-ErbB2 immunoreactivity wvas strotzger in intra-adrenal chromoblasts (small ar- rows) than in extra-adrenal chromoblasts (large arrou). Faint immunoreactivity was observed in precursory seminiferous tubule of testis (T). lMfagnzification, X 150 d: Fetal cortex wvas positive for c-ErbB2 at E16, but permanent cortex (asterisk) was negative. Immunoreactivity in ad- renomedzullary cells had becomne very faint at this stage. c-ErbB2 uwas positive in precursory tubuile of (K). Magnification, X 100. c-ErbB2 in Neuroblastoma and Adrenal Gland 667 AJP March 1995, Vol. 146, No. 3

was not due to the difference in stage because the urinary concentrations of VMA and HVA of patients at stages Ill and IV in the diffuse group were 367.9 + 63.0 and 292.8 + 39.5 pg/mg creatinine, respec- tively, and those in the negative group were 39.9 5.2 and 66.0 ± 8.8 pg/mg creatinine, respectively (P < 0.0001).

Expression of c-ErbB2 in Developing Mouse Neural Crest-Derived Tissues

The results described above suggest that the expres- sion and subcellular localization of c-ErbB2 are re- lated to the differentiation state of neuroblastoma cells. To examine whether these findings reflect the normal developmental process of neural crest-derived tissues, we have analyzed neural crest- derived tissues in the developing mouse for the ex- pression of c-ErbB2. Although the expression of the c-neu gene in developing mouse neural tissues has been studied,15 precise information about the ex- pression pattern of c-ErbB2/Neu protein after the dif- ferentiation process of peripheral neural tissues is lacking. In E13 mouse embryos, proliferating extra- adrenal adrenoblasts begin to migrate into the adre- Figure 5. Immunoperoxidase staining of c-ErbB2 in dorsal root gan- glion (DRG) of mouse atpostnatal day PO (a) and P4 (b). a: c-ErbB2 nal gland. Extra-adrenal adrenoblasts showed stron- immunoreactivity was observed in cytoplasm ofganglion cells. Neu- ger N-Myc immunoreactivity than did intra-adrenal rons in ventral horn (VH) were also positive. b: Plasma membrane and cellular processes of ganglion cells had become positive at P4 adrenoblasts (Figure 4a). At this stage N-myc immu- and immunoreactivity in cytosol had disappeared. Th-e ascending noreactivity was observed in the (asterisk) and descending tracts of spinal cord showed positive cytosol whereas this staining. antibody stains only nuclei of N-Myc-producing neu- roblastoma cells (data not shown). We have con- firmed the expression of the N-myc gene at this stage postnatal mouse, c-ErbB2 immunoreactivity was by in situ hybridization (data not shown). On the con- observed in the fasciculata layer but not in the trary, intra-adrenal adrenoblasts showed strong do- glomerulosa layer at P4 (data not shown). At P7, pamine immunoreactivity whereas extra-adrenal ad- c-ErbB2 was expressed in the reticular layer but not renoblasts did not (Figure 4b). At this stage c-ErbB2 in the glomerulosa and fasciculata layers (data not immunoreactivity was observed in the cytosol of intra- shown). adrenal adrenoblasts whereas extra-adrenal adreno- In dorsal ganglion, the c-ErbB2 immunoreactivity blasts did not show significant immunoreactivity (Fig- first appeared at E13. Intensive cytoplasmic staining ure 4c). At the electron microscopic level, there were was observed in E14, and c-ErbB2 immunoreactivity few secretory granules of which the diameter was became faint through the end of gestation. Immuno- -60 nm in extra-adrenal adrenoblasts whereas there reactivity appeared again in the cytosol of ganglion were abundant secretory granules of which the di- cells at birth (Figure 5a). At P4, the c-ErbB2 immu- ameter was 150 nm in intra-adrenal adrenoblasts noreactivity was observed in the plasma membrane (data not shown). A faint signal was also observed in and cellular processes (Figure 5b). Immunostaining fetal cortex at this stage. At E16, the c-ErbB2 immu- of c-ErbB2 in abdominal sympathetic ganglion is noreactivity was confined to the fetal cortex, and shown in Figure 6. Embryonal abdominal ganglia con- chromaffin cells lost immunoreactivity (Figure 4d). In sists of pre-aortic and post-aortic ganglia, and dif-

Figure 6. Immunoperoxidase staining of c-ErbB2 (a, C, and d) and dopamine (b) in sympathetic ganglion at E16 (a and b), Po (c), and P7 (d). a: At E16, pre-aortic ganglion cells (PRE) were positive for c-ErbB2 in cytoplasm whereas immunoreactivity ofpost-aortic ganglion (POST) was veryfaint. b: At E16, dopamine immunoreactivity was observed in pre-aortic ganglion cells but not in post-aortic ganglion cells. C: At PO, cytosolic c-ErbB2 immunoreactivity was present both in pre- and post-aortic ganglion cells. Muscle cells (M) and stromal cells in lymph node (L) were im- munoreactive. d: At P7, c-ErbB2 immunoreactivity was observed in plasma membrane and cellularprocesses ofganglion cells but not in their cy- tosol. 668 Goji et al AJPMarch 1995, Vol. 146, No. 3

iy *

.1w4 _ ' 4 g~~~~~~~~~~~~~' It, C i' 4r'½i7f 11.. &.¶ a ,< c-ErbB2 in Neuroblastoma and Adrenal Gland 669 AJP March 1995, Vol. 146, No. 3

Table 2. Clinical Profiles of the Patients Analyzedfor the Expression of c-ErbB2 in Adjacent Adrenomedullary Cells Cases analyzed Age of onset c-ErbB2 in tumor Positive* 1 7 mo Focal 2 7 mo Focal 3 7 mo Focal 4 7 mo Focal 5 7 mo Focal 6 7 mo Focal 7 8 mo Focal 8 9 mo Focal 9 1 yr 9 mo Focal 10 19 days Diffuse 1 1 7 mo Diffuse 12 7 mo Diffuse 13 4 yr 1 mo Diffuse 14 7 yr 4 mo Diffuse 15 8 mo Negative 16 1 yr 1 mo Negative 17 2 yr 0 mo Negative 18 5 yr 3 mo Negative Negative* 19 7 yr 0 mo Negative mo, months; yr, years. *Cases 1 to 18 are positive and case 19 is negative for c-ErbB2 in adrenomedullary cells.

cells than post-aortic ganglion cells at this stage (data not shown). At PO, post-aortic ganglion cells became positive for c-ErbB2 (Figure 6c). At this stage the c-ErbB2 immunoreactivity in the ganglion cells was present in the cytosol. At P7, c-ErbB2 immunoreac- tivity was localized on the plasma membrane and cel- lular processes (Figure 6d). The immunoelectron mi- Figure 7. Electron microscopic immuno.tiiobhistochemistry for c-ErbB2 in post-aortic ganglion at P4 (a) and P7 (b). a: At P4. ganglioni cells croscopic analysis was performed to define the have prominent Golgi apparatuts anid rouigh enidoplasnmic reticuluzim. intracellular localization of c-ErbB2 in sympathetic c-ErbB2 immunoreactivriti was observed oni polysomes associated with rouigh endcloplasnmic retictluim. b: At P7, c-FrbB2 inmmunoreac- ganglion cells. At P4, the c-ErbB2 immunoreactivity tivity wilas observed oni plasnma memnbranie and cellular processes (ar- was mostly present in the polysomes associated with roubeads). Magnification (a anid b). x 30,000, Bar indicates 1 It. rough endoplasmic reticulum (Figure 7a) and, at P7, c-ErbB2 immunoreactivity was confined to the ferentiation proceeds faster in pre-aortic ganglion plasma membrane of the neuronal cell body and cel- than At the immunoreac- post-aortic ganglion. E16, lular processes (Figure 7b). tivities of c-ErbB2 and dopamine were clearly present in the cytosol of the pre-aortic ganglion cells, whereas those in the post-aortic ganglion cells were very faint Expression of c-ErbB2 in Adrenal (Figure 6a, b). At E16, N-Myc immunoreactivity was Medullary Cells Adjacent to present in both pre- and post-aortic ganglion cells, Neuroblastoma Tumor and the staining became faint thereafter (data not shown). At electron microscopic level, the intracellu- Of 62 samples examined, 19 contained apparently lar micro-organelles, including rough endoplasmic normal adrenal medullary tissues adjacent to tumor. reticulum, Golgi apparatus, and secretory granules, Of these 19 samples, 18 showed strong cytoplasmic were markedly more abundant in pre-aortic ganglion c-ErbB2 immunoreactivity. The clinical profiles of

Figure 8. Imimiunoperoxidase staining o/'c-ErbB2 in adrenial glanid adjacenzt to neuroblastoma tissuce antd age-matched controls. a: Heniatoxylin- eosin staininlg ofttunior specimen of8-monith-old patient. This specinmeni showedjbcal-tipe staininlg for c-ErbB2. Tuimor cells (T)form niodules and are clearliy distinguishedfroni remainiing adrenomediullar cells (arrowheads anid asterisk) .M1agnffication, X 75. b: c-FrbB2 staining of the same specimneni. Apparently normnal adreniomnedullary cells showed stronig c-ErbB2 iwnimnoreactivity in cytosol (arrouheads anid asterisk). Adrenal cor- tex (C) was negative anid tumnior cells (7T) shoted jbcal-tlpe positivity. Magnification, x 75. C: c-ErbB2 staining of adrenial glancd of 6-month-old boy. This patient diedfrom heart disease. c-ErbB2 inimunoreactivity was obsened in cells in thefascicutlata layer (F), but adrcenonieduillary cells (M4) were negative. Mltagnificationi, X 200. d: c-ErbB2 staining of'adrenal glanid of'18-month-old girl. This patient diedfrom heart disease. c-ErbB2 inimunoreactiiity iwas observ'ed in cells in the reticular layer (R) but niot in adrenomedullary cells (M). Magnification, x200. 670 Goji et al AJP March 1995, Vol. 146, No. 3 c-ErbB2 in Neuroblastoma and Adrenal Gland 671 AJP March 1995, Vol. 146, No. 3

these patients are shown in Table II, and a typical (tyrosine to dopa), dopa example of c-ErbB2 staining is shown in Figure 8b. decarboxylase (dopa to dopamine), dopamine We have examined the expression of c-ErbB2 in 10 f-hydroxylase (dopamine to noradrenaline), and age-matched controls ranging from an 18-week em- phenylethanolamine N-methyl transferase (nora- bryo to a 7-year-old boy. In the 18-week embryo, the drenaline to ). In the abdominal sympa- c-ErbB2 immunoreactivity was observed in fetal cor- thetic ganglion of rat embryo, tyrosine hydroxylase tex but not in adrenal medulla (data not shown). In the and dopamine p-hydroxylase activities appear at postnatal human adrenal gland, c-ErbB2 immunore- E1221 (corresponding to E10 of mouse). We have activity was observed in the fasciculata layer of 1 -day- shown here that dopamine appears at E13 in adreno- old and 6-month-old children (Figure 8c) and in the blasts and E16 in sympathetic ganglion in the mouse reticular layer of 6- to 18-month-old children (Figure embryo (Figures 4 and 6). The expression of cytosolic 8d) but totally absent in adrenal medulla. The time c-ErbB2 was observed in these immature neural sequence of c-ErbB2 expression in the adrenal cortex crest-derived cells that had committed to differentia- was the same as observed in the mouse embryo as tion into each lineage. In rat adrenal chromaffin cells described above. These results suggest that the ap- the induction of phenylethanolamine N-methyl trans- parently normal adrenomedullary cells expressing ferase occurs at E1822 (corresponding to E16 of c-ErbB2 adjacent to tumor had converted their phe- mouse), and the expression of c-ErbB2 was not ob- notype to a very immature one. It is not known whether served at this stage (Figure 4d). Membrane localiza- this phenotypic conversion had occurred before or tion of c-ErbB2 was observed postnatally in mouse after tumor progression at present. dorsal root ganglion and sympathetic ganglion cells as shown in Figures 5 to 7. These data indicate that the membrane c-ErbB2 expression is a phenotype of Discussion mature peripheral neurons producing acetylcholine (dorsal root ganglion) or adrenaline (sympathetic of c-ErbB2 in primary We report here the expression ganglion). It is unlikely that c-ErbB2 is involved in the neuroblastoma tissue for the first time. The tumor cells induction of the enzymes involved in catecholamine of focal type showed cytoplasmic c-ErB2 immunore- synthesis. Peles et a123 reported that a neuroblastoma activity (Figure 1 a). This finding is contradictory to the cell line, LA-N-1, expresses functional c-ErbB2 pro- idea that c-ErbB2 is a transmembrane protein. The tein that undergoes tyrosine autophosphorylation cytoplasmic staining was not an artifact because upon binding to NDF. Shah et al14 reported that GGF these tumors expressed c-erbB2 gene transcript as cells a potential shown by RT-PCR analysis (Figure 2) and this anti- acts on immature neural crest having to differentiate into both neurons and Schwann cells, body detects a single polypeptide (p185c-ErbB2) in these tissues (Figure 3). Moreover, fetal immature and GGF suppresses neuronal differentiation while However, neural crest-derived cells showed the same staining promoting Schwannian differentiation.14 pattern (Figures 4 to 6). The immunoelectron micro- physiological ligands for c-ErbB2 expressed in neu- date, the scopic examination of mouse sympathetic ganglion ronal cells have not been identified to and cells at P4 revealed that c-ErbB2 immunoreactivity in functions of both membrane and cytosolic c-ErbB2 in cytosol resides on polysomes associated with rough neuronal cells are unknown at present. Additional endoplasmic reticulum (Figure 7a). It is not known at study is needed to explore the role of c-ErbB2 in neu- present whether or not the cytoplasmic c-ErbB2 is ronal development. functional. The apparently normal adrenomedullary cells ad- Tumor cells of the diffuse type showed membrane jacent to tumor were positive for c-ErbB2 (Figure 8b). staining for c-ErbB2 (Figure 1 b, c). The membrane- As human adrenomedullary cells do not express associated c-ErbB2 immunoreactivity was observed c-ErbB2 postnatally (Figure 8c, d), the expression of in the differentiated peripheral neurons of the mouse c-ErbB2 in adrenomedullary cells in these patients is (Figures 5 and 6). The patients of diffuse type showed unusual. Although there is no definite evidence indi- significantly higher urinary VMA and HVA secretion cating the c-ErbB2+ adrenal medulla is a precancer- compared with the focal- and negative-type patients ous region, our data raise a possibility that carcino- (Table 1). This may suggest that membrane expres- genesis of human neuroblastoma is a multistep sion of c-ErbB2 is related to catecholamine produc- process as proposed for adult , and tion or secretion. The metabolic pathway of cat- it might be possible that overexpression of cytoplas- echolamines is well defined, and the enzymes mic c-ErbB2 provides a growth advantage leading to involved in catecholamine synthesis are as follows: tumor progression. However, the possibility that the 672 Goji et al AJP March 1995, Vol. 146, No. 3

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