Int. J. Cancer (Pred. Oncol.): 79, 226–231 (1998) r 1998 Wiley-Liss, Inc. Publication of the International Union Against Cancer Publication de l’Union Internationale Contre le Cancer NEURONAL src AND trk A PROTOONCOGENE EXPRESSION IN NEUROBLASTOMAS AND PATIENT PROGNOSIS Tadashi MATSUNAGA1*, Hiroshi SHIRASAWA2, Hideki ENOMOTO1, Hideo YOSHIDA1, Jun IWAI1, Masahiro TANABE1, Kenji KAWAMURA3, Takao ETOH4 and Naomi OHNUMA1 1Department of Pediatric Surgery, Chiba University, School of Medicine, Chiba, Japan 2Department of Microbiology, Chiba University, School of Medicine, Chiba, Japan 3Division of Pediatric Surgery, Matsudo Municipal Hospital, Chiba, Japan 4Division of Surgery, Chiba Children’s Hospital, Chiba, Japan Neuroblastomas present a wide variety of clinical and biological behaviors, which are reflected by the heterogeneous expressions of protooncogenes related to the neuronal differentiation and amplification of the N-myc gene. High expression of trk A and Ha-ras in neuroblastomas has been shown to be associated with an excellent patient outcome. We have previously reported that neuron-specific src mRNA was increased in chemically differentiated neuroblastoma cell lines and in clinically observed neuroblastomas without N-myc amplification. In the present study, to clarify both the value of neuronal c-srcN2 expression as a prognostic indicator and the significance of the coexpression of these protooncogenes, we examined the expression of 3 alternatively spliced src, trk A and Ha-ras in neuroblastoma tissues from 60 patients by competitive RNA-polymerase chain reaction (PCR). The results indicate that protooncogene expression in neuroblastomas correlated with a favorable outcome for c-srcN2 and trk A. N-myc gene was amplified exclusively in tumors with low levels of trk A. Low expression of c-srcN2 and trk A might thus characterize different aggressive phenotypes due to different signal transduction pathways of neural differentiation in neuroblastoma. The combined analyses for c-srcN2 and trk A expression by RNA-PCR should provide information about the biological phenotype of a neuroblastoma within a short period of time after obtaining tumor material. Int. J. Cancer (Pred. Oncol.) 79:226–231, 1998. r 1998 Wiley-Liss, Inc. Neuroblastoma, one of the most common malignant solid cancers in childhood, is heterogeneous in its clinical behavior. The evolution of this tumor encompasses a wide spectrum of biological characteristics ranging from spontaneous regression or maturation to a highly aggressive phenotype. Infantile neuroblastomas diagnosed before 1 year of age (almost all of these are detected in Japan by a mass screening system) generally do not require intensive chemotherapy to be cured. These tumors appear to have an ability to undergo differentiation and neuronal cell death, as do developmental neuroblasts in the fetus. In contrast, most of the neuroblastomas identified by clinical symptoms at an age older than 1 year are highly aggressive and their prognosis is not favorable, even when megadose chemotherapy is performed. These tumors may have lost the ability to mature during the progression of the disease. Although the patient age at the time of diagnosis and the clinical stage of the disease are predictive of prognosis, the genetic evaluation of an individual tumor should provide important information to clarify its biological behavior. Recent advances on the role of protooncogenes in neuronal differentiation have shed light on the biological mechanisms underlying their proliferation and differentiation. Genomic amplification of N-myc, which encodes the nuclear protein regulating neuronal cell proliferation and maturation, predicts poor outcome (Brodeur et al., 1984) and is widely used as a reliable biological marker. Expression of trk A, which encodes the high-affinity nerve growth factor (NGF) receptor, and Ha-ras p21, a signal transducing protein from the cell membrane to the nucleus, has been shown to strongly correlate with favorable prognosis (Nakagawara et al., 1992, 1993; Suzuki et al., 1993; Tanaka et al., 1988, 1991, 1995). src is another signal transduction protooncogene related to neural differentiation (Brugge et al., 1985; Grady et al., 1987; Bjelfman et al., 1990; Ignelzi et al., 1994). We have previously reported a higher expression of c-src mRNA in neuroblastomas of patients with a favorable prognosis (Matsunaga et al., 1991). Pyper and Bolen (1990) have described the importance of the regulated expression of alternatively spliced neuronal src mRNAs (c-srcN1 and c-srcN2) on the role of src in the development of the human brain. We found that expression of neuronal c-srcN2 mRNA was enhanced during retinoic acid-induced differentiation of neuroblastoma cells in vitro (Matsunaga et al., 1993). On the basis of these observations, we preliminarily analyzed expression of 3 alternatively spliced src mRNAs in primary human neuroblastomas using S1 nuclease protection assays and have observed an inverse relationship between c-srcN2 mRNA expression and N-myc gene amplification (Matsunaga et al., 1994). In this study, in order to investigate the clinical significance of c-srcN2 expression as a biological predictor, we have extended our analyses to a larger number of neuroblastoma cases using the competitive RNA-polymerase chain reaction (PCR). The competitive PCR, amplifying DNA of target and control sequences simultaneously in the same reaction, is a sensitive and reliable method for analyses of gene expression and gene amplification levels (Bordow et al., 1994; Cremoux et al., 1997). We also examined expression of trk A and Ha-ras by competitive RNAPCR, and N-myc gene amplification by Southern blot hybridization to clarify the significance of the expression and amplification of these protooncogenes in neuroblastomas. MATERIAL AND METHODS Patients and tumor specimens The 60 patients with neuroblastomas analyzed were treated at Chiba University Hospital, Chiba Children’s Hospital or Matsudo Municipal Hospital between 1987 and 1997. The median follow-up period after diagnosis for the surviving children was 60 months (range: 9–128 months). The neuroblastomas were staged according to the International Neuroblastoma Staging System (INSS) (Brodeur et al., 1993). Of the 60 cases, 20 were diagnosed at an age older than 1 year and the remaining 40 were less than 1 year; 32 of the 40 infantile patients were identified by a neuroblastoma mass screening system (Table I). High molecular weight (m.w.) cellular DNAs and undegraded total RNAs were extracted from the neuroblastoma tissue obtained by biopsy or surgery prior to chemotherapy. The tumor tissues were immediately frozen after removal and stored in liquid nitrogen until tested. Prior to gene analyses, the specimens were confirmed to consist of tumor cells by pathological examination. Grant sponsors: Ministry of Education, Science and Culture and Ministry of Health and Welfare of Japan. *Correspondence to: Department of Pediatric Surgery, Chiba University, School of Medicine, 1-8-1, Inohana, Chuo-ku, Chiba 260, Japan. Fax: 043 226 2366. Received 27 October 1997; Revised 17 January 1998 NEURONAL src AND trk A IN NEUROBLASTOMA TABLE I – CLINICAL STAGE AND STATUS OF TUMORS AT DIAGNOSIS OF 60 PATIENTS WITH NEUROBLASTOMA1 Status of tumors at diagnosis Cases detected by mass screening Cases detected by clinical symptoms Clinical stage (INSS) Total 1 2A 2B 3 4 21 3 6 2 0 32 3 (1) 1 1 3 (1) 20 (18) 28 (20) 1Numbers in parentheses are cases diagnosed at an age older than 1 year. Cell lines To determine the PCR conditions, RNA from 10 neuroblastoma cell lines (IMR32, RT-BM-1, SK-N-SH, NB69, NB69N, NB69S, NB-1, GOTO, cNBI and LA-N-5) was used. SK-N-SH, IMR32, GOTO and NB-1 were obtained from the Japanese Cancer Research Resources Bank (Tokyo). LA-N-5 was kindly provided by Dr. R.C. Seeger (Los Angeles, CA). RT-BM-1 (Sugimoto et al., 1986) was kindly provided by Dr. T. Sugimoto (Miyazaki, Japan) and NB69 was kindly provided by Dr. Y. Nishi (Kanagawa, Japan). NB69N and NB69S are clonal sublines of NB69, and cNBI was described previously (Matsunaga et al., 1993). RT-BM-1 was used as a reference for c-src, trk A and Ha-ras expression, and IMR32 was used for 25-fold amplification of N-myc. Terminal differentiation of RT-BM-1 cells was induced by incubation in culture medium containing 5 µM trans-retinoic acid (Sigma, St. Louis, MO), as described previously (Matsunaga et al., 1993). RNA-PCR Total cytoplasmic RNA (5 µg) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase and random hexanucleotide primers, essentially as described previously (Matsunaga et al., 1993). Target (3 alternatively spliced src, trk A or Ha-ras) and control b2-microglobulin gene sequences were coamplified in the same reaction, using the following gene-specific oligonucleotide primers: 58-CAGACCTGTCCTTCAAGAAA-38 (src, forward); 58-TCAGCCTGGATGGAGTCGGA-38 (src, reverse); 58-TGTTCAGGTCAACGTCTCCT-38 (trk A, forward); 58-GCAGCGTGTAGTTGCCGTTG-38 (trk A, reverse); 58-TGACCATCCAGCTGATCCAG-38 (Ha-ras, forward); 58-TGTACTGGTGGATGTCCTCA-38 (Ha-ras, reverse); 58-ACCCCCACTGAAAAAGATGA-38 (b2-microglobulin, forward); 58-ATCTTCAAACCTCCATGATG-38 (b2-microglobulin, reverse). The expected sizes of the PCR products when using these sets of primers are 196 (c-srcN2), 163 (c-srcN1), 145 (c-src), 254 (trk A), 243 (Ha-ras) and 120 (b2-microglobulin) base pairs. Aliquots of cDNA corresponding to 50 ng of RNA were subjected to PCR in a final volume of 25 µl using 1 unit of AmpliTaq Gold Polymerase (Perkin-Elmer Cetus, Norwalk, CT). An initial denaturation of 3 min at 94°C was followed by 37 cycles of a 30 sec denaturing step at 94°C, a 30 sec annealing step at 57°C and a 30 sec extension step at 72°C. Finally, a further 7 min extension at 72°C was performed. These PCR conditions were determined by preliminary experiments using the 10 neuroblastoma cell lines, which indicated that the PCR products of the target and control genes were amplified in parallel according to the numbers of PCR cycles within a range from 35 to 41 cycles; 3 independent PCR studies with 37 cycles resulted in almost identical levels for all target and control genes (data not shown). The specificity of PCR products was confirmed by Southern blot hybridizations (data not shown). All PCR assays of the neuroblastoma clinical samples were performed simultaneously with cDNA from the neuroblastoma cell line RT-BM-1 and/or its chemically differentiated cells, as a reference. Following PCR, 8 µl of PCR reaction mixture was subjected to electrophoresis on 5% agarose gels for src and 2.5% agarose gels for trk A and Ha-ras. Our results obtained previously by S1 nuclease protection assays for src mRNAs (Matsunaga et al., 1993) and Northern blot hybridizations for Ha-ras mRNA (Matsunaga et al., 1991), and 227 unpublished results on trk A mRNA expression in 10 neuroblastoma cell lines and 15 neuroblastoma tissues (data not shown), are identical as to relative expression levels with those obtained using this competitive RNA-PCR. Southern blot hybridization DNA (5 µg) digested with EcoRI was separated on a 0.8% agarose gel and transferred to a nitrocellulose filter. The DNA blots were hybridized with a [a-32P]dCTP-labeled probe, the BamHIEcoRI fragment of the 2nd exon of N-myc (Kohl et al., 1986) DNA, under stringent conditions as described previously (Shirasawa et al., 1988). A UDh probe (Shirasawa et al., 1989) was used to normalize the DNA concentration. Human placental DNA or DNA extracted from IMR32 cells was used to detect a single copy or amplified copies of N-myc, respectively, in each sample. Estimation of gene expression and amplification The PCR products in gels containing a DNA staining solution (Syber green) were visualized by ultraviolet (UV) transillumination and recorded as digital images by a Kodak digital science DC40 camera, and the intensity of each band was measured by the 1D Image Analysis Application program (Eastman Kodak, Rochester, NY). Signals from Southern blot hybridizations were measured using a Bio-imaging Analyzer (BAS 2000, Fujix, Tokyo, Japan). Statistical analysis The Mann-Whitney U-test was used to evaluate the significance of protooncogene expression in localized and metastatic tumors. The probability of survival of the patients was calculated by the product limit method of Kaplan and Meier and compared using the log-rank test. Comparisons between 2 variables of c-srcN2 or trk A expression and N-myc amplification were performed by the chisquare test. RESULTS The RNA-PCR assays identified target (3 c-src, trk A or Ha-ras) and control (b2-microglobulin) genes in 60 neuroblastoma tissues as well as in 10 neuroblastoma cell lines (data not shown). Representative results are shown in Figure 1. High expression of c-srcN2 was more common in infantile and localized neuroblastomas compared to metastatic disease. As we reported previously (Matsunaga et al., 1993), the relative ratio of c-srcN2 mRNA to the total level of 3 c-src mRNAs was a more useful prognostic factor than the absolute levels of c-srcN2. To evaluate the expression of c-srcN2 in the present study, we used the PCR ratio of c-srcN2 to the total 3 c-src genes because a similar finding was obtained (data not shown). The ratio of c-srcN2 expression relative to the total 3 c-src in tumors that were classified according to localized and metastasis status of the disease is shown in Figure 2. The median c-srcN2 ratio was 0.23 (range: 0.05–0.41) in localized tumors and 0.09 (range: 0–0.23) in metastatic tumors. The localized tumors significantly expressed higher ratio of c-srcN2 ( p , 0.0001). In the present study, the expression of c-srcN2 was considered high if the c-srcN2 ratio was above that observed in the chemically differentiated neuroblastoma RT-BM-1 cells (ratio 5 0.15) and low if the ratio was 0.15 or below. The cumulative survival according to c-srcN2 expression indicated that a high ratio of c-srcN2 expression was significantly associated with longer event-free survival of the patients (7-year survival rate: 95.0 and 29.8%; x2 5 33.924, p , 0.0001, data not shown). Similarly, higher expression of trk A was commonly observed in the infantile and localized neuroblastomas compared to the metastatic tumors. The levels of trk A expression in tumors that were classified according to localized and metastasis status of the disease are shown in Figure 3. The median PCR ratio of trk A to b2-microglobulin was 0.85 (range: 0.31–1.38) in the localized tumors and 0.18 (range: 0–0.65) in the metastatic tumors. The localized tumors significantly expressed higher levels of trk A ( p , 0.0001). The expression of trk A was considered high if the 228 MATSUNAGA ET AL. FIGURE 1 – Representative results of competitive RNA-PCR analyses for expression of the target (src, trk A or Ha-ras) and control (b2-microglobulin) genes. Shown are 4 localized tumors and 4 metastatic tumors, which were staged according to the INSS (Brodeur et al., 1993). N-myc gene amplification is indicated by the symbol (A). RNA from neuroblastoma cells RT-BM-1 (0) and terminally differentiated RT-BM-1 treated by retinoic acid for 14 days (14) was also analyzed. The PCR products for src/b2-microglobulin were separated on 5% agarose gel and those for trk A/b2-microglobulin and Ha-ras/b2microglobulin were separated on 2.5% agarose gel. The PCR product sizes are 196 (c-srcN2), 163 (c-srcN1), 145 (c-src), 254 (trk A), 243 (Ha-ras) and 120 (b2-microglobulin) base pairs. Lane M, pUC19 digested by Msp I (size marker). FIGURE 3 – PCR ratio of trk A to b2-microglobulin in 40 localized tumors and 20 metastatic tumors. (X) Less than 1 year and alive; (W) less than 1 year and died of the disease; (N) older than 1 year and alive; (M) older than 1 year and recurrence or died of the disease. The PCR ratio of RT-BM-1 cells is 0.30 (broken line). FIGURE 4 – PCR ratio of Ha-ras to b2-microglobulin in 40 localized tumors and 20 metastatic tumors. (X) Less than 1 year and alive; (W) less than 1 year and died of the disease; (N) older than 1 year and alive; (M) older than 1 year and recurrence or died of the disease. FIGURE 2 – PCR ratio of c-srcN2 relative to total 3 c-src in 40 localized tumors and 20 metastatic tumors. (X) Less than 1 year and alive; (W) less than 1 year and died of the disease; (N) older than 1 year and alive; (M) older than 1 year and recurrence or died of the disease. The PCR ratio of chemically differentiated neuroblastoma RT-BM-1 cells is 0.15 (broken line). PCR ratio (trk A/b2-microglobulin) was above that observed in the RT-BM-1 cells (PCR ratio 5 0.3) and low if the ratio was 0.3 or below. The cumulative survival according to trk A expression indicated that high levels of trk A expression were significantly associated with better event-free survival of the patients as well (7-year survival rate: 90.3 and 22.3%; x2 5 31.326, p , 0.0001, data not shown). No correlation of the level of Ha-ras expression and the spread of the disease or prognosis of the patients was obvious. The levels of Ha-ras expression in localized or metastatic tumors are shown in Figure 4. The median PCR ratio of Ha-ras to b2-microglobulin was 0.51 (range: 0–0.97) for the localized tumors and 0.48 (range: 0.12–1.03) for the metastatic tumors. There was no significant association between Ha-ras expression and spread of the disease ( p 5 0.4468). The relation of c-srcN2 and trk A expression, and its relevance to the spread of the disease, age at diagnosis and outcome of the patients are shown in Figure 5. The expression patterns of c-srcN2 and trk A were very similar but did not agree in 5 cases. The coexpression of c-srcN2 and trk A was observed in 41 cases; neither c-srcN2 nor trk A was seen in 14 cases and either c-srcN2 or trk A in 1 and 4 cases, respectively. In 40 localized tumors, NEURONAL src AND trk A IN NEUROBLASTOMA FIGURE 5 – Relation of c-srcN2 and trk A expression, and its relevance to spread of the disease, age at diagnosis and outcome of the patients. (X) Less than 1 year and alive; (W) less than 1 year and died of the disease; (N) older than 1 year and alive; (M) older than 1 year and recurrence or died of the disease. 229 FIGURE 7 – Correlations of N-myc gene amplification, c-srcN2 expression and trk A expression in 20 metastatic neuroblastomas. (X) Less than 1 year and alive; (W) less than 1 year and died of the disease; (N) older than 1 year and alive; (M) older than 1 year and recurrence or died of the disease. expression to N-myc amplification was not as strong as the relation of trk A and N-myc. The relation of c-srcN2 and N-myc in 4 of the 20 metastatic tumors was not inverse, although the result is significant (x2 5 34.357, p , 0.0001). It is noteworthy that a low ratio of c-srcN2 predicted unfavorable outcomes without N-myc amplification and with high levels of trk A. DISCUSSION FIGURE 6 – Event-free survival probability for patients with neuroblastomas expressing c-srcN2 at a high ratio and trk A at high levels (c-srcN2/trk A: 1/1, 95.0% at 7 years, n 5 41), expressing either c-srcN2 at a high ratio or trk A at high levels (c-srcN2/trk A: 1/2 or 2/1, 53.3% at 7 years, n 5 5) and expressing c-srcN2 at a low ratio and trk A at low levels (c-srcN2/trk A: 2/2, 21.8% at 7 years, n 5 14; x2 5 36.847, df 5 2, p , 0.0001). coexpression of c-srcN2 and trk A was observed in 38 tumors including 31 tumors found by mass screening, and only trk A expression was observed in 2 tumors, 1 of which was detected by screening. In 20 metastatic tumors, 14 tumors expressed neither c-srcN2 nor trk A. The event-free survival probability analyzed by the combined 2 genes expression in Figure 6 discriminated 3 different subsets of patients: 1 with a favorable prognosis (both 2 genes expressed, 7-year event-free survival rate: 95.0%); 1 with intermediate prognosis (either gene expressed, 53.3%); and 1 with unfavorable prognosis (neither gene expressed, 21.8%; x2 5 36.847, df 5 2, p , 0.0001). A poor outcome was not always present for the metastatic tumors expressing both c-srcN2 and trk A. Genomic amplification of N-myc by more than 10 copies per haploid set was observed in 14 cases, all of which were at stage 4; 8 of these 14 children died of the disease. The correlation of N-myc gene amplification, c-srcN2 expression and trk A expression in 20 metastatic neuroblastomas is shown in Figure 7. N-myc amplification was restricted exclusively to the tumors expressing trk A at low levels, and only one tumor without N-myc amplification was in the group with low levels of trk A expression. Thus, trk A expression and N-myc amplification are strongly and inversely correlated (x2 5 54.783, p , 0.0001). The inverse correlation of c-srcN2 The neuronal differentiation or regression of a given neuroblastoma affects the prognosis of the patient. The modalities of treatment for neuroblastomas range from surgical removal alone to multimodal treatment including megadose chemotherapy with bone marrow transplantation, according to the aggressiveness of the individual tumor. The initial findings of N-myc gene amplification in aggressive neuroblastomas (Brodeur et al., 1984) have been followed by studies which analyzed expression of the protooncogenes related to neuronal differentiation in neuroblastomas showing different clinical courses (Tanaka et al., 1988, 1991, 1995; Nakagawara et al., 1992, 1993; Suzuki et al., 1993). To establish a treatment strategy against neuroblastoma based on genetic information, it is important to ascertain the clinical significance of the multiple protooncogenes related to neural differentiation in neuroblastomas and to overcome the difficulties presented by the available methods for gene analyses. In this study, we analyzed expression of alternatively spliced neuronal c-src, trk A and Ha-ras in neuroblastoma tissues from 60 patients using competitive RNA-PCR. Most of the infantile neuroblastomas aged less than 1 year in our study were identified by neuroblastoma mass screening. A substantial number of these tumors has been suggested to regress spontaneously because of a high incidence of infantile neuroblastomas in the screening population. However, it is considered that genetically aggressive tumors without clinical symptoms are also included in the tumors found by mass screening (Kaneko et al., 1990). In fact, 8 of the 32 patients diagnosed by mass screening in our study had gross tumor beyond midline of the body, meaning stage III tumor according to the classification system of Evans et al. (1971). Therefore, it is important to clarify the biological phenotype of the tumors identified by screening as well as the infantile neuroblastomas found by clinical symptoms. Our findings revealed that the expression pattern of c-srcN2 and trk A was predictive of the clinical behavior of the disease. Most of the localized tumors diagnosed by mass screening or by clinical symptoms expressed both c-srcN2 and trk A, and this pattern of gene expression did not always result in unfavorable outcome even in the metastatic cases. Low expression of trk A was restricted to metastatic disease and was closely related to poor outcome and MATSUNAGA ET AL. 230 N-myc amplification. On the other hand, a low ratio of c-srcN2 expression appeared to characterize a different type of aggressive neuroblastomas. The patients with a tumor expressing either c-srcN2 or trk A had an intermediate prognosis. The trk A encodes a transmembrane tyrosine-specific protein kinase which is an essential component of the high-affinity NGF receptor and is necessary for functional NGF signal transduction (Klein et al., 1991; Kaplan et al., 1991; Loeb et al., 1991). Neural crest-derived neural cells in the fetus require NGF for development, differentiation and survival. High expression of trk A has been documented to be significantly associated with favorable outcome (Nakagawara et al., 1992, 1993; Suzuki et al., 1993; Tanaka et al., 1995). Our present results using competitive RNA-PCR concur with these observations. NGF signal transduction from the cell membrane to the nucleus is mediated by many signaling proteins, including c-src and Ha-ras (Wood et al., 1992; D’Arcangelo and Halegoua, 1993). The src gene encodes a membrane-bound tyrosine-specific protein kinase, and its expression and specific kinase activity are regulated during the development of the neuronal tissues and chemically differentiated neuroblastoma cell lines (Brugge et al., 1985; Grady et al., 1987; Bjelfman et al., 1990). src mRNA is expressed in 3 distinct forms (c-src and neuron-specific c-srcN1 and c-srcN2) by alternative splicing in mammalian brain tissue (Pyper and Bolen, 1990). Although the exact functions and difference of neuronal src proteins translated by c-srcN1 and c-srcN2 mRNA are not known, the insertion of neuron-specific amino acids may add (an) important function(s) to the neuronal signal transduction pathway, since neuronal amino acids locate in the src regulatory domain which interacts with other signaling proteins. Our previous findings have suggested the relation of high ratio expression of c-srcN2 mRNA and differentiated phenotype and a favorable clinical behavior of neuroblastomas in vitro and in vivo (Matsunaga et al., 1993). The results of the present study indicate that c-srcN2 expression, as well as trk A, was significantly enhanced in tumors with a favorable outcome. However, the distribution of the low ratio of c-srcN2 and low levels of trk A expressed in the metastatic tumors was somewhat different in relation to N-myc amplification, as shown in Figure 6. It appears likely that low expression of trk A is closely related to an aggressive phenotype resulting from N-myc amplification but that expression of c-srcN2 at a low ratio in aggressive tumors is not always linked to N-myc amplification. This might be because trk A and N-myc play critical and opposite roles in the differentiation and proliferation of neuroblasts and in the malignant transformation of neuroblastomas during the fetal period (Smeyne et al., 1994; Weiss et al., 1997). However, src transduces not only the NGF signal but also other neuronal signals (Rusanescu et al., 1995; Kuo et al., 1997), which may not always take place during the same period. The Ha-ras encodes a cytoplasmic GTP-binding protein which acts as a signal transduction mediator in NGF and other signal transduction pathways (Thomas et al., 1992; D’Arcangelo and Halegoua, 1993; Rusanescu et al., 1995; Kuo et al., 1997). Tanaka et al. (1988, 1991) have demonstrated a significant association between high Ha-ras p21 and favorable outcome by an immunohistochemical technique in a large number of cases using an anti Ha-ras p21 antibody. Unexpectedly, our analyses using competitive RNA-PCR did not find significant expression of Ha-ras at high levels in the patients with favorable prognosis. Tanaka et al. (1988) described that Western blotting underestimated the amount of Ha-ras p21 because a tumor specimen contained non-malignant cells in addition to neuroblastoma cells, which resulted in the spurious decrease of Ha-ras p21 levels. The RNA-PCR assay also estimates the amount of Ha-ras transcripts in whole RNA from malignant and non-malignant cells. However, our RNA-PCR assays clearly demonstrated the difference of trk A and c-srcN2 levels among the tumors with different biological behaviors. Another explanation for this dissociation is that a post-translational stabilization of the protein also contributes to the augmentation of Ha-ras p21. Our previous finding using Northern blotting in a limited number of neuroblastomas also indicated that the tumors with a favorable outcome expressed higher levels of Ha-ras mRNA than those with unfavorable outcome, but the difference in the expression level was minimal (Matsunaga et al., 1991). We believe that the immunohistochemical technique is the best method to detect Ha-ras expression in relation to prognosis of the patients. The RNA-PCR used in the present study is very sensitive, less time-consuming and requires a smaller amount of RNA from a tumor specimen compared to S1 nuclease protection assay. While our S1 nuclease protection assay needed 40 µg of total RNA, cDNA corresponding to 50 ng of total RNA is sufficient for our PCR assay. This method enables quick analyses of multiple genes expression in the small specimens obtained by fine-needle biopsy, which is a less invasive procedure, particularly for patients with poor general condition. In conclusion, expression of c-srcN2 at a high ratio characterizes neuroblastomas with favorable prognosis, and expression of trk A at low levels identifies highly aggressive neuroblastomas including tumors with N-myc amplification. It should be possible to predict the clinical behavior of an individual tumor based on genetic information including that obtained on c-srcN2 and trk A expression by quick analyses of RNA-PCR before starting chemotherapy. ACKNOWLEDGEMENTS This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, and a research grant from the Ministry of Health and Welfare of Japan. The authors thank Drs. T. Sugimoto and Y. Nishi for providing the NB cell lines RT-BM-1 and NB69, respectively. 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