Differential Expression of Pleiotrophin and Midkine in Advanced Neuroblastomas1

Home , Pleiotrophin

(CANCER RESEARCH 55. 1792-1797. April 15. !995| Differential Expression of Pleiotrophin and Midkine in Advanced Neuroblastomas1

Akira Nakagawara,2 Jeffrey Milbrandt, Takashi Muramatsu, Thomas F. Deuel, Huaqing Zhao, Avital Cnaan, and Garrett M. Brodeur2

Division of Oncology [A. N.. G. M. B.j and Division f/BtostatÃ¬stÃ¬csund Epidemiology Â¡H.Z., A. C.ÃŒ,TheChildren's Hospital of Philadelphia, Philadelphia. Pennsylvania 19104; Departments of Pathology and Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110 [J. M.Â¡;Department of Biochemistry, Nagoya University School of Medicine. Nago\a 461. Japan IT. M.Â¡:and Departments of Medicine and Biochemistry and Molecular Biophvsics, Jewish Hospital at Washington Universitv Medical Center. St. Louis. Missouri 63110 Â¡T.F. 1).I

ABSTRACT neurite outgrowth in an autocrine or paracrine manner (7, 8). To date, there are no published reports on TRK-C or NT-3 expression Pleiotrophin (PTN) and midkine (MK) are members of a new family of neurotrophic factors whose expression is developmental!}1 regulated. PTN in neuroblastomas. MK and PTN are members of a new family of neurotrophic factors, also transforms NIH 3T3 cells, and MK is mitogenic to certain cell lines. whose expression is developmentally regulated (12-16). These hep- Neuroblastomas are tumors derived from neural crest cells, and recent arin-binding proteins have about 50% homology and no sequence studies have revealed that the biology of these tumors is at least partly regulated by neurotrophic factors and their receptors. To examine the homology with other heparin-binding proteins, such as basic and expression of PTN and M K in neuroblastoma, we analyzed their mRNA acidic fibroblast growth factors (12-16). MK is a 13-kDa molecule expression in 72 primary neuroblastomas and 11 neuroblastoma cell lines and is rich in basic amino acids and cysteine (17, 18). The gene was as well as other tissues and cell lines. PTN is highly expressed in favorable originally cloned by a differential hybridization procedure from the neuroblastomas (stages I, II, and IV-S, n = 44), whereas it is expressed at embryonal carcinoma cells which were induced to neuronal differen a significantly lower level in advanced tumors (stages III and IV, n = 28, tiation by treatment with retinoic acid (12). PTN (also known as P = 0.003). PTN is not expressed in either aggressive neuroblastomas with HB-GAM) has a molecular mass of 17 kDa and was purified inde N-iwvc amplification or in neuroblastoma cell lines. Moreover, the expres sion pattern of PTN was similar to that of TRK-A, the high affinity pendently from developing rat brain (14) and bovine uterus (15). MK is mitogenic to some cell lines (19-21) and PTN transforms NIH 3T3 receptor for nerve growth factor, in that it is correlated with a favorable prognosis (/' < 0.004). In contrast, MK is highly expressed in almost all fibroblasts (22). However, both MK and PTN act on embryonic brain primary neuroblastomas and cell lines and showed no correlation with cells and spinal ganglia as a neurotrophic factor to promote survival disease stage or N-rnyc amplification. These results suggest that differen and induce neurite outgrowth (16, 20, 21). tial expression of PTN and MK may have an important role in regulating Here we report that MK and PTN are expressed in many primary growth and differentiation of neuroblastomas. neuroblastomas, but their patterns of expression are quite different. MK mRNA is expressed in essentially all primary neuroblastomas and cell lines, independent of disease stage. In contrast, PTN mRNA is INTRODUCTION preferentially expressed in favorable stage neuroblastomas and those lacking N-myc amplification. This correlation between PTN and Neuroblastoma is one of the most common pediatrie neoplasms and originates from the sympathoadrenal lineage of the neural crest. favorable outcome suggests that it may be playing a biological role in Tumors in infants and young children have a tendency to spontane the behavior of these tumors. ously regress or to differentiate (1). However, tumors that occur in older children are frequently metastatic at diagnosis, and these children have a poor prognosis. MATERIALS AND METHODS Recently, neurotrophic factors and their receptors were found to Patients. Of 72 neuroblastomas and 5 ganglioneuromas, 63 were obtained play an important role in regulating growth, differentiation, and cell from Japanese patients and 14 from patients at Pediatrie Oncology Group death in neuroblastoma (2-8). The members of the TRK family of institutions or others in the United States. Twenty-five Japanese patients were protein-tyrosine kinases have been identified as the genes encoding identified by a mass screening program that began in 1985. Neuroblastoma the functional receptors that mediate the trophic properties of the tissues were obtained from the primary tumors of 55 untreated and 17 pre- NGF1 family of neurotrophins (9-11). In neuroblastoma. treated patients (one or two courses of high-dose cyclophosphamide and/or gpl40TRK~A, the primary receptor for NGF, is expressed in most cisplatin; Ret". 23). All diagnoses of neuroblastoma were confirmed by histo- favorable tumors (2-6, 8). Furthermore, it promotes neural differ logical assessment of a surgically resected tumor specimen. Patients were entiation in the presence of NGF and programmed cell death in its treated according to previously described protocols (3). The tumors were staged according to the system of Evans et al. (24). The median follow-up absence (3). On the other hand, aggressive tumors, especially those period after diagnosis was 32 (range, 8-116) months. Among the 17 treated with N-/TÃ•VTamplification, express very low levels of TRK-A patients, 15 had stage 111or IV disease and two had stage IV-S. Thus, there was mRNA and they usually are unresponsive to NGF (2. 3). Such a high level of confounding between treatment and stage. There were 55 tumors generally express both functional TRK-B receptor and its neuroblastomas, 17 ganglioneuroblastomas. and 5 ganglioneuromas. Neuro preferred ligand. BDNF. which promotes survival and induces blastomas and ganglioneuroblastomas were combined and analyzed as neuro blastomas. Unless stated otherwise, the five ganglioneuromas were examined Received 10/11/94; accepted 2/16/95. separately. The costs of publication of this article were defrayed in part by the payment of page Tumor Specimens and Cell Lines. The tumor specimens were obtained charges. This article must therefore be hereby marked advertisement in accordance with from the primary tumors. The normal adrenal gland was resected with an 18 U.S.C. Section 1734 solely to indicate this faci. 1This work was supported in part by Ronald McDonald Children's Charities ipsilateral neuroblastoma from a 1-year-old patient. The normal kidney tissue was obtained from an area adjacent to Wilms' tumor tissue from a 5-year-old (G. M. B.. J. D. M.), NIH Grants CA39771 and CA05887 (G. M. B.). the Mental Retardation Research Center Grant HD26979-0452. (H. Z.. A. C.. G. M. B.). and the patient. The surgical specimens were immediately frozen and stored at â€”70Â°C University of Pennsylvania Cancer Center Core Grant CAI6520 (H. Z.. A. C.). until used. Eleven human neuroblastoma cell lines (NGP. SMS-KAN, SMS- ~ To whom requests for reprints should be addressed, at Division of Oncology, The Children's Hospital of Philadelphia. 324 South 34th Street. Philadelphia. PA 19104. SAN, NMB, SMS-KCN, NLF, LA-N-6, SK-N-SH, SH-SY5Y, SMS-LHN, and 1The abbreviations used arc: NGF. nerve growth factor: BDNF. brain-derived NBL-S), the A875 human melanoma cell line, and the PC 12 rat pheochromo- neurotrophic factor: MK. midkine: PTN. pleiotrophin: du. density units. cytoma cell line have been described before (7). 1792

PTN - 1.5 kb

MK â€¢â€¢â€¢â€¢â€¢â€¢ - 1.0 kb

Fig. 1. Expression of PTN and MK mRNA in primary neuroblastomas and neuroblastoma cell lines. Northern blot hybridization was performed ÃŸ-actin as described in "Materials and Methods." PTN IIMIIIIII i* and MK are expressed differentially in advanced tumors and cell lines.

N-myc ampi. ------+ +

Stage II IVS III IV IV IVS

Southern Blot and Northern Blot Analyses. The cDNA probes used were Kodak NTB-2 emulsion. The sections were observed under the light micro a 487-base pair human MK cDNA fragment (from T. M.; Ref. 18) and a scope with dark-field condensers. 2.4-kilobase human PTN cDNA (from T. F. D.; Ref. 25). There was no Statistics. Both PTN and MK mRNA expression had skewed distributions. cross-reactivity between the probes under the conditions used in this study. The Therefore all analyses were done either on the natural logarithm of the density human N-myc probe was a gift from J. Michael Bishop (26). N-wiyc amplifi unit scale (r tests, ANOVAs) or using nonparametric statistics. The distinction cation was measured by Southern blot analysis (26). Total RNA was prepared between high and low levels of expression of PTN and MK was based on the and the mRNA level was determined by Northern blot analysis, as described value on the histograms that gave the best natural separation (data not shown), previously (2, 3). regardless of stage, N-myc amplification, or survival. Kaplan-Meier survival All bands of N-myc DNA, as well as MK, PTN, and /3-actin RNA, were curves were calculated, and survival distributions were compared using the log analyzed by scanning the autoradiographs on an Apple Scanner. One RNA rank test. Cox regression models were used to explore associations between sample was analyzed for PTN expression but then degraded and could not be PTN and MK expression and survival, as well as to explore the prognostic analyzed for MK expression. The intensity of mRNA expression was measured value of each in the presence of other prognostic variables such as N-myc by a computer-based densitometry program (Densitometer-on-a-Disc; compli amplification. ments of IMAGENETICS and AMOCO Technology Co., Naperville, IL). mRNA expression of MK and PTN was normalized to ÃŸ-actinand expressed as arbitrary du. Thus, the results for expression of the two neurotrophins, and RESULTS between the primary tumors, cell lines, and tissues, can be directly compared. A level of <1() du indicates that a weak band was barely visible, as opposed to Expression of PTN and MK in Neuroblastomas and Other no band at all, but the precise level of expression was at the limits of detection Tissues. Examples of the mRNA expression of PTN and MK are over background and could not be quantitated accurately. shown in Fig. 1, and the distribution of expression by stage and N-myc In Situ Hybridization. In situ hybridization procedures were based on a amplification is shown in Table 1. Because they were skewed, Table previous report (27) with some modifications. Slides were equilibrated to room 1 gives both summary nonparametric statistics and transformed after temperature, and the sections were fixed in 4% paraformaldehyde in 0.1 M normalization values. For one patient, there were no MK values sodium phosphate buffer for 20 min. After washing with PBS. the sections available. The distribution based on the histogram of all of the values were treated with 10 fig/ml proteinase K in 50 mM Tris-HCl-5 mM EDTA of both PTN and MK expression was bimodal, and a cutoff value of (EDTA, pH 8.0) for 7.5 min at room temperature. They were postfixed in 4% 150 du completely separates the high and low values (Fig. 2). An paraformaldehyde, acetylated with acetic anhydride in 0.1 M triethanolamine, rinsed with PBS, dehydrated in an ascending alcohol series, and air dried. The overall ANOVA test shows that PTN levels were different in the sections were hybridized for 16 h with 35S-labeled RNA probes (antisense or different stages (P = 0.0125). In particular, there was a significant sense) at a concentration of 5 X IO5 cpm/75 fil/slide, as described previously difference between the PTN expressions in the early stages (median, (27). After hybridization, the slides were washed, air dried, and coated with 103; IQR 16-579) and advanced stages (median, 14; IQR 7-29;

Table 1 Distribution of PTN and MK expression by stage and N-myc amplification

expressionMean mRNAMedian

(IQR") (95% CI*) (IQR) (95% CI) CategoryStage (No.)19 du189(15-730) du1 du1197(52-2331) du385(147-1006) I 12 (43-286) Stage II 13 67 (24-122) 64(13-322) 487(38-1663) 271 (75-981) Stage IV-S 12 103(19-640) 105 (32-339) 1505(179-1899) 568(170-1903) Stage III 14C 15 (9-199) 18 (3-99) 574 (54-2094) 364(116-1149) Stage IV 14 13(6-25) 8 (2-32) 895(70-3169) 573(186-1760) Stage I + II + IV-S 44 103(16-579) 93 (48-177) 1269(48-2091) 386(211-705) Stage III + IV 28e 14(7-29) 12(4-34) 574 (54-2384) 461 (217-977) N-myc amplification (-) 63 65 (14-484) 56(29-105) 713 (43-2094) 360 (220-59(1) N-myc amplification (+) 9e 9(6-12) 6(1-23) 2137(527-3910) 1201 (338-4266) GanglioneuromasPatients 5PTNMedian 368 (273-408)mRNA 220(27-1814)MK 78(50-107)expressionMean 100(22^151) " IQR.*interquartile ranges, i.e. , 25th and 75th percentilcs; CI, confidence interval, ' Mean and 95% CI based on log scale values transformed back to du. ' One patient's MK data were not available. 1793

10,000 MK was expressed at a very high level in almost all neuroblasto mas. An overall ANOVA showed no significant differences between 5,000 the stages, both overall and grouped as early versus advanced stages. The tumors with N-myc amplification appeared to have higher MK expression; however, the difference did not achieve significance "ST 2,000 (P = 0.10). Ganglioneuromas had relatively low MK expression. All 11 neuroblastoma cell lines expressed very high levels of MK mRNA compared to primary tumors. For comparison, we analyzed other cell lines and tissues (Table 2). The rat pheochromocytoma cell line PC12 is known to express the Â°Â° _ o TRK-A receptor and is responsive to NGF. It expressed a low level of OO O PTN, but it did not express MK at all. The melanoma cell line A875 expressed neither PTN nor MK. The infant adrenal tissue expressed a low level of both PTN and MK. Normal kidney tissue from a child oÂ°o o showed a high level of PTN mRNA, but not MK. Two Wilms' tumors expressed high levels of both PTN and MK transcripts. In three 50 hepatoblastomas and three primitive neuroectodermal tumors, the expression pattern of PTN and MK was variable (Table 2).

O O Â° Survival and Expression of PTN and MK. Higher PTN was 20 Q associated with prolonged survival, as expected, based on the differ OÃ’O OO OM OBB OÂ«Â» ence in PTN among the stages of disease (Fig. 3A). The group with OB OB PTN expression of more than 150 du (n = 26) had a 5-year survival l II \VS III IV GN rate of 93%, while the group with PTN expression of less than 150 du (n = 46) had a 5-year survival rate of 43% (P = 0.0023, log rank test). Stage PTN expression was associated with survival in a Cox regression model (P < 0.0005). MK expression was also associated with a 100005,0002,0001,00050020010050200*o difference in survival (Fig. 3ÃŸ).The group with MK expression higher than 150 du (n = 45) had a 55% survival rate at 5 years, while â€¢Â° the group with MK expression lower than 150 du (n = 26) had a 92% *^ â€¢ survival rate at 5 years (P = 0.031). MK expression was also *OÃ§)O Q associated with survival in a Cox regression model (P = 0.029). Â° OOâ€¢8 *OB The most significant prognostic factor for survival was N-wryc oo.o0 8 Â£Â§Â£ amplification (P < 0.0005). All nine patients with more than one copy died, as opposed to 8 of 68 patients whose tumors had one copy per *00 haploid genome. TRK-A expression which had been analyzed previ 0o0 ously (3) was strongly prognostic (P < 0.0005). Also, patient age was â€¢0â€”o prognostic, with younger patients faring substantially better (P = 0.011). Finally, patients with favorable stages (I, II, and IV-S)

oo Table 2 Expression of MK and PTN in cell lines and various tissues Â°. â€¢ <& oog8 00oo0 0 0 tissuesCellCell lines and (du)32029503550582061202650440420049903880207000<10<101310960130700<101020460<10expressionPTN(du)00<100000<10<10<10<1021011430640250140080<10<10390 o0 linesNeuroblastomaNeuroblastomaNeuroblastomaNeuroblastomaNeuroblastomaNeuroblastomaNeuroblastomaNeuroblastomaNeuroblastomaNeuroblastomaNeuroblastomaPheochromocytomaMelanomaNormal 0 00f ,.

IVS III IV QN

Fig. 2. Densitometric analysis of expression of PTN and MK in mRNA in tumor samples from 72 and 71 patients with neuroblastomas, respectively, according to disease stage. Data obtained from five ganglioneuromas (GN) are also shown. The levels of expression of PTN and MK were normalized to that of ÃŸ-actinand measured in arbitrary du. Horizontal lines, group means; O, and â€¢,patients whose tumor samples had no tissuesNormaland tumor amplification of N-myc; D, and â€¢patients whose tumor samples had N-myc amplication; (infant)Normaladrenal gland â€¢and â€¢patients who have died. (child)Wilms'kidney tumorWilms' tumorHepatoblastomaHepatobtastomaHepatoblastomaPrimitive P = 0.0006). The patients with ganglioneuromas had high levels of expression. In addition, expression of PTN in nine neuroblastomas with N-m_yc amplification was very low as compared with tumors without N-myc amplification (P = 0.011). All 11 neuroblastoma cell tumorPrimitiveneuroectodermal tumorPrimitiveneuroectodermal lines, which had been established from aggressive tumors, also neuroectodermal tumorNGP"SMS-KAN"SMS-SAN"NMB"SMS-KCN"NLF"LA-N-6SK-N-SHSH-SY5YSMS-LHNNBL-SPC12A875W-lW-2H-lH-2H-3P-lP-2P-3mRNAMK showed very low or absent expression of PTN (Table 2). " Neuroblastoma cell line with N-nryc amplification. 1794

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1995 American Association for Cancer Research. DIFFERENTIAL EXPRESSION OF PTN AND MK IN NEUROBLASTOMAS AgÃ PTN(n=26)II High separately, was not a significant predictor once any strong predictor I lili !Il was in the model. â€¢/i ,93%l," II l n o In Situ Hybridization of PTN. A neuroblastoma tissue sample |,il was obtained from the adrenal primary of a 1-day-old girl with stage '' ' II neuroblastoma. The tumor sample was frozen and later processed to il, Low PTN(n = 46) analyze the in vivo expression pattern of PTN. As shown in Fig. 44, n Sn 1p= the antisense PTN probe hybridized to almost all tumor cells in the oCL'Sâ€¢|5100n75-50-25-n-; cluster. A control sense probe did not hybridize at all (data not shown). Fig. 43 shows the result of PTN in situ hybridization in an

.002

O 2 4 6 8 10 Years since Diagnosis

Bg1ooÃSurvival100-75-50-25-26)"TU MK(n = l III "tjU92%<-ÃŽLM. liULUJL L l.-.Ul LiLL J

'!',I H i II I ilL i High MK(n =45)J

K C0Â¿p

= .03 n -Low 10 Years since Diagnosis

Fig. 3. Cumulative survival curves of patients with neuroblastoma, according to expression of PTN (A, n = 72) and MK (B, n = 71). The Kaplan-Meier curves show the probability of survival in terms of the level of expression of PTN and MK. High levels of PTN expression were defined as values >150 du and low levels as values <150 du. High levels of MK expression were defined as values >150 du and low levels as values <150 du. had a better prognosis than those with advanced stages (P < 0.0005), but histology was not prognostic, and prior treatment was not either, once stage was accounted for. PTN expression was prognostic even in the presence of other prognostic variables. Table 3 gives several Cox regression models Fig. 4. In situ PTN mRNA expression in primary neuroblasÃ®omas.A, dark-field with PTN and other prognostic variables as well as MK expression photomicrograph of a section of stage II neuroblastoma hybridized in situ with a 35S- and other prognostic variables. Table 3 shows that PTN expression labeled antisense PTN RNA probe (nuclei counterstained). Strong hybridization signals were found uniformly over almost all tumor cells in a cluster. This tumor expressed a high remained prognostic even in the presence of N-myc amplification, and level of PTN mRNA by Northern blot analysis. No signal above background was seen that the best-fitting model included N-myc amplification, stage at with the sense control probe. B, dark-field photomicrograph of a section of stage IV-S neuroblastoma with N-mvc amplification, hybridized in situ with a 35S-labeled antisense diagnosis, and PTN expression. Indeed, this model fit better than PTN RNA probe. No specific hybridization signal was present in most tumor cells. This including TRK-A. MK expression, while prognostic when considered tumor did not express PTN mRNA by Northern blot analysis.

Table 3 Cox models with stage, N-myc amplification, and expression of MK, PTN, and TRK-A

ModelABCDEFGHIVariableN-myc P0.0010.0040.23Variable likelihood-43.589-45.801-46.477-46.605-52.314-49.859-43.129-39.289-38.541 amplificationN-myc (log)MK amplificationTRK-A (log)PTN (log)TRK-A (log)MK (log)Stage (log)PTN (2cat.")Stage (log)MK (2cat.)N-myc (log)TRK-A amplificationN-myc (log)Stage (log)PTN amplificationN-myc (2cat.)Stage (log)TRK-A amplificationP<0.0005<0.0005<0.0005<0.00050.0030.0010.004<0.00050.013VariablePTN(2 cat.)P<0.00050.170.0010.130.0050.0390.360.0160.013VariablePTN(log)P PTN (log) 0.003Log cat., categories. 1795

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1995 American Association for Cancer Research. DIFFERENTIAL EXPRESSION OF PTN AND MK IN NEUROBLASTOMAS aggressive stage IV neuroblastoma with N-myc amplification. Hybrid with N-myc amplification. Interestingly, these tumors also have a ization of antisense PTN was not seen in any area of the tumor. These high expression of another neurotrophin, BDNF, and its receptor, and other in situ hybridization results (data not shown) correlated well TRK-B (7, 8). with the overall PTN mRNA expression level in each tumor. The pattern of expression of PTN in favorable neuroblastomas is similar to that of TRK-A (2-6) which encodes the high-affinity NGF receptor. Neuroblastoma cells expressing a high level of TRK-A DISCUSSION differentiate in the presence of NGF, but die in the absence of NGF in PTN was highly expressed in favorable neuroblastomas, which the primary culture (3). Furthermore, both PTN and TRK-A expres often show spontaneous regression and differentiation. Mature gan- sion are very low or absent in neuroblastomas with N-mvc amplifi glioneuromas also expressed high levels of PTN transcripts. In con cation, which have a poor prognosis. Thus, expression of PTN and/or trast, PTN expression was extremely low in stage III and IV tumors, TRK-A may be useful as prognostic markers for neuroblastoma, and especially those with N-myc amplification. Also, there was a tendency they may play an important role in the biological and clinical behavior for neuroblastoma cell lines without amplification to have trace of these tumors. amounts of PTN expression compared to no detectable expression in N-myc-amplified cell lines. MK was expressed at high levels in almost ACKNOWLEDGMENTS all neuroblastoma primary tumors and cell lines, and aggressive neuroblastomas seem to have a relatively increased amount of MK We thank S. Horning for technical assistance and K. Kadomatsu (Depart transcripts. Thus, neuroblastomas clearly show quite different patterns ment of Biochemistry, Nagoya University) and P. Kotzbauer (Departments of of expression for these two closely related genes. Pathology and Internal Medicine, Washington University School of Medicine) PTN and MK are members of the same family of neurotrophic for their useful advice. We are grateful to Drs. Keiichi Ikeda, Sachiyo Suita, factors and they share about 50% homology (16). Both are cysteine- Hiroshi Ohgami, Hideko Tasaka, SumiÃ³ Miyazaki, Yoshifumi Sera, Hiroshi rich, basic proteins that are secreted and have heparin-binding activity Akiyama, and Kiyoshi Kawakami (Kyushu Neuroblastoma Study Group in (14, 15, 19). The expression of PTN and MK genes is developmen- stitutions), Dr. Takashi Yokoyama (Hiroshima University Hospital), Dr. Akira tally regulated (12, 16, 28), and they are both expressed in a variety Kuwano (Yamaguchi University Hospital), Dr. Kazuhiro Kume (Matsuyama of tissues in the mid-gestation mouse embryo. Their expression de Red Cross Hospital), Drs. Paul Bowman, Randall Graver, Joseph Dickerman, Donald Fernbach, Vita Land, Ruprecht Nitchke, and Teresa Vietti (Pediatrie creases in later embryogenesis, but then increases again postnatally in Oncology Group institutions), and Dr. Audrey Evans (Children's Hospital of restricted organs or tissues. In mouse brain, MK is expressed more Philadelphia) for contributing neuroblastoma samples for these studies. intensely than PTN during mid-gestation, but postnatally PTN expres sion is more intense than MK. Both proteins have neurotrophic activity on embryonic brain cells and dorsal root ganglion cells (16, REFERENCES 20, 21, 29, 30). MK is inducible by the treatment with retinoic acid 1. Evans, A. E., Gerson, J., and Schnaufer, L. Spontaneous regression of neuroblastoma. (12), whereas PTN is not (25, 30). Furthermore, PTN, but not MK, has Nati. Cancer Inst. Monogr., 44: 49-54, 1976. 2. Nakagawara, A., Arima, M., Azar, C. G., Scavarda, N. J., and Brodeur, G. M. Inverse recently been known to be induced by platelet-derived growth factor relationship between trk expression and N-mvr amplification in human neuroblasto (25). These findings suggest that, although PTN and MK are homol mas. Cancer Res., 52: 1364-1368, 1992. 3. Nakagawara, A., Arima-Nakagawara, M., Scavarda, N. J., Azar, C. G., Cantor, A. B., ogous, they have rather different biological functions. and Brodeur. G. M. Association between high levels of expression of the TRK gene It is interesting that PTN is expressed in ganglioneuromas as well and favorable outcome in human neuroblastoma. N. Engl. J. Med., 328: 847-854, as in neuroblastomas with the potential to differentiate. It is possible 1993. 4. Suzuki, T.. Bogcnmann, E., Shimada, H-, Stram, D., and Seeger, R. C. Lack of that PTN acts on neuroblastoma cells to potentiate the neuronal and/or high-affinity nerve growth factor receptors in aggressive neuroblastomas. J. Nati. Schwannian differentiation. On the other hand, PTN has been found to Cancer Ins!., 85: 377-384, 1993. be secreted from human breast carcinomas and implicated as a tumor 5. Kogner, P., Barbany, G., Dominici, C., Castello, M. A., Raschella, G., and Persson, H. Coexpression of messenger RNA for TRK protooncogene and low affinity nerve growth factor (31). Furthermore, PTN transforms NIH 3T3 cells and growth factor receptor in neuroblastoma with favorable prognosis. Cancer Res, 53: induces tumors in nude mice (22). Thus, the role and function of PTN 2044-2050, 1993. in neuroblastomas is still unclear. The candidate molecule for the 6. Borrello, M. G., Bongarzone, I., Pierotti, M. A., Luksch, R., Gasparini, M., Collini, P., Pilotti, S., Rizzetti, M. G., Mondellini, P., DeBernardi, B., DiMartino, D., PTN receptor has recently been reported as syndecan-3 (32). Garaventa, A., Brisigotti, M., and Tonini, G. P. TRK an RET protooncogene expres Analysis of PTN receptor expression and function may give further sion in human neuroblastoma specimens: High-frequency of TRK expression in non-advanced stages. Intl. J. Cancer, 54: 540-545, 1993. insights into the role of PTN in neuroblastomas. 7. Nakagawara, A., Azar, C. G., Scavarda, N. J., and Brodeur, G. M. Expression and Although MK expression in normal tissues and organs is limited, it function of TRK-B and BDNF in human neuroblastomas. Mol. Cell. Biol., 14: is frequently expressed in Wilms' tumors as well as a variety of other 759-767, 1994. 8. Nakagawara, A., Arima-Nakagawara, M., Azar, C. G., Scavarda, N. J., and Brodeur, human tumors including stomach, colon, pancreatic, lung, and esoph- G. M. Clinical significance of expression of neurotrophic factors and their receptors ageal cancers (33). The coexpression of PTN and MK was reported in in neuroblastoma. In: A. E. Evans, J. L. Biedler, G. M. Brodeur, G. J. D'Angio, and A. Nakagawara (eds.), Advances in Neuroblastoma Research 4, Vol. 4, pp. 155-161. malignant breast tissues (34). However, normal breast tissues ex New York: Wiley-Liss, 1994. pressed only PTN. In contrast, MK, but not PTN, was expressed in 9. Hempstead, B. L., Martin-Zanca, D., Kaplan, D. R., Parada, L. F., and Chao, M. V. lung cancers, although PTN, but not MK, was expressed in normal High-affinity NGF binding requires coexpression of the irk proto-oncogene and the low-affinity NGF receptor. Nature (Lond.), 350: 678-683, 1991. lung tissues (35). These data provide further support for the differen 10. Klein, R., Jing, S., Nanduri, V., O'Rourke, E., and Barbacid, M. 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Akira Nakagawara, Jeffrey Milbrandt, Takashi Muramatsu, et al.

Cancer Res 1995;55:1792-1797.

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