Expression of the Neuronal Form of Pp60c Clinical Stage And

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(CANCER RESEARCH 50, 6908-6914, November I, 1990] Expression of the Neuronal Form of pp60c in Neuroblastoma in Relation to Clinical Stage and Prognosis1

Catarina Bjelfman, Fredrik Hedborg, Irja Johansson, Magnus NordenskjÃ¶ld, and Sven PÃ¡hlman2

Departments of Pathology; University of Uppsala, University Hospital fC. B.. F. H., I. J., S. P.], and Pediatrics, University Hospital IF. //./. Uppsala, and Department of Clinical Genetics, Karolinska Hospital, Stockholm [M. N.J, Sweden

ABSTRACT tumors of neural crest derivation with varying clinical findings and also varying degrees of differentiation. It is usually a highly The expression of the protooncogene c-src has been studied in speci malignant childhood tumor. The tumor cells are arrested at mens of childhood tumors with special reference to neuroblastoma and other tumors of neuronal origin. For comparison i--.vÂ»-cgeneexpression various stages of neuronal differentiation, and the grade of was studied in seven neuroblastoma and neuroepithelioma cell lines. The differentiation correlates to clinical course (28, 29). The more structurally distinct neuronal product of the gene, pp60' *"\ expressed differentiated tumors have cell elements of ganglionic and glial during normal development in neuroblasts and neurons, was identified by appearance. They are therefore referred to as ganglioneuroblas- immunoblotting technique together with the fibroblast form, pp60c~"c. tomas and tend to be less aggressive than the poorly differen While pp60c""cwas found in most tumors studied, the neuronal form was tiated tumors which have a monomorphic blast appearance, restricted to neuroblastomas (23 of 27) and retinoblastomas (3 of 3) and often with formation of pseudorosettes (29). Ganglioneuroma could not be detected in the other childhood tumors. A dominance of the neuronal form, pp60c"rcN, was exclusively found in the infant cases is an entirely benign tumor which is included in the neuro blastoma tumor group in this presentation, since it is believed of neuroblastoma (9 of 12), estimated to have good prognosis. These results indicate that pp60c~*rcNmightbe a diagnostic marker in primitive to be the end stage of maturing neuroblastoma (30). For prog childhood tumors. When expressed in higher amounts than pp60c~"rc, nostic and therapeutic purposes neuroblastomas can be divided pp60' ~"Nmay be a positive prognostic marker in neuroblastoma, espe into clinical stages 1-4 and 4s according to the International cially useful in the evaluation of infants. In addition, lack of pp60c""cS Neuroblastoma Staging System (31). The stage 4s neuroblas seems to be incompatible with low stage neuroblastoma. tomas, although a disseminating disease, can spontaneously revert. Parallel to the disappearance of the multiple tumors, the remaining tissue differentiates from neuroblasts to mature non- INTRODUCTION proliferative cells resembling mature ganglion cells (32). pp60c"sr%the cellular homologue of the transforming gene Furthermore, the age of the patient is prognostically of great product pp60%srcof Rous sarcoma virus, is a tyrosine-specific importance: infants usually have low stage, or stage 4s disease, kinase (1-3) found at low levels in most cell types. High levels with good prognoses (28, 33). Histologically, the infant tumors of pp60c"srchave been found in neural tissues (4-9), platelets usually show poor differentiation at the time of diagnosis (28). (10), peripheral blood lymphocytes (10), monocytes (11, 12), In spite ofthat, most of these patients can be cured by nonrad- and chromaffin cells (13). In tumor cells, high expression of ical surgical excision of the tumor (34). Both localized infant pp60Â°"srchasbeen found in cultured neuroblastoma and small- tumors and widespread 4s tumors have shown remarkable cell lung carcinoma cell lines (14-16). High pp60csrt protein capacity for histolÃ³gica! maturation with time (32). A smaller and kinase activity levels have also been found in colon carci number of infants, however, have highly malignant tumors with nomas (17-20), rhabdomyosarcoma (17), and breast cancer (17, the same clinical findings and poor prognosis as the vast ma 21). jority of the older children with neuroblastoma (28, 33). In A substantial fraction of the src protein in tissues of the CNS' children after infancy, the histology of the tumor at the time of is structurally distinct from the protein expressed in non-neural diagnosis is of greater prognostic importance. In spite of mod tissues (7, 22). This neuronal form of pp60c srl, pp60c srcN,is ern highly aggressive therapy, the survival rates in this patient modified in the NH: terminus by addition of six extra amino category have not improved very much, and figures >40% for acids, due to alternative splicing of the c-src gene transcript (7, a 5-year survival are seldom seen (35-38). Approximately 20% 23, 24). Expression of pp60csrcN has, with the exception of all neuroblastomas have an amplified N-myc gene, which is of AtT20 mouse pituitary tumor cells (25), so far only been strongly correlated with rapid progression of the disease and found in neuronal cells. However, in the rat PNS, expression of pp60e"sri:Nappears to be considerably lower than in CNS (26). poor prognosis, as reviewed by Brodeur (39). A distinct but related group of tumors are the peripheral neuroblastomas or During fetal development different regions of the mouse brain neuroepitheliomas which are highly malignant tumors usually show varying src expression, both with respect to total src protein levels and the relative levels of pp60c"srcand pp60c"srcN located in peripheral nerves in the extremities of adolescents and young adults. Askin tumor is considered to be a variant (27). form, located in the chest wall. Clinically the term neuroblastoma is used for a group of In a previous study of human neuroblastoma and small-cell Received 4/30/90; accepted 8/2/90. lung carcinoma cell lines, we found a positive correlation among The costs of publication of this article were defrayed in part by the payment c-src protein level, c-src kinase activity, and neuronal/neuroen- of page charges. This article must therefore be hereby marked advertisement in docrine differentiation measured as neuron-specific enolase accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by The Swedish Cancer Society, The Children expression (15). We also found that the most highly differen Cancer Foundation of Sweden, HKH Kronprinsessan Lovisas fÃ¶reningfor bar- tiated neuroblastoma cell lines, which also had the highest src nasjukvard, Hans von Kantzows. and Ollie och Elof Ericssons stiftelser. 2To whom requests for reprints should be addressed, at Department of kinase activity levels, expressed both pp60c sri and pp60c'srcN. Pathology. University Hospital, S-751 85 Uppsala. Sweden. However, pp60c"srcNcould not be detected in the small-cell lung 3The abbreviations used are: CNS, central nervous system; PNS, peripheral nervous system; SDS, sodium dodecyl sulfate; PBS. phosphate-buffered saline; carcinoma cell lines with high src kinase activity. In this study, PAGE, polyacrylamide gel electrophoresis; Mab, monoclonal antibody. we have extended the analysis of human neuroblastoma and 6908

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. c-src EXPRESSION IN NEUROBLASTOMA neuroepithelioma cell lines and quantified the expression of Brugge, University of Pennsylvania, Philadelphia, PA) at a 1:100 pp60c srcand pp60c srcN.Furthermore, we analyzed specimens of dilution in PBS containing 0.1% gelatin. After five washings in PBS several neuronal human tumors, i.e., neuroblastoma, ganglio- with 0.1% gelatin, the filters were incubated at room temperature for 2 h with rabbit anti-mouse immunoglobulin antibodies (Dakopatts, Co neuroblastoma, ganglioneuroma, retinoblastoma, and Askin tu penhagen, Denmark) (1:200 dilution), followed by five washings as mor, as well as various other childhood tumors including Wilms' tumor, hepatoblastoma, rhabdomyosarcoma, pancreob- above. Goat anti-rabbit IgG (heavy and light chains) alkaline phosphatase-conjugated antibodies (BioRad Laboratories, Richmond, CA) di lastoma, and teratoma, for the expression of pp60c src and pp60c SIXN.Wealso examined the expression of pp60c srcNrela luted 1:3000 in PBS with 0.1% gelatin were incubated with the filters for 2 h at room temperature. Finally, the filters were washed five times tive to pp60csrc as well as the total c-src protein levels in and incubated with alkaline phosphatase color development reagents neuroblastomas and related these findings to age at diagnosis, nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate clinical stage, N-myc amplification, and prognosis to evaluate p-toluidine salt (BioRad) in 0.1 M NaHCO., and 1.0 mM MgCU, pH whether pp60csrcN could be used as a prognostic/diagnostic 9.8, until the bands were fully visible. In blots of tumor homogenates, pp60c "' and pp60c srcNwere identified by their molecular weights, using marker in this disease. the three cell lines SK-N-MC (only pp60c irc),SH-SY5Y (50% of each ire form), and LA-N-5 (80% of the neuronal form) (Fig. IÃŸ)ascontrols. Quantification of pp60c""' and pp60c'*"N in Immunoblots. To deter MATERIALS AND METHODS mine the proportion of pp60c "' and pp60c lrcNineach cell line the filters Patients. Specimens from primary tumors, immediately frozen in used for immunoblotting were, after development with alkaline phos liquid nitrogen or on dry ice at the time of surgery, were obtained from phatase color development reagents to detect the two forms of pp60s", 27 infants and children with neuroblastomas or ganglioneuroblastomas, incubated with I25l-labeled protein A (Radiochemical Centre, Amer- 8 children with ganglioneuromas, 3 with retinoblastomas, 6 with sham, England) (0.25 ^Ci/ml) for 2 h. The filters were washed five Wilms' tumors, 6 with other childhood tumors, and one adult with times in PBS, dried, and subjected to autoradiography. The bands corresponding to pp60'""' and pp60c srcNwere excised from the filters esthesioneuroblastoma. The neuroblastoma/ganglioneuroma speci mens represent a consecutive series and is taken from specimens col and the protein contents were quantified by gamma counting. Total lected in Sweden from the fall of 1986 to the summer of 1990. A pp60src levels in tumor specimens were quantified using the same detailed clinical description of this material will be presented else method, although these blots were not of the same quality as those with where.4 the cell line material and thus did not, in general, permit a reliable Cell Lines. Human neuroblastoma cell line SH-SY5Y, neuroepithe quantification of the two separate pp60src forms. We have therefore lioma cell line SK-N-MC (kindly provided by Dr. June Biedler, Sloan made a semiquantification by estimating visually which of the two Kettering Institute, New York), and neuroblastoma cell line IMR-32 forms was most abundant. For quantification of the total c-src protein (American Type Culture Collection. Rockville, MD) were routinely levels in tumor specimens, neuroblastoma cell line SH-SY5Y was used grown in Eagle's minimum essential medium supplemented with 10% as a standard and to verify that the method was linear for the detected fetal calf serum, penicillin (100 lU/ml), and streptomycin (50 ng/ml). ire protein levels (data not shown). The tumors were grouped into three Neuroepithelioma cell line Paju (kindly provided by Dr. Leif Anders- groups: those with pp60m levels similar to that of SH-SY5Y cells (++), son, Helsinki, Finland) and neuroblastoma cell lines LA-N-1, LA-N-2, those with lower but clearly measurable levels (+), and tumors with and LA-N-5 (kindly provided by Dr. Robert Seeger, University of pp60src levels that gave counts just above background (bg). In a repre California, Los Angeles) were grown in RPMI 1640 medium supple sentative experiment the following cpm intervals were obtained for each mented with fetal calf serum, penicillin, and streptomycin as above. group: ++, 1500-2500: +. 500-1500: bg, <500. Preparation of Tumor Tissue and Cultured Cells. All tumor samples were coded and analyzed without knowledge of the diagnosis. Small pieces (approximately 2x2x2 mm) of tumor tissue were sonicated in RESULTS 300 Mlof 10 HIMTris-HCl buffer, pH 7.2, containing 0.16 M NaCl, 1% (w/v) Triton X-100, 1% sodium deoxycholate, and 0.1% SDS supple Expression of pp60c"sriand pp60c""cNin Human Neuroblastoma mented with 1 m.MEDTA and 1 mM ethyleneglycol bis(/i-aminoethyl ether)-yV,7V,yV",A/'-tetraaceticacid. DNase I (2 /Â¿'ofl mg/ml) was added and Neuroepithelioma Cell Lines. Seven human neuroblastoma and neuroepithelioma cell lines were examined by immunoblot to each sample before sonication. The sonicated samples were centri- technique for the presence of pp60csrc and pp60csrcN (Fig. \A). fuged in an Eppendorf centrifuge for 10 min and the supernatant was By combining an enzymatic and a I25l-protein A detection recovered. Total supernatant protein (0.3 mg), as determined by a system we were able to quantify the proportions of pp60'SIXand modified Lowry procedure (40), was used in each blot. pp6f> S"Nin each cell line as well as the total c-src protein level. Cells from human neuroblastoma and neuroepithelioma cell lines were washed in PBS, harvested, and lysed in 300 ^1of 10 mM Tris-HCl The fibroblast form, pp60l"rc, could be detected in all cell lines buffer. pH 7.2, containing 0.16 M NaCl, 1% (w/v) Triton X-100, 1% analyzed, while the two neuroepithelioma cell lines, Paju and sodium deoxycholate, and 0.1% SDS supplemented with 1 mM EDTA SK-N-MC, lacked the neuronal form, pp60c 5rcN.As indicated and 1 mM ethyleneglycol bis(/i-aminoethyl ether)-A'.A',A'',A"-tetraace- in Fig. \A and seen in Fig. \B, the proportions of pp60csrt and tic acid buffer prior to homogenization. The samples were then treated pp60c srcNvaried in the tested neuroblastoma cell lines, with the as the tumor samples before subjecting to immunoblotting. Total highest and lowest levels of pp60c'SIVNfound in LA-N-5 and protein (0.5 mg) was used in each blot. IMR-32 cells, respectively. Furthermore, the five neuro Immunoblotting. Prior to SDS-PAGE, each sample was diluted 1:1 with 2x sample buffer containing 10% /3-mercaptoethanol and was then blastoma cell lines had higher total c-src protein levels than the boiled for 5 min. The material was subjected to SDS-PAGE according two neuroepithelioma cell lines (Fig. IB). The c-src kinase to the method of Laemmli (41) using a 7.5% polyacrylamide gel. The activity levels in the seven cell lines were also determined, proteins were electrophoretically blotted onto nitrocellulose filters. The according to methods described by Bjelfman et al. (42). With filters were blocked for 5 h at room temperature in 2% nonfat dried small variations between cell lines, the activity levels reflected milk (Semper, Stockholm, Sweden) dissolved in PBS containing 0.05% the amount of total c-src protein, indicating that there are no Tween-20. The filters were then incubated at 4Â°Covernight with the pronounced differences in specific c-src kinase activity in the anti-pp60src antibody Mab 327 (hybridoma kindly provided by Dr. Joan tested cell lines (data not shown), which is in agreement with 4 F. Hedborg. I. Johansson. P. Kogner. B-O. Samuelsson. A. Bekassy, A. data previously reported (16, 43). Three of the cell lines (SK- Kreuger. L. Olsen. and S. Pahlman. manuscript in preparation. N-MC, SH-SY5Y, and LA-N-5) were selected for their expres- 6909

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Fig. 1. A, analysis of pp60"rc and pp60c!rcN in seven human neuroblastoma and neuroepithelioma cell linos (SH-SY5Y, LA-N-I, LA-N-2, LA-N-5, IMR-32. SK-N-MC. and Paju). using immunoblot technique (see "Materials and Meth ods"). Total protein (0.5 mg) was used in each lane. After the src proteins were separated by SDS-PAGE, the proteins were blotted onto a nitrocellulose filter. 3 8 The filter was subjected to sequential incubation with Mab 327, rabbit anti-mouse immunoglobulin antibodies, and goat anti-rabbit IgG (heavy and light chains) alkaline phosphatase-conjugated antibodies. After development, the proteins were identified by their molecular weights by using prestained molecular weight mark ers. B, quantification of pp60clre and pp60c"cN in human neuroblastoma cell lines SH-SY5Y. LA-N-1, LA-N-2. LA-N-5. and IMR-32 and neuroepithelioma cell lines SK-N-MC and Paju using 1:'l-protein A as the detection system. The filter shown in A was incubated with 12'l-protein A and subjected to autoradiography pp60Â»rc_ to confirm a specific labeling of pp60c*reand pp60c"IN. Bands corresponding to these two proteins were excised from the filter and the '"I content of the filter pieces was determined in a gamma counter. Shaded columns, p60""; while columns, pp60c*reN. Fig. 2. A and B, analysis of pp60c *" and pp60c ""N in human tumor biopsies, sion profile of ppoO1'""1'and pp60csrcN (Fig. \B) to serve as using immunoblot technique. The human neuroblastoma cell lines SH-SY5Y and LA-N-5 and neuroepithelioma cell line SK-N-MC were used as controls. Tumor controls for detection of the two proteins in tumor specimens. biopsies identified by numbers (abscissa) were prepared as described in "Materials Expression of pp60'"src and pp60c""'s in Human Childhood and Methods" and were subjected to SDS-PAGE. Approximately 0.3 mg of total Tumors. Specimens from neuroblastoma, ganglioneuroblas- protein was used in each lane. Immunoblotting was performed as described in toma, ganglioneuroma, retinoblastoma, Askin tumor, and es- Fig. I. C, only SH-SY5Y cells used as controls. thesioneuroblastoma were examined for the expression of pp60c"rc and pp60c"sri'N(Fig. 2). pp60c"rc was detected in most tomas/neuroepitheliomas (44), the lack of ppoO1"""1"*in the tumor specimens studied, while the neuronal form, pp60c'srcN, analyzed case is in accordance with the c-src expression in the was restricted to neuroblastoma, ganglioneuroblastoma, and two tested neuroepithelioma cell lines mentioned above. retinoblastoma. In the neuroblastoma group, 23 of 27 samples We also analyzed some childhood tumors of non-neuronal tested had detectable pp60csrcN levels (Table 1). All three reti- origin for the expression of pp60'"rc and pp60c"srcN(Fig. 2). This noblastomas analyzed expressed both forms of the c-src protein, group consisted of Wilms' tumors, teratomas, hepatoblastoma, although the neuronal form was considerably more abundant rhabdomyosarcoma, and pancreoblastoma. pp60csrc was de tected in all but one, but no tumor expressed pp60c"srcN(Table (Table 2). Interestingly, an esthesioneuroblastoma from an adult (Table 2) and all 6 ganglioneuromas with detectable levels 3). of c-src protein expressed only pp60c~srcandnot pp60c srcN(Table Expression of pp60c"srcand pp60c"srcNin Neuroblastomas Re 1). Also, an Askin tumor (chest wall location and positive for lated to Age, Stage, N-m>'c Amplification and Prognosis. Expres neuron-specific enolase) showed the same pattern (Table 2). sion of pp60c srcNin normal, nontransformed cells is restricted Because an Askin tumor is a subgroup of peripheral neuroblas- to neuroblasts and neurons and is still highly expressed in 6910

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. c-src EXPRESSION IN NEUROBLASTOMA Table 1 pp60"" and ppoO""'' expression in neuroblastoma, ganglioneuroblastoma, and ganglioneuroma specimens

at PatientsInfants diagn.a4 time37 stageNB-2bNB-2aNB-2bNB-2bNBcomment1 amplification pp60"cN>cc level++++++++++++++++++++++++++++++++++++bg++N (si 8mos)Alive444551586066787984858788103Dead

mo-2 mo38 +cyt.Surg.Surg.Surg. relapsePrematureSpinalintraspinal mo3 mo34 =NN>cN>cN>cONc mo0 mo26 compressionLocal mo2 mo25 x3Surg.Surg.Surg. 24mos.Extensiverelapses at 10 and

mo4 mo17 1NB-1NB-3NB4NB-3NB-3NB-2bNB-1NB-2aNB-4SNB-4NB-4GNB-3GNB-3NB-4NB-4GNB-4NB-4NB-4NB-4GNB-4NB-4NB-3GNGNGNGNGNGNGNGNTreatmentSurg. mo12 mo15 mo17 mo36 rad.Surg.+ cyt. + treatmentExtensivecytostatic NONN>cN>cN>cN>cN>cN>clOx= mo3 mo4 +cyt.Surg. treatment.1cytostatic plannedABMTGoodskeletal relapse,

mo1 mo4 +cyt.Surg. cyt.treatm.Goodresponse to mild

mo18 mo4 +cyt.Surg. cyt.treatm.Goodresponse to mild

mo16 mo8 +cyt.Surg.Surg.Surg. cyt.treatm.Respiratory,response to mild

mo0 mo1 mo0 mo6wk1 ofdisease395255Noninfants mo5 rad.Cyt.+ cyt. + hepaticfailureAbd.renal,

mo10 DDODNEDRelapseRes.1mo4 +surg.Cyt. b.m.Abd.turn. + ON40x mo8yr6yr26m6yr23 mo25 +surg.Cyt. skel.Accidentalturn. 4- b.m. + NONON50xc > mos)Alive618995Dead(>18

mo9 +surg.Cyt. totraumaQuickdiagn. due

mo10 +surg.Cyt. relapseRecent disDODDODDODDODDODDODDODDODNEDNEDNEDNEDNEDNEDNEDNEDFollow-upmo12 +surg.Cyt. surg.No c =Nc ofdisease3238414849566869Ganglioneuroma3740535965737780Age mo31 x2Cyt.+ surg. remissionLate =NCc mo6.5 mo8 +ABMTSurg.+ surg. relapseNo

yr4.5 mo9 +cyt.Cyt. remissionNo N40X = vr7yr6yr3.5 mo7 +surg.Cyt. remissionQuick NDcc70x Dbgbg+++bg++N mo28 +ABMTCyt.+ surg. relapseLate

mo7 +surg.Cyt. relapseNo yrSyr5.5 mo17 +surg.Cyt. remissionLate c>Nc mo42 x3Surg.Surg.Surg.Surg.Cyt.+ surg. relapseMisdiagn. =NccCCNDccNDProt.

yr8yrlOyr11 mo39 mo34 mo25 yr6 mo24 yr29 mo15 +surg.Surg.Surg.Cyt. turn.shrink,initially, no DbghgND cyt.Pretreatm.on mo4yr15 mo1 1mo20 moPrognosisNEDNEDNEDRelapseNEDNEDNEDRem.OITOITOITNEDNEDDODDOmoDiagn. + surg.Clinical diagn.: infant NBN-myc " diagn.. diagnosis; prot. level, total c-Ãrc-proteinlevel, value for SH-SY5Y set as ++ (see "Materials and Methods"); NED, no evidence of disease, off treatment; NB. neuroblastoma: surg., surgery : cyt., cytostatic treatment; N, pp60"rcN; c. pp60Â°"":rad., radiation: rem., remission: ABMT. autologous bone marrow transplantation: OIT. on initial treatment: treatm., treatment; DOD, dead of disease: abd. turn., abdominal tumor: b.m.. bone marrow infiltration: skel., skeletal mÃ©tastases;GNB, ganglioneuroblastoma; res. dis., residual disease; bg, background level; ND, not detectable; GN, ganglioneuroma: turn, shrink., tumor shrinkage.

Table 2 pp6ff'tre and ppoff '"* expression in retinoblastoma, Table 3 pp60"" expression in childhood tumors of non-neuronal derivation esthesioneuroblastoma, and Askin tumor specimens Patient5057626370861314161718Age0 level"++bgbg+â€¢+ND++bgbg Patient12354DiagnosisENB*RBRBRBAskin level"NTMNTM-1- mo23 yr2 mo7 mo1 yr3yrSyr9 tumorpp60mcN>cN>cN>ccProtein tumorWilms' tumorWilms' Â°Total c-Ãrc-proteinlevel, value for SH-SY5Y set as ++ (see "Materials and DcCCCProtein tumorWilms' Methods"). mo4 tumorWilms' * ENB, esthesioneuroblastoma; RB, retinoblastoma; c, pp60c '"; NT, not tested; yr3yr3yrDiagnosisTeratomaPancreoblastomaRhabdomyosarcomaHepatoblastomaTeratomaWilms' tumorWilms' N. tumorpp60"ccÂ»ccCCCN " Total c-src protein level; value for SH-SY5Y set as ++ (see "Materials and terminally differentiated, postmitotic CNS neurons (7), which Methods"). indicates that pp60csrcN is a late neuronal differentiation *c, ppoO"*"; bg, background level; ND. not detectable. marker. Since the grade of differentiation in neuroblastoma is of prognostic importance, pp601'srcNexpression could be a po fore estimated and compared to three important prognostic tential prognostic marker for this tumor. The ratio between parameters: age at diagnosis, clinical stage, and N-myc ampli pp60c srcand pp60c srcNin the neuroblastoma tissues was there- fication. 691l

Thirty-five neuroblastomas, ganglioneuroblastomas, and blastomas have an excellent prognosis. It is noteworthy that ganglioneuromas were tested for the expression of pp60c"rc and most of these tumors, in spite of their primitive morphology, pp6(TsrcN. pp60csrc could be detected in 32 samples and had the highest expression of pp60'"rcN, since expression of this pp60csrcN in 23 (Table 1). Twelve tumors were obtained from protein is a putative sign of neuronal differentiation. In addi infants with stages 1-3 neuroblastomas. Although the obser tion, these tumors had high total src protein levels. Three vation time was short in some cases, all of these patients were infants with recently diagnosed disease, one with stage 2b (case estimated to have a favorable outcome. All specimens contained 87) and two with stage 3 disease (case 84 and 85), have in this both forms of c-src protein and 9 tumors expressed more of the presentation also been considered to have good prognosis. neuronal form than pp60csrc. Only one patient was considered These patients also had high expression of pp60c srcNand high to be at high risk and, accordingly, was given extensive treat total src protein levels. Prognostic evaluation of such patients ment (case 78). This tumor expressed the two forms in equal can be difficult however, and if the estimated outcome will turn amounts. Three additional infants (cases 52, 55, and 79) had out to be correct, these patients could exemplify a clinical highly malignant, stage 4 disease. All of these tumors expressed applicability of pp60c"srcNas a prognostic parameter. The three less pp60c srcNthan pp60c~src.Two have died and both had an cases of neuroblastomas expressing only pp60'sro represented amplified N-myc gene. Another infant also died of her disease highly malignant tumors of older children lacking N-myc am (case 39). This tumor was of the 4s type with massive congenital plification. Thus, these results indicate that pp60c sr'Ncould be spread in the abdomen, skin, meninges, and bone marrow. used as a diagnostic marker for neuroblastoma. When expressed Death occurred soon after birth and was caused by multiple to a higher extent than pp60c src,pp60c "rcNcould also be used organ (respiratory, hepatic, renal) failure due to the heavy tumor as a favorable prognostic marker for these patients. burden. This tumor expressed pp60c~srcNina pattern very similar Stage 4s neuroblastoma is an intriguing nosological entity. to that of the neuroblastoma cell line LA-N-5, which shows a In general, this disease is characterized by good prognosis and clear dominance of this form over pp60CSIX(Table 1; Fig. 2A). spontaneous clinical remission, often paralleled by neuronal All three specimens in which only pp60c srccould be detected differentiation of the tumor cells. However, in some cases an (cases 38, 49, and 56) were from highly malignant tumors of overwhelming tumor growth may lead to fatal respiratory prob stage 4 in children after infancy. All three patients have died of lems, as in the 4s case presented here. This tumor, while lethal the disease. These three tumors did not have an amplified N- due to concurrent respiratory problems, expressed predomi myc gene. Although being highly malignant stage 4 tumors, nantly the neuronal form pp60c srcN.This observation supports they could not be classified as the most aggressive tumors in the notion that disturbances in growth and differentiation prop this group. Morphologically, the degree of differentiation varied erties in stage 4s neuroblastomas are different from, and less from ganglioneuroblastoma (two cases) to undifferentiated profound than, the disturbances seen in true stage 4 neuro neuroblastoma (one case). The remaining malignant tumors in blastoma tumors. older children expressed both forms of c-src protein in either Notably, none of the four highly aggressive infant tumors equal amounts (four cases) or more of the pp60c srcform (three (cases 52, 55, 78, and 79) had predominantly the neuronal form cases). Three of four tumors with N-myc amplification and of the src protein. Two of the patients have died and both of detectable c-src protein levels (cases 52, 55, and 68) expressed these had genetic evidence of truly aggressive tumors (N-myc more pp60c srcthan pp60csrcN. The fourth case expressed the amplification and homogeneously staining regions.4) None of two forms in equal amounts (case 95) (Table 1). The specimen the other two tumors had an amplified N-myc gene but were from the fifth case (case 48) with N-myc amplification was nevertheless considered highly aggressive based on clinical pa highly necrotic, which probably explained why no pp60c"src rameters. One patient is considered cured after extensive treat protein could be detected. pp60c"''r';Ncould not be detected in ment. The other patient is presently in remission and awaiting any of the 8 ganglioneuromas tested, while pp60'Slx was found an autologous bone marrow transplant treatment in the near in 6 of the tumors (Table 1). future. Analysis of the expression of the two forms of the src The immunoblots performed on tumor specimens did not gene product therefore seems to give additional support to the permit a reliable quantification of the proportion of the two c- prognostic classification of infant neuroblastoma. This could src proteins by I25l-protein A labeling of the filters. However, also be exemplified by case 52. The patient presented at the age we were able to semiquantify the total amount of c-src protein of 5 months with an abdominal tumor and bone marrow infil in most tumor samples, using the pp60csrc level in the SH- tration but no skeletal mÃ©tastases.Initially, it might therefore SY5Y cell line as a standard (Table 1). These results indicated have been uncertain whether the tumor stage should have been that the neuroblastomas expressing both forms of c-src protein classified as 4s with fair prognosis or as stage 4 with much had the highest total c-src protein content. Furthermore, the worse prognosis. In this case, however, both N-myc and src infant neuroblastomas had more protein than most of the protein data pointed in the latter direction. tumors of older children. Ganglioneuromas, the non-neuronal Despite the short observation time for some patients in the childhood tumors, and neuroblastomas expressing only noninfant age group, all but one seemed to have a poor prog pp60l srchad the lowest levels of the protein (Tables 1 and Table nosis including the three with tumors lacking detectable expres 3). sion of pp60c srcN(cases 38, 49, and 56). Although lack of expression of pp60c 5lxNtheoretically would indicate poor neu ronal differentiation, the histology of these tumors varied, two DISCUSSION being ganglioneuroblastomas and one neuroblastoma. Clini In this study we show that the expression of pp60csrcN is cally, disease has progressed in the three patients at varying restricted to the neuroblastoma group of tumors and retinob- rates, only one of them (case 49) being among the most aggres lastomas, while pp60c"srccould be detected in most neuronal sive cases. Thus, within this age group, pp60c 5rcNexpression in and non-neuronal tumors tested. The study results further the tumors varies considerably and seems to add little infor supports the common observation that low stage infant neuro mation in terms of prognostic evaluation. 6912

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. C-m EXI'RKSSION IN NEI ROBI.ASTOMA The benign tumor, ganglioneuroma, expressed only pp6f/src. the entire pp60csrcN population is available. In order to more Since our results in the other neuroblastomas indicated that firmly establish a clinical use of pp60" srcN-diagnostics, methods presence of pp60c~srcNcould be related to positive outcome and for more accurate quantification of pp60c "rcNin tumor speci- that postmitotic neurons still express this protein, one could expect ganglioneuromas to express pp60c~srcN.One explanation for the lack of detectable levels of pp60c srcNin these tumors ACKNOWLEDGMENTS could be a matter of concentration, especially if the analyzed specimens contained few ganglion cells and predominantly glial We thank Dr. Per Kogner (Stockholm, Sweden), Dr. Bengt-Olov cells. However, this is probably not the only explanation, since Samuelsson (GÃ¶teborg,Sweden), Dr. Albert Bekassy (Lund, Sweden), histopathological examination revealed that some of these tu and their colleagues at the Departments of Pediatrics, Pediatrie Sur mors contained almost only ganglion cells (data not shown). gery, and Pathology for assistance in collecting tumor specimens and Furthermore, in the rat PNS, expression of pp60c"s'cNis consid supplying clinical data. erably lower than in the CNS and in some ganglions not even detectable (26). In the CNS the expression of pp60c~srcN(and pp60c"src)is developmentally regulated and, for instance in the REFERENCES midbrain of mouse, pp60csrcN shows a drastic decrease in 1. Hunter, T.. and Sefton, B. M. Transforming gene product of Rous sarcoma expression relative to pp60c~srcduring the postnatal period (27). virus phosphon laies tyrosine. Proc. Nati. Acad. Sci. USA, 77: 1311-1315, 1980. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1990 American Association for Cancer Research. Expression of the Neuronal Form of pp60c-src in Neuroblastoma in Relation to Clinical Stage and Prognosis

Catarina Bjelfman, Fredrik Hedborg, Irja Johansson, et al.

Cancer Res 1990;50:6908-6914.

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