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Journal of Clinical Oncology, Vol 24, No 10 (April 1), 2006: pp. 1546-1553 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.01.6196 Association of High-Level MRP1 Expression With Poor Clinical Outcome in a Large Prospective Study of Primary Neuroblastoma
From the Children's Cancer Institute Australia for Medical Research, Sydney, Australia; Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, IL; and Department of Statistics, University of Florida, and Children's Oncology Group Statistics Department, Gainesville, FL Address reprint requests to Murray D. Norris, PhD, Children's Cancer Institute Australia for Medical Research, P.O. Box 81, Randwick, 2031, Sydney, Australia; e-mail: m.norris{at}unsw.edu.au
PURPOSE: We have previously shown in a retrospective study that expression of the multidrug transporter gene MRP1 (ABCC1) is associated with outcome in neuroblastoma. We have now undertaken a prospective analysis to examine the independent prognostic significance of MRP1 expression in a large cohort of primary untreated neuroblastomas. PATIENTS AND METHODS: Two hundred nine diagnostic neuroblastoma samples from patients prospectively enrolled onto the Pediatric Oncology Group biology protocol 9047 were analyzed for expression of the MRP1, MDR1, MYCN, and TRKA genes using real-time polymerase chain reaction. Expression levels were correlated with established prognostic indicators and disease outcome. RESULTS: MRP1 expression was detected in all tumors analyzed, and levels were significantly higher in tumors with versus without MYCN amplification (P < .0001). High levels of MRP1 were highly predictive of both event-free survival (EFS; P < .001) and overall survival (OS; P < .001). High-level MYCN and low-level TRKA were also predictive of poor outcome. MDR1 expression demonstrated no prognostic significance. After adjustment for the effect of statistically significant prognostic indicators in multivariate models, MRP1 expression retained significant prognostic value for both EFS (hazard ratio = 3.0; P = .0011) and OS (hazard ratio = 2.5; P = .0095), whereas MYCN amplification did not have prognostic significance. CONCLUSION: The results of this prospective study confirm our earlier findings and support a clinically relevant role for MRP1 gene expression in neuroblastoma. These findings have implications for the biology, prognosis, and treatment of this disease and provide evidence that MRP1 is a bone fide molecular target for reversing chemotherapy resistance in aggressive drug-refractory neuroblastoma.
The pediatric malignancy neuroblastoma, which is the most common solid tumor of young children, arises in cells of neural crest origin. The majority of patients present with widely disseminated disease at diagnosis, and despite highly intensive treatment, the prognosis for children greater than 1 year of age with metastatic disease is dismal.1 A number of prognostic factors have been identified for this disease including age at diagnosis, tumor stage, unfavorable histology, and specific molecular genetic tumor aberrations.2 In particular, amplification of the MYCN oncogene has been shown to be a powerful adverse prognostic indicator.2,3 The risk of treatment failure in patients whose tumors display MYCN amplification is high, and this subset of children is generally treated with intensive multimodality therapy that includes chemotherapy, surgery, radiation, and myeloablative regimens with stem-cell rescue.4 For both children and adults with cancer, the development of multidrug resistance is one of the major causes of treatment failure. This phenomenon, whereby cells exposed to one or more of a range of cytotoxic drugs, including the commonly used vinca alkaloids, anthracyclines, and epipodophyllotoxins, develop cross resistance to other structurally unrelated drugs, has been well described in the literature.5 Some of the best-characterized mechanisms responsible for conferring a multidrug resistance phenotype include members of the ATP-binding cassette superfamily of multidrug transporters. We, and others, have previously demonstrated a close correlation between expression of the MYCN and multidrug resistance–associated protein 1 (MRP1) genes in primary neuroblastoma and cell lines.6,7 Likewise, the presence of MRP1 RNA determined by Northern analysis correlated with MYCN amplification and expression.8 We have also shown in a retrospective analysis of 60 primary untreated neuroblastomas that high MRP1 expression is strongly associated with reduced overall survival (OS) and event-free survival (EFS).9 We have now undertaken a prospective study of MRP1 gene expression in a large number of primary untreated neuroblastomas from a cohort of patients enrolled onto Pediatric Oncology Group (POG) biology protocol 9047. Expression levels of MRP1, as well as MYCN, MDR1, and TRKA, were related to well-established prognostic indicators, including age at diagnosis, MYCN oncogene amplification, clinical stage, and ploidy, and to clinical outcome. The results confirm our earlier findings that MRP1 is a powerful independent prognostic indicator of outcome in this disease.
Patients and Tumor Samples All patients were enrolled onto POG Neuroblastoma Biology Study 9047. Consecutively accrued neuroblastoma patients had their tumors snap frozen after diagnosis and stored in the POG Neuroblastoma Tumor Bank. As they accrued, batches of approximately 50 consecutive frozen tumor samples were shipped from the POG reference lab to the Children's Cancer Institute Australia for RNA isolation and gene expression analysis. After RNA isolation and cDNA synthesis from these batches, samples were stored frozen until the complete cohort was recruited. Gene expression studies were then undertaken at one time on this entire cohort to ensure uniform analysis. The final cohort of patients with follow-up data comprised 209 patients (205 patients were enrolled onto POG 9047 between 1996 and 1998, and the remaining four patients were enrolled between 1994 and 1995). Specimens for which identification numbers were unable to be linked to the clinical data were excluded from the analysis. The protocol was approved by individual institutional review boards, and informed consent was obtained for every patient registered on the study. The diagnosis of neuroblastoma was based on histologic examination of tumor specimens. In addition to at least a single bone marrow aspiration and single bone biopsy, extent of disease for all patients was evaluated with computed tomography scan and/or magnetic resonance imaging, technetium-99 bone scan, and skeletal radiographs.
MYCN and Tumor Cell Ploidy Analysis
Treatment
Real-Time Polymerase Chain Reaction Gene Expression Analysis
Western Blot Analysis of MRP1 Expression
Study Design and Statistical Analysis For each of the genes analyzed, a continuous range of PCR values was obtained for the cohort of tumors. Expression in an individual tumor was categorized as either low or high as follows. For MRP1, tumors with expression levels that were in the upper decile were categorized as having high expression because this cut point corresponded closely to the level of expression in the SKNSH cells, the cell line chosen as a reference point in our original analysis using conventional semiquantitative PCR.9 The statistical methodology used to identify the optimal cut point to maximize the difference in outcome between groups with low versus high MRP1 expression is described in detail in London et al.21 For MYCN and MDR1, tumors were categorized as having high expression based on a range of cut points, including the median, upper quartile, and upper decile PCR values. For the TRKA gene, tumors were categorized as having absent expression if the PCR value was less than 100 arbitrary units (range, 9 to 122,000 arbitrary units). The clinical characteristics and outcome of the patients were masked from the laboratory staff who performed the gene expression analysis. Laboratory analyses were undertaken at Children's Cancer Institute Australia, and statistical analyses were performed at the Children's Oncology Group (formerly POG) Statistics and Data Center in Gainesville, Florida.
Clinical Characteristics A total of 209 neuroblastomas were analyzed from patients enrolled onto POG biology protocol 9047. The clinical characteristics and outcome of the study population based on established prognostic indicators are listed in Table 1. The median follow-up time was 4.2 years, and the estimated 5-year EFS and OS rates for the entire cohort were 73% ± 3% and 77% ± 3%, respectively. As anticipated, infants, who composed 46% of the patients, had a significantly better EFS and OS compared with children older than 1 year. Eleven percent of tumors were MYCN amplified, and MYCN amplification, unfavorable tumor stage (INSS stages 3 and 4), and a diploid DNA content were all powerful predictors of poor EFS and OS. TRKA gene expression was significantly lower in MYCN-amplified tumors compared with tumors without gene amplification (P < .001; Fig 1A), and as previously reported,9,22 high-level TRKA was also strongly associated with improved EFS and OS.
MRP1, MYCN, and MDR1 Gene Expression Using real-time PCR analysis, we detected MRP1, MYCN, and MDR1 gene expression in all 209 tumors, although the levels of expression varied considerably. As anticipated, MYCN expression was significantly higher in MYCN-amplified tumors compared with tumors without gene amplification (P < .0001; Fig 1A). Confirming our previous studies,7,9 MRP1 expression was also significantly higher in MYCN-amplified tumors compared with tumors lacking MYCN amplification (P < .0001), whereas no such relationship existed for MDR1 expression (P = .176; Fig 1B). Of the 209 patients with known MYCN status and in whom MRP1 expression was determined, 13 patients had tumors displaying high MRP1 expression without MYCN oncogene amplification. Linear regression analysis revealed that there was no correlation between expression of the MYCN and MDR1 genes in the overall cohort of patients (r = 0.04; P = .62); however, in contrast, we observed a significant correlation between MYCN and MRP1 expression (r = 0.69; P < .0001).
Gene Expression and Outcome
The prognostic significance of MRP1 expression was also examined in subsets of patients with localized (nonmetastatic) disease (INSS stages 1, 2, and 3). Although by comparison with patients having metastatic (stage 4) disease, this cohort would otherwise be expected to have a relatively favorable outcome, a high level of MRP1 expression was associated with a significantly worse outcome (3A Fig), with a 5-year EFS rate of 56% ± 19% compared with 90% ± 5% for patients with low-level MRP1 expression in their tumors (P < .001). Similarly, for patients without MYCN amplification, low-level MRP1 expression was associated with a better outcome compared with patients with high MRP1 levels (81% ± 3% v 46% ± 14%, respectively; P < .001; Fig 3B). Furthermore, even for patients with metastatic (stage 4) disease, high levels of MRP1 expression identified a subset of patients with a particularly poor outcome compared with patients with low levels of MRP1 expression (< 14% ± 13% v 44% ± 7%, respectively; P < .001; Fig 3C). In all cases, similar effects were also observed for OS (data not shown).
Multivariate analysis was used to determine the independent prognostic significance of MRP1 gene expression. Tumor stage (1, 2, or 4S v 3 or 4), age at diagnosis (< 1 v  1 year), MYCN oncogene status (MYCN amplified v single copy), dichotomized MRP1 gene expression, dichotomized MYCN expression level, dichotomized TRKA expression level, and dichotomized MDR1 expression level were tested in a Cox proportional hazards model, and statistically significant variables were retained (model A, Table 2). In this model, MRP1 expression remained a highly significant independent indicator of both poor EFS (relative risk = 3.0; 95% CI, 1.5 to 5.7) and OS (relative risk = 2.5; 95% CI, 1.3 to 5.2). Model B is simply model A plus the forced addition of a term for MYCN status in the model. Although tumor stage and age also contributed significant predictive power in determining neuroblastoma outcome, there was little evidence of independent prognostic impact for MYCN status (model B, Table 2). Within the subset of the MYCN single-copy patients, MRP1 was more highly statistically significant than in the overall cohort (model C, Table 2).
The independent prognostic significance of MRP1 expression was also determined in clinically relevant subgroups in models adjusting for age, stage, MYCN status, and expression of TRKA, MDR1, and MYCN genes, whereby only statistically significant factors were retained. In patients with stage 4 disease (n = 63) or with stage 4 disease but excluding children less than 1 year old (n = 52), MRP1 expression was independently significantly prognostic for both EFS (P = .0002 and P = .0350, respectively) and OS (P < .0001 and P = .0107, respectively). In the subset of all stage 4 patients, the only other variable that was independently statistically significant was age (P = .0239 and P = .0294 for EFS and OS, respectively); whereas, in the subset of stage 4 patients aged greater than 1 year, no variables other than MRP1 expression were statistically significant in the multivariate model for either EFS or OS. In a multivariate model within the subset of patients with localized disease, MRP1 expression was independently significantly prognostic for EFS (P = .003), although not for OS, with no variables other than MRP1 expression being statistically significant in the multivariate model for EFS. The model for OS retained only one statistically significant term, MYCN status (P = .0246), although it should be noted that there is low power within this cohort because of the relatively small sample size. In a multivariate analysis of patients without MYCN amplification (n = 184), MRP1 expression demonstrated independent prognostic significance for both EFS (P < .0001) and OS (P = .0067). In addition to MRP1, age and stage were independently statistically significantly prognostic for EFS (P = .0135 and P = .0005, respectively) and OS (P = .0165 and P = .0001, respectively). Thus, although age and stage are clearly useful in stratifying MYCN-nonamplified patients, MRP1 provides further important information beyond that provided just by age and stage.
MRP1 Western Analysis
The results of this prospective study demonstrate that MRP1 gene expression is a powerful prognostic indicator for children with neuroblastoma. High levels of MRP1 expression were strongly associated with MYCN oncogene amplification and predictive of poor outcome not only in the overall cohort, but also in specific subgroups of patients who might otherwise be predicted to have either good or poor outcome, including patients with localized disease, non–MYCN-amplified patients, and patients with stage 4 disease. Importantly, after multivariate analysis in which the major well-established prognostic markers for disease outcome in neuroblastoma were included as variables, MRP1 expression demonstrated independent prognostic significance, whereas MYCN amplification did not. These findings contrasted with those of the MDR1 gene, where, irrespective of the cut point used, expression of this gene failed to predict clinical outcome. These results corroborate our earlier findings performed in a retrospective fashion in a smaller cohort of tumor samples9 and indicate that this multidrug transporter has an important role to play in mediating the clinical behavior of this tumor. The importance of confirming MRP1 expression as a powerful independent prognostic marker in neuroblastoma lies in its role in mediating resistance to multiple cytotoxic drugs. This study opens the way for MRP1 tumor levels to be used in the clinic as a guide to altering patient treatment. By demonstrating that MRP1 expression provides new and important insights into prognosis over and above those provided by age, stage, and MYCN status, this study should allow better risk assessment and stratification of patients in clinically relevant subgroups than what has been possible to date. Moreover, the findings of this study can now be incorporated directly into clinical practice either by replacing MRP1 substrate drugs in the combination chemotherapy of relevant at-risk patients or potentially by adding modulators of this multidrug transporter to standard chemotherapeutic protocols. These direct clinical implications make MRP1 a relatively unique molecular marker by comparison with other prognostic variables identified for neuroblastoma. MYCN oncogene amplification is the most well-known and established clinical biologic marker used in determining neuroblastoma prognosis, with numerous reports confirming its association with rapid tumor progression, advanced clinical stage, and poor outcome.1,3,23 The presence of this prognostic factor is often used as the basis for bone marrow transplantation,4 and devising more effective therapy for these high-risk neuroblastoma patients remains a significant challenge. In this regard, previous research in our laboratory has demonstrated that antisense oligonucleotide downregulation of MYCN in neuroblastoma cells both in vitro and in vivo16,24,25 leads not only to decreased tumorigenicity but also to decreased MRP1 expression. This, in turn, is accompanied by significantly increased sensitivity to cytotoxic drugs that are substrates for MRP1.24,25 Furthermore, studies involving the MRP1 promoter provide direct evidence of MYCN regulating MRP1.26 Collectively, these results provide strong evidence for MRP1 being an MYCN target gene and, hence, provide a critical link between the malignant and drug-resistant phenotypes of this disease. MYCN gene amplification is known to occur in 20% to 25% of primary untreated neuroblastomas.1,27 In this current study, however, only 11% of patients were found to be MYCN amplified compared with 22% in our original report associating MRP1 expression with clinical outcome.9 This under-representation in the present study can be related to the frequent diagnosis of stage 4 disease based on bone marrow sampling rather than tumor biopsy, reducing the number of advanced-stage tumor samples available for analysis. Given the strong correlation between MYCN amplification and MRP1 overexpression, those MYCN-amplified patients lost to the current study would most likely only further strengthen the association between high MRP1 expression and poor patient outcome. Despite the relatively low proportion of MYCN-amplified tumors, the current results demonstrate that assessment of MRP1 expression should be helpful in resolving some of the biologic heterogeneity that exists not only in aggressive late-stage disease, but also in patients with low-risk neuroblastoma. Apart from its value as an independent prognostic marker, this multidrug transporter also represents a potentially valuable molecular target for modulating the drug resistance phenotype of neuroblastoma and, ultimately, improving the outcome of patients with this disease. There are currently at least nine multidrug resistance–associated protein family members that have been described, and although the precise physiologic roles of the multidrug resistance–associated protein family are largely unknown, MRP1 seems to be critically involved in protecting cells against oxidative stress.28,29 MRP1 knockout mice demonstrate hypersensitivity to anticancer agents and an impaired response to inflammatory stimuli that can be linked to decreased secretion of leukotriene C4 from mast cells.30 Importantly, although MRP1 knockout mice display increased sensitivity to xenobiotics, they are nevertheless healthy and fertile, indicating that this transporter is not essential to life.30 Despite the role of MRP1 in defense against oxidative stress, the prognostic impact of MRP1 expression in neuroblastoma is most likely related to its role as a drug efflux pump. In common with P-glycoprotein, MRP1 is able to confer resistance to natural product drugs such as vinca alkaloids, anthracyclines, and epipodophyllotoxins. In addition, however, MRP1 confers resistance to heavy metals and is also a glutathione-S-conjugate efflux pump that mostly transports anionic phase II conjugates and has been shown to be involved in a number of glutathione-related cellular processes.31 Although MRP1 does not seem to able to confer resistance to alkylating agents such as cisplatin and cyclophosphamide, these drugs are known to undergo conjugation, and available evidence suggests that critical genes in this detoxification pathway can be coordinately induced with MRP1.32-34 Therefore, it will be interesting to analyze the expression of these genes in primary neuroblastoma and correlate their levels with those of MRP1. In the current study, we were able to demonstrate, in a small subset of neuroblastoma samples with sufficient material available for analysis, a significant association between high MRP1 RNA and protein levels. Interestingly, Lu et al35 recently examined MRP1, P-glycoprotein, and LRP protein levels in a cohort of 70 primary untreated neuroblastoma patients and found that, of these three drug-resistance proteins, only MRP1 was significantly associated with patient outcome. These results provide further evidence of the importance of MRP1 in neuroblastoma. In considering a role for MRP1 in clinical drug resistance, we have previously shown, in aggressive neuroblastoma, an increase in MRP1 expression after chemotherapy in a patient for whom tumor was available both at diagnosis and after treatment.9 In addition, a recent study investigated the contribution of basal levels of MRP1 and P-glycoprotein to drug sensitivity by examining the response to cytotoxic drugs of mouse cells in which these genes had been made nonfunctional through targeted disruption.36 Both transporters contributed markedly to drug resistance, although MRP1 conferred significant hypersensitivity to an even broader range of drugs than P-glycoprotein. Therefore, the results suggest that low-level MRP1 expression is an important parameter clinically and that modulators of this transporter could be useful in a wider range of tumors than has previously been believed. In particular, lung cancer has been highlighted as being a particularly attractive cancer for evaluating MRP1-specific inhibitors given the high levels of MRP1 expression but low levels of MDR1 expression observed in this malignancy.5 Moreover, studies that we have recently completed, in which mice harboring MYCN-driven transgenic neuroblastomas lacking the MRP1 gene, show greatly extended life span after treatment with MRP1-substrate drugs, including vincristine and etoposide, compared with neuroblastomas having wild-type MRP1, which strongly support the clinical relevance of MRP1 expression in conferring drug resistance (Burkhart et al; manuscript in preparation). In conclusion, this study demonstrates that high-level MRP1 expression is a powerful independent prognostic indicator in neuroblastoma and provides a link between the malignant and drug-resistant phenotypes of this childhood disease. Although MYCN is critical to the malignant process of neuroblastoma and amplification of this gene is commonly used as a basis for stratification to more intensive treatment regimens, the mechanism by which this oncogene influences neuroblastoma outcome has remained elusive. This study not only provides a possible explanation for this underlying mechanism but also highlights MRP1 as a potential valuable molecular target for the development of inhibitors that may be useful in reversing tumor-based drug resistance.
The authors indicated no potential conflicts of interest.
We thank the Pediatric Oncology Group Biology Committee for approving this study and providing tumor samples from the neuroblastoma tumor bank. The Children's Cancer Institute Australia for Medical Research is affiliated with the University of New South Wales and Sydney Children's Hospital.
This work was supported by grants from the National Health and Medical Research Council (Australia; M.H., G.M.M., and M.D.N.) and the Cancer Council New South Wales (Australia; M.H., G.M.M., and M.D.N.), Grant No. CA29139 from the National Cancer Institute, National Institutes of Health (S.L.C. and W.B.L.), and the Children's Oncology Group. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Brodeur GM: Neuroblastoma: Biological insights into a clinical enigma. Nat Rev Cancer 3:203-216, 2003[CrossRef][Medline] 2. Brodeur GM, Castleberry RP: Neuroblastoma, in Pizzo PA, Poplack DG (eds): Principles and Practices of Pediatric Oncology. Philadelphia, PA, Lippincott-Raven, 1997, pp 761-791 3. Seeger RC, Brodeur GM, Sather H, et al: Association of multiple copies of the N-myc oncogene with rapid disease progression of neuroblastomas. N Engl J Med 313:1111-1116, 1985[Abstract] 4. Bown N: Neuroblastoma tumour genetics: Clinical and biological aspects. J Clin Pathol 54:897-910, 2001 5. Gottesman MM, Fojo T, Bates SE: Multidrug resistance in cancer: Role of ATP-dependent transporters. Nat Rev Cancer 2:48-58, 2002[CrossRef][Medline] 6. Bader P, Schilling F, Schlaud M, et al: Expression analysis of multidrug resistance associated genes in neuroblastomas. Oncol Rep 6:1143-1146, 1999[Medline] 7. Bordow S, Haber M, Madafiglio J, et al: Expression of the multidrug resistance-associated protein (MRP) gene correlates with amplification and overexpression of the N-myc oncogene in childhood neuroblastoma. Cancer Res 54:5036-5040, 1994 8. Goto H, Keshelava N, Matthay KK, et al: Multidrug resistance-associated protein 1 (MRP1) expression in neuroblastoma cell lines and primary tumors. Med Pediatr Oncol 35:619-622, 2000[CrossRef][Medline] 9. Norris M, Bordow S, Marshall G, et al: Expression of the gene for multidrug-resistance-associated protein and outcome in patients with neuroblastoma. N Engl J Med 334:231-238, 1996 10. Shapiro DN, Valentine MB, Rowe ST, et al: Detection of N-myc gene amplification by fluorescence in situ hybridization: Diagnostic utility for neuroblastoma. Am J Pathol 142:1339-1346, 1993[Abstract] 11. Look AT, Hayes FA, Nitschke R, et al: Cellular DNA content as a predictor of response to chemotherapy in infants with unresectable neuroblastoma. N Engl J Med 311:231-235, 1984[Abstract] 12. Alvarado CS, London WB, Look AT, et al: Natural history and biology of stage A neuroblastoma: A Pediatric Oncology Group study. J Pediatr Hematol Oncol 22:197-205, 2000[CrossRef][Medline] 13. Bowman LC, Castleberry RP, Cantor A, et al: Genetic staging of unresectable or metastatic neuroblastoma in infants: A Pediatric Oncology Group study. J Natl Cancer Inst 89:373-380, 1997 14. Strother D, van Hoff J, Rao PV, et al: Event-free survival of children with biologically favourable neuroblastoma based on the degree of initial tumour resection: Results from the Pediatric Oncology Group. Eur J Cancer 33:2121-2125, 1997[CrossRef][Medline] 15. Katzenstein HM, Bowman LC, Brodeur GM, et al: Prognostic significance of age, MYCN oncogene amplification, tumor cell ploidy, and histology in 110 infants with stage D(S) neuroblastoma: The Pediatric Oncology Group experience—A Pediatric Oncology Group study. J Clin Oncol 16:2007-2017, 1998[Abstract] 16. Burkhart CA, Cheng AJ, Madafiglio J, et al: Effects of MYCN antisense oligonucleotide administration on tumorigenesis in a murine model of neuroblastoma. J Natl Cancer Inst 95:1394-1403, 2003 17. Winer J, Jung CKS, Shackel I, et al: Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro. Anal Biochem 270:41-49, 1999[CrossRef][Medline] 18. Kaplan EL, Meier P: Nonparametric estimation from incomplete observations. J Am Stat Assoc 53:457-481, 1958[CrossRef] 19. Peto R, Peto J: Asymptotically efficient rank invariant test procedures. J R Stat Soc A 135:185-198, 1972 20. Cox DR: Regression models and life tables. J R Stat Soc B 34:187-220, 1972 21. London WB, Castleberry RP, Matthay KK, et al: Evidence for an age cut-off greater than 365 days for neuroblastoma risk group stratification in the Children's Oncology Group. J Clin Oncol 23:6459-6465, 2005 22. Nakagawara A, Arima-Nakagawara M, Scavarda NJ, et al: Association between high levels of expression of the TRK gene and favorable outcome in human neuroblastoma. N Engl J Med 328:847-854, 1993 23. Schwab M: MYCN in neuronal tumours. Cancer Lett 204:179-187, 2004[CrossRef][Medline] 24. Haber M, Bordow S, Gilbert J, et al: Altered expression of the MYCN oncogene modulates MRP gene expression and response to cytotoxic drugs in neuroblastoma cells. Oncogene 18:2777-2782, 1999[CrossRef][Medline] 25. Norris M, Bordow S, Haber P, et al: Evidence that the MYCN oncogene regulates MRP gene expression in neuroblastoma. Eur J Cancer 33:1911-1916, 1997[CrossRef][Medline] 26. Manohar CF, Bray JA, Salwen HR, et al: MYCN mediated regulation of the MRP1 promoter in human neuroblastoma. Oncogene 23:753-762, 2004[CrossRef][Medline] 27. Maris JM, Matthay KK: Molecular biology of neuroblastoma. J Clin Oncol 17:2264-2279, 1999 28. Homolya L, Varadi A, Sarkadi B: Multidrug resistance-associated proteins: Export pumps for conjugates with glutathione, glucuronate or sulfate. Biofactors 17:103-114, 2003[Medline] 29. Renes J, de Vries EE, Hooiveld GJ, et al: Multidrug resistance protein MRP1 protects against the toxicity of the major lipid peroxidation product 4-hydroxynonenal. Biochem J 350:555-561, 2000[Medline] 30. Borst P, Evers R, Kool M, et al: A family of drug transporters: The multidrug resistance-associated proteins. J Natl Cancer Inst 92:1295-1302, 2000 31. Kruh GD, Belinsky MG: The MRP family of drug efflux pumps. Oncogene 22:7537-7552, 2003[CrossRef][Medline] 32. Kigawa J, Minagawa Y, Cheng X, et al: Gamma-glutamyl cysteine synthetase up-regulates glutathione and multidrug resistance-associated protein in patients with chemoresistant epithelial ovarian cancer. Clin Cancer Res 4:1737-1741, 1998[Abstract] 33. Kuo MT, Bao JJ, Curley SA, et al: Frequent coordinated overexpression of the MRP/GS-X pump and gamma-glutamylcysteine synthetase genes in human colorectal cancers. Cancer Res 56:3642-3644, 1996 34. Ishikawa T, Bao JJ, Yamane Y, et al: Coordinated induction of MRP/GS-X pump and gamma-glutamylcysteine synthetase by heavy metals in human leukemia cells. J Biol Chem 271:14981-14988, 1996 35. Lu QJ, Dong F, Zhang JH, et al: Expression of multidrug resistance-related markers in primary neuroblastoma. Chin Med J 117:1358-1363, 2004[Medline] 36. Allen JD, Brinkhuis RF, van Deemter L, et al: Extensive contribution of the multidrug transporters P-glycoprotein and Mrp1 to basal drug resistance. Cancer Res 60:5761-5766, 2000 Submitted February 14, 2005; accepted January 24, 2006.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
Ct method, which is a comparative method that involves taking the Ct value, defined as the cycle number that a particular sample achieves when it crosses a set fluorescence threshold intensity, normalized to the beta2-microglobulin control Ct value in each case and expressed relative to a calibrator.
