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Journal of Clinical Oncology, Vol 22, No 13 (July 1), 2004: pp. 2681-2690 © 2004 American Society of Clinical Oncology. DOI: 10.1200/JCO.2004.07.192 Coamplification of DDX1 Correlates With an Improved Survival Probability in Children With MYCN-Amplified Human NeuroblastomaFrom the Children's Hospital, Pediatric Hematology and Oncology, University of Marburg, Marburg, Germany Address reprint requests to Holger Christiansen, MD, The Children's Hospital of Marburg, Pedriatric Oncology and Hematology, Neuroblastoma Research Laboratory, Deutschhausstrasse 12, 35037 Marburg, Germany; e-mail:Holger.Christiansen{at}staff.uni-marburg.de
PURPOSE: Amplification of the MYCN oncogene at chromosome 2p24-25 identifies an aggressive subtype of human neuroblastoma with a poor clinical outcome. Differences in amplicon structure and coamplification of genes telomeric and centromeric to the MYCN oncogene have previously been described. A relevant role of gene coamplification for neuroblastoma pathogenesis remains elusive. PATIENTS AND METHODS: We analyzed 98 primary neuroblastoma tumors with MYCN amplification for coamplification of seven additional genes at chromosome 2p24-25 (DDX1, NAG, NSE1, LPIN1, EST-AA581763, SMC6, and SDC1). Two semiquantitative multiplex polymerase chain reactions were used to obtain the amplification status of the target genes in relation to a reference gene on chromosome 2q (Inhibin-beta-b). Furthermore, mRNA expression pattern of coamplified genes in a subset of tumors was analyzed. RESULTS: Our results show that the frequency of gene coamplification on 2p24-25 in neuroblastoma is correlated directly to the physical distance to MYCN. Coamplification is correlated to an upregulated gene expression for DDX1 and NAG. Coamplification of the DDX1 gene within 400kb telomeric to MYCN identifies a subgroup of advanced stage neuroblastoma tumors with a more favorable outcome (P = .027, log-rank test). A high expression level of DDX1 is associated with a trend towards a better survival probability (P = .058, log-rank test). CONCLUSION: Our results indicate that DDX1 coamplification correlates with a better prognosis and improved patient survival in MYCN-amplified neurobastoma.
Neuroblastoma is the most common extracranial solid cancer in childhood and responsible for approximately 15% of all childhood cancer deaths. The 6-year overall survival probability of all patients with neuroblastoma is approximately 60%. Amplification of the MYCN oncogene is the most extensively studied biologic marker for an unfavorable prognostic outcome in patients with neuroblastoma.1–3 The overall survival probability for patients with MYCN amplification is less than 30% in the first 6 years after diagnosis (authors' own data of 216 patients with MYCN-amplified neuroblastoma). Because of the poor prognosis, these patients are treated by combination of high-dose chemotherapy, surgical therapy, and different experimental therapy strategies depending on the therapy protocol. However, some MYCN-amplified tumors can be distinguished by a better response to the combined treatment resulting in a better prognosis for the individual patient, as 7% of patients with MYCN-amplified neuroblastoma survive more than 72 months after initial diagnosis and survival probability after 72 months is more than 95% for these patients (authors' own unpublished data of 1,443 patients registered from 1981 to 2000). Thus, amplification of the MYCN gene does not seem to be a death warrant per se, because long-time survival is possible for patients even with MYCN amplified neuroblastoma.4 Amplification of MYCN can be found in double minutes and homogenously staining regions.5 The role of coamplification of additional genes close to the MYCN locus on 2p24-25 for the progression of the disease or resistance to therapy in neuroblastoma tumors is still unclear. Coamplification of different genes in close distance to MYCN had been reported previously.6–11 Furthermore, a diversity in amplicon structure and size was observed in more detailed genomic analyses.12 To get further information of the amplicon structure and the clinical relevance of coamplified genes within MYCN-amplified tumors, we investigated seven genes located in different distances telomeric and centromeric to MYCN on 2p24-25 for coamplification in a cohort of 98 primary neuroblastoma tumors with known MYCN gene amplification and a long-time follow-up.13 DDX1 (DEAD/H-BOX 1) is a putative RNA helicase containing the characteristic D(Asp)-E(Glu)-A(Ala)-D(Asp)-box conserved sequence motif. Proteins of this family have been described to be involved in RNA processing, and thus to influence transcription, splicing, translation, and intracellular transport of different RNA subfamilies.14 Genes of the DEAD/H protein family were found to be involved in tumorigenesis and cellular proliferation,15 as well as in antiproliferative processes.16 The preferential expression pattern of DDX1 in cells of neuronal origin and the coamplification with MYCN in neuroblastoma and retinoblastoma cell lines was first described in 1993.17 Several authors described coamplification of DDX1 in neuroblastoma cell lines and primary tumors as well.6,8–10,18–20 Some authors described a trend towards a worse prognosis for patients with DDX1 coamplified tumors.6–11 Because of the relative homogeneity regarding prognosis of the neuroblastoma patients in these studies, statistical significance was not obtained. The NAG (neuroblastoma amplified gene) gene locus encodes for a 155.9 kDa protein, including domains for nuclear-localization, coil-coil-, and leucine-zipper-motifs. The function of NAG is yet unknown. Coamplification of NAG within the MYCN amplicon has been reported first by Wimmer et al.10,21 Recently, Scott et al11 characterized the amino acid sequence and the genomic structure of NAG systematically, and found an association of NAG coamplification and lower stage disease in human neuroblastoma with MYCN amplification. Non-SMC element 1(NSE-1) is described to be a structural maintenance complex component of a smc5/smc6 complex involved in chromosome structure organization, DNA repair, and cellular proliferation.22 LPIN1 is a nuclear protein required for normal adipose tissue development, differentiation, and metabolism processes.23 As the region in close vicinity centromeric to MYCN is lacking yet known genes, we investigated the coamplification of the expressed sequence tag (EST) AA581763 that is located 87kb centromeric to MYCN. This EST was identified as a spliced EST in a kidney tumor cDNA library (NCI-CGAP_Kid6, Homo sapiens EST library) predicting an encoded gene in this region. Structural maintenance complex protein 6 (SMC6) is a chromatin bound protein described to act as a regulating factor in cellular proliferation and cell division processes.24 Thus, a second gene for a protein belonging to the same DNA organizing SMC-complex is located in a close distance of 3000 kb around MYCN, as it is shown for NSE1.22 Syndecan1 (SDC1) is a cell surface proteoglycan acting as a receptor for the extracellular matrix and represents a protein critical for WNT1-induced tumorigenesis in the mouse mammary gland.25,26 In this study, we investigated a large cohort of MYCN-amplified human neuroblastomas with a long mean follow-up time of 48.6 months for the patients alive. Our results show a direct dependency of coamplification frequency on the physical distance to MYCN. Coamplification of DDX1 and NAG are both correlated to an upregulated mRNA expression. Furthermore, our data provide statistical evidence that coamplification of DDX1 correlates with an improved survival probability for patients with MYCN-amplified neuroblastoma.
Patients We studied primary tumor specimens from 98 children with MYCN-amplified neuroblastoma diagnosed in Germany from 1981 to 2000. The investigated cohort is representative for the group of MYCN-amplified neuroblastomas during this time period, regarding age at diagnosis, disease stage, and the follow-up time. All neuroblastoma diagnoses were confirmed by histologic assessment of a tumor specimen obtained at surgery. The tumors were classified according to the International Neuroblastoma Staging System criteria.27 Our study group consisted of four stage 1 patients, one stage 2 patient, 25 stage 3 patients, 60 stage 4 patients, and eight stage 4S tumors. The median patient age at diagnosis was 20.5 months (range, 0.03 to 164.9 months). The median follow-up time for all 98 patients was 20.9 months (range, 0.36 to 138.9 months). The median follow-up time of the patients that died of the disease was 15.8 months (n = 63) compared with 42.2 months for patients alive (n = 35). All patients were treated according to previously described protocols with confirmed consent for therapy and study procedures.28,29 Because of limited availability of comparable amounts of tumor tissue, a subset of 19 neuroblastoma specimens was selected for gene expression analyses to ensure material homogeneity used for RNA isolation and cDNA synthesis in order to get comparable results. The median age at diagnosis of this subset was 25.8 months (range, 1.3 to 78.6 months). The median follow-up time for the 19 patients was 20.6 months (range, 2.3 to 92.5 months).
Tissue Preparation
DNA Extraction
RNA Extraction
Reverse Transcription
MYCN Amplification (Southern Blot; reference method)
Primer Design
Multiplex PCR The first multiplex-PCR for evaluation of the genomic amplification status contained the primer pairs of MYCN, DDX1, NAG, LPIN1, SDC1, and Inhibin-beta-b on chromosome 2q13 as single copy reference. The second multiplex-PCR contained the primer pairs of NSE1, EST-AA581763, SMC6, and again MYCN as amplification status control and Inhibin-beta-b as single copy reference. The amplified PCR fragments (10 µL of the PCR reaction) were separated in a 2% agarose gel by electrophoresis, identified by ethidium bromide staining, and documented by the Image Master VDS software (Amersham Biosciences, Piscataway, NJ). Relative band intensity of each specific PCR-band was calculated with Image Master VDS software. The amplification status was expressed as relative band intensities of the genes of interest on 2p24-25 to the band intensitiy of the reference gene Inhibin-beta-b (ratio, target gene/inhibin-beta-b). To define the single copy status of the band intensity for each gene, we used human placental and kidney DNA as single copy reference. Fifteen separate PCR reactions were done with either placental and kidney DNA. Mean and standard deviation for the ratios of the single copy status for each gene were determined. Double of the standard deviation above the mean ratio was defined as threshold. A positive amplification status was defined as a band intensity ratio (target gene/Inhibin-beta-b) above the threshold. To verify our assays, we compared the amplification status of MYCN determined by the PCR-threshold in both multiplex PCRs for all 98 tumors with a reference method (southern blot, described above). All of the investigated neuroblastoma tumors were defined as MYCN amplified in both of our PCR-assays.
Real-Time PCR
Threshold cycles were determined using the iCycler PCR detection system and automated computer software (Biorad; Laboratories, Hercules, CA) following the manufacturer’s protocols. Expression analysis of beta-actin and the target genes were perfomed simultaneously in one experiment. Each experiment was performed twice. The mean Ct-values of each sample were used to perform mathematical analysis. Relative expression pattern of the target genes to beta-actin in each sample and in relation to the mean of the whole investigated population was performed using the 2-D
Distance Calculation
Statistical Analysis
Multivariate analysis was perfomed using the Cox regression analysis. Statistical analyses were performed with the SPSS version 10.0 software (SPSS Inc, Chicago IL). P values of
Gene Coamplification Frequency Within the MYCN Amplicon Correlates With the Physical Distance to MYCN Ninety-eight primary neuroblastoma tumor samples with known MYCN amplification were investigated with two multiplex PCR for coamplification of seven genes on chromosome 2p24-25 (Fig 1). All tumors showed positive MYCN-amplification status in Southern blot analysis (data not shown). In accordance to Southern blot analysis, MYCN-amplification status was found to be positive in both multiplex PCRs in all 98 tumor samples.
Besides MYCN amplification 64 (65.3%) of the investigated tumors showed coamplification for DDX1, 42 (42.9%) for NAG, 21 (21.4%) for NSE1, five (5.1%) for LPIN1, 89 (90.8%) for EST-AA581763, 12 (12.2%) for SMC6, and four (4.1%) for SDC1. No tumor sample was found to be coamplified for all seven investigated genes. The most extensive amplicon size included both marginal genes (LPIN1 and SDC1), but showed a deleted region containing the SMC6 locus. With consideration of current gene mapping data, our results show that the frequency of gene coamplification on 2p24-25 in human neuroblastoma is directly depending on the physical distance to MYCN in our population of 98 MYCN amplified neuroblastomas (correlation coefficient, –0.857; P = .007, Spearman-Rho test; Fig 2).
Coamplification Status of the Investigated Genes Does Not Correlate to Stage or Age at Diagnosis As clinical parameters such as age at diagnosis and stage of disease are of interest, even in MYCN amplified neuroblastoma in respect to prognosis, we looked for statistical correlations of these parameters to the coamplification status of the investigated genes. We found no significant correlation between coamplification of an investigated gene and a clinical parameter as stage (stages 1, 2, and 4S v 3 and 4, or stages 1, 2, 3, 4S v 4) or age at diagnosis (< 12 months v > 12 months; data not shown), with exception of SMC6, which was found to be coamplified more frequently in infants (age < 12 months at diagnosis) compared with older patients (P = .017, Fisher’s exact test).
Coamplification of DDX1 Correlates Significantly With an Enhanced Survival Probability in Patients With MYCN Amplification
Survival analysis of 1,443 neuroblastoma tumors of different stage, age, and MYCN amplification status indicate no more statistical relevant change in survival probability after a 6-year follow-up time (probability to die of the disease after surviving 72 months after diagnosis < 1%; authors' own data, not shown). Thus, to focus on a definitively observed cohort, we excluded patients with a short time follow-up (< 72 months) and perfomed survival analysis only for patients diagnosed before 1996 (Fig 3; n = 51; minimum follow-up time, 72 months after initial diagnosis). In this group, DDX1 coamplification identifies patients with a 35% chance of long-time survival. In contrast, all patients without DDX1 coamplification died within 28 months after initial diagnosis (Fig 3; P = .01, Kaplan-Meier analysis). As survival rates of DDX1 coamplified or noncoamplified patients showed no difference within the first 24 months after diagnosis, the prognostic relevance of DDX1 coamplification status was of particular interest for patients with MYCN-amplified neuroblastoma that survived the first 2 years after diagnosis. A reason for this finding might be the advanced stage of disease for most patients at the time of diagnosis and the intensive therapy protocol for MYCN-amplified neuroblastoma patients independent of the gene coamplification status. A re-evaluation of survival analysis for the mentioned patients (survivors 24 months after diagnosis, n = 46) resulted in a survival probability for the next 4 years of 78.9% with DDX1 coamplification compared with 30.4% without DDX1 coamplification (P < .0005, Kaplan and Meier analysis). Multivariate Cox regression analysis showed that prognostic relevance of DDX1 coamplification is independent of age at diagnosis (P = .033) or stage of disease (1, 2, 3, and 4S v 4; P = .05) in our investigated group (Table 2). Thus, coamplification of DDX1 defines a better prognostic subtype of MYCN-amplified neuroblastoma independent of clinical prognostic factors.
However, patients with DDX1 coamplified MYCN-amplified neuroblastomas are still at increased risk when compared with patients with MYCN-nonamplified tumors. Survival analysis of 1,149 neuroblastoma patients without MYCN amplification diagnosed from 1981 to 2000 out of our database revealed a median survival of 187.8 months (95% CI, 138.3 to 236.3 months) and a 6-year survival probability of 60.4% (compare with Table 1). The 64 patients with MYCN amplification and DDX1 coamplification have a worse prognosis compared with the patients without MYCN amplification (P = 1.8 x 10–9, Kaplan-Meier analysis). The 34 patients with MYCN amplification but without DDX1 coamplification show even worse prognosis compared with patients without MYCN amplification, respectively (P = 5.6 x 10–19, Kaplan-Meier analysis).
Amplification Correlates With Elevated Gene Expression Level for DDX1 and NAG
Expression of DDX1 mRNA Is Associated With an Improved Survival
It is generally accepted that the MYCN oncogene represents the core of the chromosomal region on 2p24-25, frequently amplified in human neuroblastoma tumors as no genes in this region have been reported to be amplified independent of the MYCN gene locus.10,12 Mechanisms of gene amplification are complex. Thus, it is yet unclear whether it is possible for a highly proliferating clone of cells to control the size and structure of resulting amplicons, as we can see a variety of amplicon sizes, rearrangments, deletions, and resulting structures in MYCN amplicons in neuroblastoma tumors.12 In accordance to these previous observations, we can see a high number of gene coamplification combinations in our investigated population defining differences in amplicon size and structure and, in a subset of amplicons, deleted regions. Most of the investigated MYCN amplicons (66 of 98) span shorter distances telomeric and centromeric of the MYCN locus, including DDX1, NAG, and EST-AA581763. Genes located in farther distances from MYCN are coamplified less frequently (Fig 2). For all investigated genes, we can show that frequency of gene coamplification within the 2p24-25 amplicon in neuroblastoma is directly correlated to the physical distance to MYCN. These results underline the role for MYCN as the core gene within the 2p24-25 amplicon, and make a superordinated, controlled determination of the amplicon size and structure unlikely. Coamplification of DDX1, NAG, SDC1, LPIN1, and NSE1 within the MYCN amplicon have been reported previously7,8,33 and our data are in accordance with the reported coamplification frequencies and maximal amplicon sizes. Despite these genes, we can first describe the coamplification of SMC6 and a locus in centromeric vincinity to MYCN as EST-AA581763. Interestingly, SMC6 belongs to the same DNA organizing SMC complex as NSE1,22 thus, two genes encoding for proteins involved in DNA damage repair lie within 3,000 kb distance telomeric and centromeric to MYCN and are coamplified in a subset of tumors.34 As expected, investigated genes located most distantly to MYCN in our study (SDC1 and LPIN1) are coamplified in only 4% and 5%, respectively. Thus, we could show that these genes statistically earmark the maximal expansion of the MYCN amplicon, as almost 95% of the investigated neuroblastoma tumors showed amplicon sizes of smaller extension. We hypothesize that genes in farther distances telomeric or centromeric to MYCN have to be found even more rarely coamplified with MYCN. Reiter and Brodeur12 found a high frequency of rearrangements in investigated MYCN amplicons. They hypothesized that most of the coamplified sequences belonging to normally functional genes would lack their genomic function, because genes would not lie within a physiological genomic context in the DNA amplicons compared with their context (eg, promotor regions) in the wild type chromosomal region. They defined a 130 kb core region for the MYCN amplicon containing the MYCN gene and a region of 125 kb centromeric and 5 kb telomeric to MYCN that is amplified in 32 (97%) of 33 MYCN amplified tumors. However, recent studies by Chen et al35 could show that rearrangements, even at the MYCN locus, occur late in amplicon formation, as they could be found less frequently compared with the according unrearranged sequences in the same cell line. These results indicate that even the case of existing rearranged sequences within an amplicon structure does not exclude the existence of coamplified functional regions in far distance to the amplicon’s core. Based on recent BLAT alignment data, DDX1 and NAG are the closest yet known functional sequences telomeric to MYCN on 2p24-25, but they do not lie within the formerly defined core region. The coamplification frequency for both genes based on our data is in accordance to this finding, as both genes are coamplified less often compared with sequences within the defined core region (Fig 2). We found EST-AA581763 coamplified in 91% of all 98 investigated cases according to the close genomic localization 87.3 kb centromeric to MYCN and thus, within the defined amplicon core region. Conspicuously, the centromeric gene SCM6 is coamplified less often than the telomeric genes DDX1 or NAG, although it is assigned in a closer distance to MYCN. This finding might be explained as a result of the high variety of amplicon borders and rearrangements described outside the defined core region by Reiter and Brodeur.12 Another argument for functionality of coamplified genes is given by the frequent finding of an upregulated mRNA expression level of the respective amplified gene.35 For neuroblastoma tumors, most data concerning correlation of gene amplification and expression exist for MYCN, DDX1, and NAG.9,36,37 Our expression analysis of 19 out of the 98 primary neuroblastomas with MYCN amplification also revealed significantly higher mRNA expression levels for DDX1 and NAG in tumors with coamplification for the according gene, compared with noncoamplified tumors. It is thought that genetic alterations, deletions of chromosomal regions, amplification, and coamplification of genes in tumor cells could lead to a growth advantage for the malignant cell clone through either loss of tumor suppressor genes or alteration of gene products with oncogenic potential. Both aberrations qualify the malignant clone to overcome physiological growth control mechanisms. Thus, it does not seem to be conclusive that coamplification and resulting overexpression of a gene might be responsible for a better prognosis in malignant disease. However, we can show that coamplification of DDX1 within the MYCN amplicon correlates with an improved long-term survival probability in neuroblastoma patients (Table 1; Fig 3). These data are, in part, contrary to previous investigations of smaller sample studies that describe no correlations or trends towards a worse prognosis in case of DDX1 coamplification (n = 13, 16, 27, 35, 66, and 45 respectively).6–11 Interestingly, the survival analysis of 66 neuroblastoma patients recently published by De Preter et al10 shows a trend toward a better prognosis for patients in the groups of DDX1 and DDX1/NAG coamplified neuroblastomas, which is in accordance with our investigation. However, their analysis did not reach statistical significance, probably because of the shorter follow-up time and the smaller cohort size (n = 66). DDX1 is a putative RNA helicase containing the characteristic D(Asp)-E(Glu)-A(Ala)-D(Asp)-box highly conserved sequence motif. The DDX family proteins share function by altering RNA secondary structure, thus they are found to influence translation initiation, splicing, and ribosome and splicesome assembly. Godbout et al19 identified a homologous region in the amino acid sequence for DDX1 to heterogenous nuclear ribonucleoprotein-U (hnRNP-U), a RNA binding and processing protein.38 Furthermore, DDX1 has recently been identified as an interaction partner for heterogenous nuclear ribonucleoprotein K (hnRNP-K), and the authors can show that the ability of RNA unwinding of DDX1 is dependent on complex formation with hnRNP-K in vitro.39 hnRNP-K is a multifunctional protein involved in regulation of transcription, translation, nuclear transport, and signal transduction.40–42 Recent findings identify translational silencing of differentiation regulating proteins as a major specific function of active hnRNP-K. This translation control activity is regulated by c-Src kinase.43 Thus, DDX1 might be directly involved in RNA processing by its local homology to hnRNP-U, and in translational silencing processes by its interaction with hnRNP-K. Our data also provide a trend towards a better prognosis in patients with neuroblastomas coamplified for NAG. Scott et al11 recently characterized the NAG transcript and genomic structure. These authors found a significant association between coamplification of NAG and lower stage of the MYCN-amplified tumors. These data are in accordance to our findings for a better survival in the NAG-coamplified group of tumors, although there is no correlation in MYCN-amplified neuroblastoma between stage and survival, per se. In our group of 98 neuroblastoma patients, we can see a significantly better outcome for patients with tumors of stage 1, 2, 3, and 4S at the time of diagnosis compared with stage 4 (n = 98; P = .016, Kaplan and Meier analysis; Table 2). In contrast to our DDX1 coamplification data, this analysis loses statistical significance with exclusion of patients with a short time-to-follow-up period (< 72 months; n = 51; P = .44, Kaplan and Meier analysis). As coamplification of a gene involved in mechanisms of cell proliferation and/or division (eg, RNA processing like DDX1) may lead primarily to a growth advantage for the according cells, it may also lead to a better chemotherapy response in these tumors. Thus, more than a direct antiproliferative effect on tumor growth, enhanced chemosensibility should be discussed as a more likely reason for the better outcome of patients with DDX1 coamplification. Comparable findings in childhood acute lymphoblastic leukemia demonstrate that amplification of the AML1 gene or involvement of AML1 in a gene fusion product, as a result of a translocation t(12;22), results in a better response to chemotherapy treatment of the leukemic cells and thus a better prognosis for the patients.44–49 These data emphasize the possibility for a factor-mediating clonal growth advantage to sensitize cells to chemotherapy treatment. Taken together, our data show that coamplification frequency of genes in primary human neuroblastoma tumors with MYCN amplification is directly correlated with the physical distance to the core region of the amplified chromosomal region on chromosome 2p24-25. Our findings strongly imply a role for DDX1 in tumorigenesis and further tumor development of MYCN-amplified human neuroblastoma tumors. Thus, we show that coamplification of DDX1 identifies a subgroup of patients with a significantly better survival probability. Furthermore, the prognostic impact of DDX1 coamplification is statistically independent of tumor stage and patient's age at diagnosis. One possible mechanism for our findings might be a functional interaction of DDX1 with hnRNP-K in influencing the translational control of genes involved in cellular differentation processes. More information about the regulation of these differentiation influencing pathways is needed for future therapeutic approaches that may aim at influencing DDX1 and hnRNP-K pathways contributing differentiation induction in human neuroblastomas. Our findings are of substantial clinical interest for patients with MYCN-amplified neuroblastoma for evaluation of survival probability and reevaluation after initial treatment. However, further confirmation of our correlated data is needed to evaluate a possible practical usage for the coamplification and expression status of DDX1 as an additional prognostic marker for MYCN-amplified neuroblastoma.
The authors indicated no potential conflicts of interest.
We thank our colleagues from about 100 German pediatric centers for providing us with neuroblastoma tumor material since 1981. We thank Prof Martin Eilers (IMT Marburg) for critical reading of the manuscript, Prof Berthold and Dr Hero (University of Cologne) for providing clinical data, and Prof Lampert (University of Gießen) for support of our research group.
This work contains parts of the doctoral theses of Patricia Imisch. Authors' disclosures of potential conflicts of interest are found at the end of this article.
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Leuk Lymphoma 41:615–623, 2001[Medline] Submitted July 25, 2003; accepted April 2, 2004.
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Copyright © 2004 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
Ct method.
.05 were regarded as significant. 
72 months (n = 51) after initial diagnosis and (C) for DDX1 mRNA expression in a subset of patients (n = 19).
