High-Level Expression of EPHB6, EFNB2, and EFNB3 Is Associated with Low Tumor Stage and High Trka Expression in Human Neuroblastomas1

Vol. 5, 1491–1496, June 1999 Clinical Cancer Research 1491

High-Level Expression of EPHB6, EFNB2, and EFNB3 Is Associated with Low Tumor Stage and High TrkA Expression in Human Neuroblastomas1

Xao X. Tang, Audrey E. Evans, Huaqing Zhao, ated with favorable NB and that low-level expression of Avital Cnaan, Wendy London, Susan L. Cohn, EPHB6, EFNB2, and EFNB3 correlates with aggressive Garrett M. Brodeur, and Naohiko Ikegaki2 MYCN-amplified NB. Thus, EPHB6, EFNB2, and EFNB3 may have biological relevance in NB. Further investigation Divisions of Oncology [X. X. T., A. E. E., G. M. B., N. I.] and on the biology of these genes may help provide insight into Biostatistics [H. Z., A. C.], The Children’s Hospital of Philadelphia, Abramson Research Center, Philadelphia, Pennsylvania 19104-4318; the treatment of NB. University of Florida, Pediatric Oncology Group, Statistical Office, Gainesville, Florida 32601 [W. L.]; and Department of Pediatrics, INTRODUCTION Northwestern University and Children’s Memorial Hospital, Chicago, 3 Illinois 60614-3394 [S. L. C.] NB is a common pediatric tumor of neural crest origin. The tumor occurs frequently in infants and young children, with the primary lesion in the adrenals or the sympathetic chain. It is ABSTRACT known that NB exhibits a wide range of clinical, genetic, and Neuroblastoma (NB) is a common pediatric tumor of biological heterogeneity. Previous studies suggest that there are neural crest origin that is biologically and clinically hetero- three distinct subsets of NB (1–4). Of those, two subsets show geneous. EPH family receptor tyrosine kinases and ephrin apparently opposite outcomes; low-risk NB is curable with ligands play fundamental roles in neurodevelopmental pro- minimal or no treatment, whereas high-risk NB is usually lethal cesses. Recently, we found that NB cell lines expressed sev- despite the most aggressive treatments, including bone marrow eral EPHB and EFNB transcripts, which encode EPHB transplantation. Patients with low-risk NB are likely to be in- subgroup receptors and ephrin-B subgroup ligands, respec- fants having low-stage disease (stage 1, 2, or 4S). These NBs are tively. To explore the role of EPHB receptors and ephrin-B near triploid in karyotype, rarely show chromosomal abnormal- ligands in the biology of NB, we examined the expression of ities, and have a high TrkA expression. In contrast, patients with EPHB and EFNB transcripts in 47 primary NB specimens. high-risk NB are more likely to be Ͼ1 year of age and have Multiple EPHB and EFNB transcripts were expressed in all advanced-stage disease (stage 3 or 4). High-risk NBs are near of the NB tumors examined, suggesting the involvement of diploid in karyotype and have numerous structural chromosomal these transcripts in modulating the biological behavior of abnormalities, including MYCN amplification and deletion or NB. Higher levels of EPHB6, EFNB2, and EFNB3 expres- allelic loss of chromosome 1p. TrkA expression is low or absent sion were found in low-stage tumors (stage 1, 2, and 4S) than in the high-risk NB, but those tumors with MYCN amplification .(often express TrkB (5 ؍ P ,0.0013 ؍ in advanced-stage tumors (stage 3 and 4; P respectively). Expression of TrkA,a Between these two NB subsets is the intermediate-risk NB ,0.027 ؍ and P ,0.0048 well-established prognostic marker of favorable NB, was subset, which accounts for about 25–30% of total NB cases. positively correlated with EPHB6, EFNB2, and EFNB3 ex- Intermediate-risk NBs are characterized by patients with ad- ,respec- vanced-stage disease (stage 3 and 4), a near diploid karyotype ,0.0001 ؍ and P ,0.0019 ؍ pression (P < 0.0001, P tively). MYCN-amplified tumors expressed lower levels of and no MYCN amplification. About 25–50% of them have EPHB6, EFNB2, EFNB3, and TrkA transcripts compared to deletion or allelic loss of 1p or other chromosomes. TrkA expression is generally low in this group. The 3-year survival for ,0.0048 ؍ P ,0.0023 ؍ P ,0.0006 ؍ nonamplified tumors (P respectively). These data suggest that high- intermediate-risk NB is between 25–50%, suggesting that this ,0.0001 ؍ and P level expression of EPHB6, EFNB2, and EFNB3 is associ- subset of NB may be heterogeneous. EPH family receptor kinases and their ephrin ligands are involved in fundamental developmental processes in the nervous system, including axon guidance (6), axon fasciculation (7), Received 11/23/98; revised 3/8/99; accepted 3/15/99. neural crest cell migration (8), acquisition of brain subregional The costs of publication of this article were defrayed in part by the identity (9), and neuronal cell survival (10). Moreover, evidence payment of page charges. This article must therefore be hereby marked suggests that some members of the EPH family and their ligands advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. are involved in angiogenesis and oncogenesis (11–14). The EPH 1 Supported by NIH Grants F32 CA75748 (to X. X. T.), NS34514 (to family is the largest subfamily of receptor protein tyrosine G. M. B.), and CA70958 (to N. I.) and by the Children’s Cancer Re- kinases, consisting of 14 known members (15). Similarly, ephrin search Foundation. 2 To whom requests for reprints should be addressed, at Division of Oncology, The Children’s Hospital of Philadelphia, Abramson Research Center Suite 902, 324 South 34th Street, Philadelphia, PA 19104-4318. Phone: (215) 590-5243; Fax: (215) 590-3770; E-mail: ikegaki@ 3 The abbreviations used are: NB, neuroblastoma; RT, reverse transcrip- kermit.oncol.chop.edu. tion; RT-PCR, reverse transcription-PCR.

ligands constitute a large family, which includes eight members at 55°C for an additional 60 min. RNase H (2 units; Life (15). Technologies, Inc.) was then added to each RT reaction, fol- Ephrins are cell surface-bound ligands and are divided into lowed by incubation at 37°C for 20 min. Finally, the RT reaction two subgroups (ephrin-A and ephrin-B), depending on how they was diluted by addition of 30 ␮l of water and stored at Ϫ20°C. are anchored to the cell membrane (16). The ephrin-A ligands Semiquantitative RT-PCR Assay. Two ␮l of the RT are glycosylphosphatidylinositol-linked proteins and are en- reaction were subjected to PCR amplification. PCR primers coded by the EFNA genes. In contrast, ephrin-B ligands are were biotinylated at their 5Ј ends, and the biotinylated PCR transmembrane proteins and are encoded by the EFNB genes. products were detected using a chemiluminescence detection The EPH family receptors can also be divided into two sub- procedure (see below). To correct variations in RT, PCR, and groups, based on the relatedness of their extracellular domain chemiluminescence detection steps, the expression of the house- sequences and on their ability to bind to the two subgroups of keeper gene GAPD was used as the internal control. GAPD ephrins. The EPHA subgroup interacts preferentially with eph- primers were added together with gene-specific primers in the rin-A ligands, whereas the EPHB subgroup interacts preferen- same PCR reaction. GAPD has been used as an invariant control tially with ephrin-B ligands (16). for transcript expression analysis in NB (19, 20). Furthermore, We reported previously that EPHB2 transcripts were levels of GAPD expression were similar between low-stage and highly expressed in NB cell lines (17). Subsequently, we found advanced-stage NB as well as MYCN-amplified and nonampli- that transcripts encoding several EPHB receptors and their eph- fied tumors.4 rin-B ligands were coexpressed in these NB cell lines.4 In this The semiquantitative RT-PCR assay used in this study met study, we examined the expression of transcripts encoding five two important criteria: (a) PCRs were to be in the exponential members of the EPHB subgroup and three members of the phase of amplification for both the internal control (GAPD) and ephrin-B subgroup in 47 NB tumor specimens representing all the transcripts of interest. The number of PCR cycles was clinical stages. We found that transcripts encoding multiple determined accordingly to satisfy this criterion; and (b) the EPHB receptors and ephrin-B ligands were expressed at various signal intensity of GAPD and the transcripts of interest was to be levels in all of the NB tumors examined. High levels of EPHB6, within the linear range of X-ray film detection as well. To EFNB2, and EFNB3 expression were associated with low-stage achieve this, a mixture of biotinylated and nonbiotinylated NB, and their expression was positively correlated with TrkA GAPD primers at a ratio of 1:49 was used in the PCR reaction expression. In contrast, MYCN-amplified tumors expressed because the expression of GAPD is higher than that of EPHB lower levels of EPHB6, EFNB2, EFNB3, and TrkA transcripts and EFNB. This modification solved the overexposure problem compared to nonamplified tumors. The implications of these of GAPD signals to X-ray film compared to those of experi- observations in the biology of NB are discussed. mental transcripts and helped quantification of signals from the control and experimental transcripts be performed accurately. MATERIALS AND METHODS Detailed description of the semiquantitative RT-PCR assay will 5 NB Tumor Samples. Forty-seven NB tumor specimens be described elsewhere. Briefly, PCR amplifications were per- ␮ ␮ were obtained from the Tumor Bank of The Children’s Hospital formed in volumes of 20 l and contained 0.4 M primer, 200 ␮ ␮ of Philadelphia and from the Tumor Bank of The Pediatric M deoxynucleotide triphosphates, 2 l of the RT reaction, 50 Oncology Group. These include 10 stage 1 tumors, 8 stage 2 mM KCl, 2 mM MgCl2,10mM Tris-HCl (pH 8.3), and 1 unit of tumors, 5 stage 4S tumors, 12 stage 3 tumors, and 12 stage 4 AmpliTaq Gold (PE Applied Biosystems). PCR conditions were tumors. Among these tumors, two stage 3 tumors and four stage 95°C for 12 min followed by 20 cycles of 95°C for 30 s, 55°C 4 tumors had MYCN amplification. for 30 s, and 72°C for 5 min, followed by 72°C for 10 min. RNA Extraction and RT. Total cellular RNA was pre- Nucleotide sequences of EPHB1, EPHB2, EPHB3, EPHB4, pared by either the Totally RNA kit (Ambion) or by the method EPHB6, EFNB1, EFNB2, EFNB3, and GAPD primers were described by Auffray and Rougeon (18). Total RNA (1 ␮g) reported elsewhere (21). TrkA PCR primers have the following Ј extracted from primary NB specimens was mixed with 35 ng of nucleotide sequences: sense primer, 5 -TGCCTGCCTCTTC- Ј Ј random hexamers and 250 ng of oligothymidylic acid (15-mer) CTTTCTA-3 ; antisense primer, 5 -GTGGTGAACACAG- Ј in a volume of 10 ␮l. The resultant mixture was heated at 70°C GCATCAC-3 . ␮ ␮ for 10 min and chilled on ice. Each RT reaction was carried out PCR products (10 l of a total of 20 l of PCR products) were ␮ subjected to 6% PAGE. DNA bands were electrotransferred onto in a total volume of 30 l containing the RNA-primer mixture, ϩ 10 mM DTT, 500 ␮M deoxynucleotide triphosphates, 50 mM nylon membrane (Hybond N ; Amersham) and immobilized to the membrane by baking the filter for 30 min at 80°C, followed by 1 Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 1 unit of PRIME RNase inhibitor (3 PRIME35 PRIME, Inc.), and 200 units of min of UV irradiation. The biotinylated PCR products were then Superscript II reverse transcriptase (Life Technologies, Inc.). detected using the Southern Light chemiluminescence detection The RT was initially allowed to take place at 25°C for 10 min. procedure (Tropix, Inc.). Quantification of RNA transcript expres- The reaction temperature was then gradually increased to 55°C sion was performed by densitometric analysis on X-ray films. The at a rate of 1°C/20 s. The RT reaction was allowed to continue

5 A. Eggert, G. M. Brodeur, and N. Ikegaki. A relative quantitative RT-PCR protocol for TrkB expression in neuroblastoma using GAPD as 4 X. X. Tang and N. Ikegaki, unpublished data. an internal control, manuscript in preparation.

Fig. 1 Expression of EPHB2, EPHB3, EPHB4, and EPHB6 transcripts in primary NBs. Expression levels of EPHB transcripts in NB were determined by semiquantitative RT- PCR (see “Materials and Methods”), using GAPD transcripts as an internal control. Nor- malized expression of each transcript was determined by taking the ratio between the densitometric units of transcripts of interest and those of GAPD. The Y axis represents the normalized expression level of EPHB transcripts, and numbers on the X axis represent tumor stages. The human fetal brain control is indicated as F.B. on the X axis. f, transcript levels expressed by low-stage tumors. F, transcript levels expressed by advanced-stage tumors. Œ, transcript levels expressed in MYCN-amplified tumors that are all in advanced stages. Differ- ential expression of EPHB transcripts in low- stage (stage 1, 2, and 4S) and advanced-stage (stage 3 and 4) tumors was assessed by a t test on the mean expression values between the two groups. P is shown when there is a statistically significant difference between the two groups. Differential expression of EPHB3 and EPHB6 in MYCN-amplified and nonamplified tumors was statistically significant, as indicated by the Ps.

expression of a given transcript was then normalized by taking the Semiquantitative RT-PCR analysis was used to examine ratio between the densitometric unit of the transcript and that of the EPHB and EFNB expression in NB tumors (see “Materials and internal control, GAPD. Methods”). Human fetal brain RNA was included in the RT- This semiquantitative RT-PCR analysis requires much PCR analysis as a positive control because transcripts encoding less RNA and allowed us to examine the expression of 15 or EPH family receptors and ephrin ligands are known to be highly more transcripts from a few micrograms of RNA sample. expressed in the developing nervous system (17, 22, 23). As This was critical because in some cases, we could only obtain described below, multiple EPHB transcripts (EPHB2, EPHB3, a very limited amount of tumor RNA. Moreover, the semi- EPHB4, and EPHB6) were expressed at various levels in all quantitative RT-PCR analysis provided results consistent NBs examined. EPHB1 expression was found to be either absent with those obtained by Northern blot analysis, indicating the or very low (data not shown). validity of this method for a quantitative analysis of tran- Expression of EPHB2, EPHB3, and EPHB4 Transcripts script expression (21). in NB. As shown in Fig. 1, all NBs examined expressed Statistical Analysis. A t test was used to examine pos- EPHB2, EPHB3, and EPHB4 transcripts at various levels. sible associations between clinical stages and the expression of There was no association between the expression levels of genes of interest as well as differential expression of EPHB and these transcripts and the clinical stage of NB. In addition, EFNB transcripts in MYCN-amplified and nonamplified NBs. there was no differential expression of EPHB2 or EPHB4 in The Pearson correlation coefficient (r) and P for each gene pair MYCN-amplified and nonamplified tumors. However, there examined were calculated. Analysis was performed using Stata was a trend in which MYCN-amplified tumors expressed version 5.0 (State Corp., College Station, TX). lower levels of EPHB3 than nonamplified tumors (P ϭ 0.0391; Fig. 1). RESULTS Association between High-Level EPHB6 Expression We found previously that EPHB2 and EFNB transcripts and Low-Stage NB, and Low-Level Expression of EPHB6 in were expressed in NB cell lines (17).4 These observations MYCN-amplified Tumors. Like EPHB2, EPHB3, and prompted us to examine the expression of EPHB2 transcripts as EPHB4, EPHB6 transcripts were also expressed at various lev- well as EFNB1, EFNB2, and EFNB3 transcripts encoding eph- els in all 47 NBs examined (Fig. 1). However, EPHB6 was rin-B1, ephrin-B2, and ephrin-B3, respectively, in primary NB. found to be expressed at higher levels in low-stage tumors (stage Because all three members of the ephrin-B subgroup can inter- 1, 2, and 4S) than in advanced-stage tumors (stage 3 and 4). act with all members of the EPHB receptor subgroup, the This association was statistically significant (P ϭ 0.0013; expression of EPHB1, EPHB3, EPHB4, and EPHB6 was also Fig. 1). In addition, MYCN-amplified tumors were found to examined. The expression of EPHB5 was not examined because express lower levels of EPHB6 than nonamplified tumors a human EPHB5 gene has not been identified. (P ϭ 0.0006; Fig. 1).

Fig. 2 Expression of EFNB1, EFNB2, and EFNB3 transcripts in primary NBs. The expression of EFNB1, EFNB2, and EFNB3 transcripts in primary NBs was determined as described in the Fig. 1 legend. f, transcript levels expressed by low-stage tumors. F, transcript levels expressed in advanced-stage tumors. Œ, transcript levels expressed in MYCN-amplified tumors that are all in advanced stages. The association between high-level expression of EFNB2 and EFNB3 and low-stage NB was statistically significant, as indicated by the Ps. Differential expression of EFNB2 and EFNB3 in MYCN-amplified and nonamplified tumors was statistically significant, as indicated by the Ps.

Expression of EFNB Transcripts in NB and Association DISCUSSION of High-Level EFNB2 and EFNB3 Expression with Low- A number of growth/differentiation factor receptors are Stage NB. EFNB1, EFNB2, and EFNB3 transcripts were known to influence the biological behavior of various tumors also detected at various levels in the same set of NB samples through autocrine and/or paracrine activation by interacting with (Fig. 2). There was no association of EFNB1 expression with their cognate ligands. Trk family receptor protein tyrosine ki- NB stage, and there was no differential expression of EFNB1 nases and their ligands, neurotrophins, have been suggested to expression in MYCN-amplified and nonamplified tumors. In play important roles in the biological behavior of NB. High contrast, higher levels of EFNB2 expression were found to be TrkA expression without concomitant expression of its ligand, associated with low-stage NB (P ϭ 0.0048; Fig. 2). More- nerve growth factor, is associated with low-stage, favorable- over, MYCN-amplified tumors expressed lower levels of prognosis NB (3). In contrast, the coexpression of TrkB and its EFNB2 than nonamplified tumors, and this differential ex- ligand, brain-derived neurotrophic factor, is associated with pression of EFNB2 was statistically significant (P ϭ 0.0023; Fig. 2). Similarly, high levels of EFNB3 expression were advanced-stage, unfavorable-prognosis NB, especially in NBs associated with low-stage NB (P ϭ 0.027; Fig. 2). As found with MYCN amplification (5). in EPHB6 and EFNB2, MYCN-amplified tumors expressed In this study, we found that transcripts encoding multiple lower levels of EFNB3 than nonamplified tumors. This dif- members of the EPHB receptor kinases and the ephrin-B ligands ferential expression of EFNB3 was statistically significant as were expressed together in primary NB. It should be mentioned well (P ϭ 0.0048; Fig. 2). that coexpression of multiple EPHB and EFNB transcripts was Correlation of EPHB6, EFNB2, and EFNB3 Expression also detected in NB cell lines (data not shown), indicating that with TrkA Expression. Because the expression of EPHB6, NB cells expressed these transcripts. The coexpression of EPHB EFNB2, and EFNB3 was found to be associated with low-stage and EFNB transcripts suggests that there are multiple autostimu- NB tumors, we examined whether their expression was correlation loops of EPHB receptors mediated by their ephrin-B lated with TrkA expression, a well-established prognostic ligands in NB. These autostimulation loops may, in turn, mod- marker of favorable NB (24–26). This analysis was designed to ulate biological behaviors of NB cells. evaluate the potential contribution of EPHB6, EFNB2, and To further explore the biological significance of EPHB EFNB3 expression to a favorable outcome of NB. The exami- receptors and their ephrin-B ligands in NB, we examined nation of TrkA in our study cohort of NB also served as a control whether their expression was associated with the clinical stage for sampling bias (see “Discussion”). As shown in Fig. 3, TrkA of NB and with well-established favorable and unfavorable expression was positively correlated with the expression of prognostic markers of NB, namely TrkA expression and MYCN EPHB6, EFNB2, and EFNB3 (P Ͻ 0.0001, P ϭ 0.0019, and amplification, respectively. The examination of TrkA expression P ϭ 0.0001, respectively). also served as a control for sampling bias because TrkA expres-

TrkA and low-stage NB was statistically significant, there was an overlap in the expression of these genes between low- and advanced-stage NB. This reflects the fact that the NB staging system is not a perfect prognostic parameter for risk assessment of NB. In fact, one of our goals is to identify such prognostic markers that clearly distinguish high-risk groups from low-risk groups in the general NB population. This raised the question of whether the expression of EPHB6, EFNB2, and EFNB3 could predict the outcome of NB, especially of intermediate-risk NB. In our study cohort, there were only 12 patients in this category. Nonetheless, there was a trend in which high expression of EPHB6, EFNB2, and EFNB3 was associated with a favorable outcome and low expression was associated with a poor outcome in the intermediate-risk NB group. Future studies in larger study cohorts will be required to determine whether the expression of EPHB6, EFNB2, and EFNB3 is predictive of NB outcome. In contrast to the positive correlation between EPHB6, EFNB2, and EFNB3 expression and TrkA expression, we found an inverse relationship between MYCN amplification and the expression of these genes. MYCN-amplified tumors expressed significantly lower levels of EPHB6, EFNB2, and EFNB3 than nonamplified tumors. TrkA expression in these MYCN-amplified tumors was also very low (P ϭ 0.0001; data not shown). As expected, MYCN-amplified tumors expressed substantially higher levels of MYCN than nonamplified tumors (data not shown). A similar finding has also been made for cell adhesion molecule CD44. High levels of CD44 expression are associated with low tumor stage and favorable outcome of NB, and MYCN- Fig. 3 Correlation between TrkA expression and the expression of amplified tumors express lower levels of CD44 than nonampli- EPHB6, EFNB2, and EFNB3 in primary NBs. Normalized expression fied tumors (27, 28). levels of EPHB6, EFNB2, and EFNB3 in primary NBs were plotted The inverse relationship between MYCN amplification and against those of TrkA in the corresponding tumors. The Pearson correlation coefficient (r) and P for each pair were calculated using Stata the expression of genes associated with favorable NB, including version 5.0 software. TrkA, CD44, EPHB6, EFNB2, and EFNB3, is an intriguing observation. However, mechanisms that account for this phe- nomenon remain elusive. Nonetheless, there are at least two possible explanations. High levels of MYCN expression in sion has been investigated in large study cohorts, and its expression MYCN-amplified tumors may negatively regulate the expression pattern in NB has been well established (24–26). In fact, our data of these genes that would provide NB with the favorable phe- on TrkA expression in NB in this study were consistent with the notype. Alternatively, the process leading to MYCN amplifica- previous finding: high TrkA expression was associated with low- tion may cause repression of these genes. Whichever the case, stage, favorable NB (P ϭ 0.0011; data not shown). These data suppression of genes that are associated with the favorable suggest that there is no apparent sampling bias in our study cohort; phenotype in MYCN-amplified NB may result in poor outcome. thus, the expression pattern of EPHB6, EFNB2, and EFNB3 tran- This feature, along with the other known effect of MYCN, scripts in the 47 NB specimens examined is likely to reflect that in namely, augmentation of NB cell growth (29), may ultimately the general NB population. cause the progression of MYCN-amplified NB. As described earlier, we found that higher levels of The association of high EPHB6, EFNB2, and EFNB3 ex- EPHB6, EFNB2, and EFNB3 expression were associated with pression with low-risk NB and the low EPHB6, EFNB2, and low-stage NB, and this association was statistically significant. EFNB3 expression in MYCN-amplified tumors raise some in- In addition, the expression of EPHB6, EFNB2, and EFNB3 was triguing possibilities as to their roles in the biology of NB. positively correlated with TrkA expression. Collectively, these EPHB6 lacks tyrosine kinase activity due to a nucleotide mu- data suggest that high-level expression of EPHB6, EFNB2, and tation at the region corresponding to the ATP acceptor site (30, EFNB3 is associated with a favorable outcome of NB. Although 31), suggesting that EPHB6 acts as a dominant negative member it is not clear if this association indicates a causal relationship, of the EPHB subgroup. Thus, EPHB6 might negatively regulate it is worth examining whether high-level expression of EPHB6, the kinase activity of other EPHB members by reducing the EFNB2, and EFNB3 could suppress aggressive behaviors of NB density of unoccupied ephrin-B ligands on the cell surface, in experimental model systems. which are otherwise available to the other EPHB receptors. On the It should also be mentioned that whereas the association other hand, the cytoplasmic domain of ephrin-B1 has been shown between high-level expression of EPHB6, EFNB2, EFNB3, and to suppress the transformation activity of several activated protein

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Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 1999 American Association for Cancer Research. High-Level Expression of EPHB6, EFNB2, and EFNB3 Is Associated with Low Tumor Stage and High TrkA Expression in Human Neuroblastomas

Xao X. Tang, Audrey E. Evans, Huaqing Zhao, et al.

Clin Cancer Res 1999;5:1491-1496.

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