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Clinical Cancer Research Vol. 11, 8661-8668, December 15, 2005
© 2005 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Expression of Multidrug Resistance–Associated Proteins Predicts Prognosis in Childhood and Adult Acute Lymphoblastic Leukemia

Sabine L.A. Plasschaert1, Eveline S.J.M. de Bont1, Marike Boezen2, Dorina M. vander Kolk3, Simon M.J.G. Daenen3, Klaas Nico Faber4, Willem A. Kamps1, Elisabeth G.E. de Vries5 and Edo Vellenga3

Authors' Affiliations: Departments of 1 Pediatric Oncology and Hematology, 2 Epidemiology, 3 Hematology, 4 Gastroenterology and Hepatology, and 5 Medical Oncology, University Medical Center Groningen, Groningen, the Netherlands

Requests for reprints: Edo Vellenga, Division of Hematology, Department of Internal Medicine, University Medical Center Groningen, Hanzeplein 1, 9713 GZ Groningen, the Netherlands. Phone: 31-50-3612354; Fax: 31-50-3614862; E-mail: E.Vellenga{at}int.umcg.nl.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Patients with acute lymphoblastic leukemia (ALL) are treated with a variety of chemotherapeutic drugs, which can be transported by six multidrug resistance–associated proteins (MRP). These MRPs have strongly overlapping functional activities. The aim of this study was to investigate the expression levels of MRP1 to MRP6 and study their effect on prognosis.

Experimental Design: The mRNA expression levels of MRP1 to MRP6 were analyzed by quantitative real-time PCR in leukemic blasts of 105 de novo ALL patients (adults, n = 49; children, n = 56) including 70% B-lineage and 30% T-lineage ALL patients.

Results: Adults showed a higher expressions of MRP1 (P = 0.008), MRP2 (P = 0.026), and MRP3 (P = 0.039) than children. Interestingly, this difference disappeared when patients were categorized based on clinical outcome. Relapsed patients showed a higher expression of all MRP genes, except MRP4. For the total group of ALL patients, the expressions of MRP1, MRP2, MRP3, MRP5, and MRP6 predicted relapse. Moreover, high expression of all MRP genes, except MRP4, was associated with a reduced relapse-free survival in children and adults (MRP1, P = 0.005; MRP2, P = 0.008; MRP3, P = 0.001; MRP5, P = 0.016; MRP6, P = 0.037).

Conclusions: The present study shows that a subset of ALL patients with high MRP expression has an unfavorable prognosis independently of age.

The prognosis of acute lymphoblastic leukemia (ALL) has improved considerably during the last years. However, children persistently have a better survival than adults (1, 2). This is linked to a higher tolerance of children for chemotherapy with the result that they can receive more intensive treatment. Additionally, intrinsic cellular differences might exist between childhood versus adult ALL cells. This is supported by studies suggesting that the malignant transformation originates at different levels in the lymphoid stem cell compartment (3, 4). These differences might be translated in an altered drug sensitivity profile (5). Chemosensitivity is mainly regulated by the ATP-binding cassette superfamily of membrane transporters. Of this family, especially P-glycoprotein, breast cancer resistance protein, multidrug resistance–associated protein 1 (MRP1), and MRP2 have been studied in ALL. Thus far, high P-glycoprotein activity has been associated with poor clinical outcome mainly in adults (6–9). For MRP1 and MRP2, no correlation with prognosis has been observed in childhood ALL (7, 8, 10, 11) whereas the effect of breast cancer resistance protein on prognosis is yet unknown (12). Currently, eight MRP genes have been identified, of which the MRP transporters (MRP1-6) are known to be involved in extruding substrates that are generally used in the treatment of ALL, including doxorubicin, vincristine, etoposide, 6-mercaptopurine, and methotrexate (13–22). In view of the strong overlapping activities of the different MRPs, studying only one or two of these transporters can potentially underestimate the clinical relevance of these proteins. The aim of this study was to investigate the mRNA expression of all six relevant MRP genes (MRP1-6) in childhood and adult ALL and study their effect on patient prognosis. Our results show that a subset of patients with high MRP expression has an unfavorable prognosis independently of age.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients. During the period of 1989 to 2002, 189 patients were newly diagnosed with ALL at the University Medical Center Groningen. Bone marrow and peripheral blood samples of these patients were collected and cryopreserved after informed consent. Patients were included in the study if a sufficient number of bone marrow or peripheral mononuclear cells were available. The majority of the childhood samples were collected between 1999 and 2002 and most of the adult patient samples were obtained between 1996 and 2002. The clinical data from all patients were obtained, including age at diagnosis, gender, WBC count at diagnosis, lactate dehydrogenase (LDH), immunophenotype, cytogenetics, and clinical outcome. Complete remission was defined as <5% leukemic blasts in bone marrow, absence of blasts in peripheral blood, and absence of leukemic blasts in spinal fluid or other extramedullary sites. Sufficient patient material was available from 105 patients. The Dutch Childhood Leukemia Study Group did the immunophenotyping and cytomorphology of pediatric samples. Adult patient samples were immunophenotyped by the central laboratory of the University Medical Center Groningen.

Children were treated according to the Dutch Childhood Leukemia Study Group protocol ALL-9, except for three patients who were treated according to protocol ALL-8 (23). Childhood patients received vincristine, dexamethasone, and asparaginase as induction therapy. Maintenance therapy consisted of 6-mercaptopurine and methotrexate alternated with vincristine and dexamethasone. High-risk patients (WBC count > 50 x 109/L, mediastinal mass, central nervous system involvement, unfavorable cytogenetics or T-cell immunophenotype) additionally received daunorubicin during induction therapy and intensification treatment with one cycle comparable with induction treatment and a second cycle consisting of cytosine arabinoside and cyclophosphamide. The adult patients received a regimen including prednisone, vincristine, doxorubicin, cytosine arabinoside, and asparaginase as induction and consolidation therapy. Maintenance therapy consisted of 6-mercaptopurine and methotrexate in combination with cytosine arabinoside and etoposide or cyclophosphamide and mitoxantrone. All but five patients received an intensification course with cytosine arabinoside and etoposide (24).

Mononuclear cells from bone marrow or peripheral blood were isolated on Ficoll-Isopaque (Nycomed, Oslo, Norway) density gradient by centrifugation. The cells were cryopreserved in RPMI 1640 supplemented with 10% FCS and 10% DMSO (Merck, Amsterdam, the Netherlands) and stored at –196°C. The percentage of blasts in patient material was 86 ± 15% (mean ± SD).

RNA isolation. Total cellular RNA was isolated from ALL blasts using RNeasy Mini Kit including DNase digestion (Qiagen, Hilden, Germany). From some samples total cellular RNA was extracted using 1 mL of Trizol reagent (Life Technologies, Breda, the Netherlands). The amount of RNA was measured by photometry. Subsequently, 1 µg RNA was reverse transcribed in 20 µL reverse transcriptase buffer containing 10 mmol/L DTT, 0.5 mmol/L each of dATP, dGTP, dCTP, and dTTP, 200 units of Moloney murine leukemia virus reverse transcriptase, 5 units of RNase inhibitor, and 10 ng/µL pd(N)6 random primers (MBI Fermentas, St. Leon-Rot, Germany).

Quantitative PCR was done using the ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA). Primers and TaqMan probes for the MRPs were used as previously described (25, 26). For glyceraldehyde-3-phosphate dehydrogenase, the primers and probes were forward, 5'-GGTGGTCTCCTCTGACTTCAACA-3'; reverse, 5'-GTGGTCGTTGAGGGCAATG-3'; and TaqMan probe, 5'-ACACCCACTCCTCCACCTTTGACGC-3'. We used 4 µL of a 22.5-fold diluted cDNA in each PCR reaction in a final volume of 20 µL containing 900 nmol/L of sense and antisense primers, 200 nmol/L of the TaqMan probe, 5 mmol/L MgCl2, KCl, and Tris-HCl, 0.2 mmol/L dATP, dCTP, dGTP, dTTP, and dUTP, and 0.5 units of AmpliTaq DNA polymerase (qPCR Core Kit, Eurogentech, Seraing, Belgium). TaqMan probes were labeled by a 5' FAM (6-carboxy-fluorescein) reporter and a 3' TAMRA (6-carboxyl-tetramethyl-rhodamine) quencher. The PCR program was 95°C for 10 minutes, followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C.

The expression of the MRP genes was standardized for expression of glyceraldehyde-3-phosphate dehydrogenase. Serial cDNA dilutions of a mixture of all patient samples were used to generate standard curves. The expression of each gene in each sample was analyzed in duplicate. The regression coefficients of the standard curves were ≥0.99.

Statistical methods. Because the levels of MRP expression were not normally distributed, nonparametric methods were used. The Mann Whitney U test was done to compare MRP expression between two groups. For more than two groups, the Kruskal-Wallis test was employed. With the Spearman rank test, the correlation between MRP expression and other continuous variables was determined. The ability to discriminate between patients who will remain in complete remission and patients who will relapse was investigated by receiver operating characteristic (ROC) curves, and the area under the curve (AUC) was calculated. Kaplan-Meier statistics and log-rank tests were calculated to estimate the significance of differences between survival curves. Median values were used as cutoffs for high versus low MRP expression. Relapse-free survival was defined as the time interval between the documented date of complete remission until the date of relapse. Second malignancy, early death, and toxic death in remission were censored at the time of occurrence. Relapse-free survival was censored at the date of last contact for patients with no report of relapse. Patients who died during induction were not included in the analysis of relapse-free survival.

All P values are given for two-sided tests and P ≤ 0.05 was considered significant. Analyses were done using SPSS 11.0 for Windows software (SPSS, Chicago, IL).


   Results
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 Materials and Methods
 Results
Discussion
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Patient characteristics. A total of 105 patients with de novo ALL were studied with ages ranging between 2 and 60 years. Table 1 compares the patient characteristics between the studied patients (n = 105) and the patients that did not participate in our study (n = 84) due to lack of sufficient leukemic cells. No differences were observed in age, gender, WBC, LDH, immunophenotype, and unfavorable chromosomal aberrations [t(9;22), t(4;11), or 11q23 rearrangements] between the two groups. The t(12;21) was more often observed in the group of patients that did not participate in our study. Furthermore, the childhood study group contained more patients who relapsed than the group of patients that were not included in the study. The inclusion of more children with an unfavorable outcome allowed a better opportunity to investigate the effect of MRP expression on prognosis. In adults, the patients who were studied and those who were not had similar outcomes.


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  		Table 1. Patient characteristics at diagnosis

 
In total, 56 children were treated according to the Dutch Childhood Leukemia Study Group protocol. Forty-five adults received treatment according to the adult protocol and four children under the age of 17 years received this treatment as well. The overall survival was 82% in children with a median follow-up of 3.6 years for the total group of childhood patients. For the children who remained in complete remission, the median follow-up was 3.8 years. The overall survival for adults was 48% with a median follow-up of 2.0 years for the total group. The group of adult patients who remained in complete remission had a median follow-up of 3.9 years.

Expression of multidrug resistance–associated protein in acute lymphoblastic leukemia. The expression of the MRP genes in leukemic blasts of children and adults was studied and showed a large variability in expression level (Table 2). MRP1 expression could be detected in 48 children (86%) and 48 adults (98%). Similarly, MRP2 expression was present in 49 children (87%) and 46 adults (94%). The lowest expression was found for MRP3, which could only be shown in 29 childhood (52%) and 40 adult (82%) patients. MRP4, MRP5, and MRP6 were observed in almost all patients (98%, 99%, and 98% respectively). When comparing the MRP expression between children and adults, the expression levels of MRP1, MRP2, and MRP3 were higher in adults than in children (P = 0.008, P = 0.026, and P = 0.039, respectively). To investigate whether or not coexpression of genes is encountered in the same individual patients, the correlation of the expressions of the six MRP genes was calculated. Correlations were observed between the expressions of the six MRP genes in ALL blasts but with low Spearman's correlation coefficients, ranging from 0.23 to 0.56. This suggests that high expression of one MRP in individual patients was correlated with increased expression of multiple MRPs.


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  		Table 2. MRP expression in ALL

 
Multidrug resistance–associated protein expression and survival. The median MRP1 expression was higher in patients who relapsed than in patients who remained in complete remission (P = 0.002). The same pattern was observed for MRP2 (P = 0.002), MRP3 (P = 0.001), MRP5 (P = 0.004), and MRP6 (P = 0.009; Table 2). When the children were analyzed separately, a higher expression level was observed in relapsed patients for MRP1 (P = 0.028), MRP3 (P = 0.013), and MRP5 (P = 0.016). The remaining MRPs (MRP2 and MRP6) showed a trend towards a higher expression in relapsed patients (Table 2). In adults who relapsed, MRP2 and MRP6 expressions were higher compared with adults who did not relapse (P = 0.012 and P = 0.015, respectively). The expressions of MRP1, MRP3, and MRP5 were also higher in relapsed adults but this was not statistically significant (Table 2). MRP4 expression was similar in all patients who remained in complete remission and all patients who relapsed. Because only two adults did not achieve complete remission, a possible effect of MRP expression on complete remission rates could not be calculated in a reliable manner. Additionally, we compared MRP expression when patients were categorized based on clinical outcome. Interestingly, MRP1 to MRP6 expression was comparable in children and adults who eventually relapsed. By ROC curves, the ability of MRPs to discriminate between patients who will remain in complete remission versus patients who will relapse was analyzed. The ROC curves and AUCs for both children and adults are shown in Fig. 1 and Table 3. All individual MRP genes, except MRP4, predicted whether patients would relapse. The AUCs were comparable for the five MRPs, which indicates that they can all predict prognosis with comparable power. A high expression of all five MRPs did not lead to a stronger prediction of relapse than the MRPs individually. When the children were analyzed separately, the same MRPs could predict which patients would relapse with statistical significance for MRP1, MRP3, and MRP5 (MRP1, P = 0.028; MRP3, P = 0.018; MPR5, P = 0.016). Of the traditional predictors, initial WBC count also discriminated for outcome (AUC, 0.79; P = 0.007). ROC curves for adults showed the prognostic effect of the same MRPs, of which MRP2 and MRP6 were statistically significant (MRP2, P = 0.014; MRP6, P = 0.015). For the adults, initial WBC count, age, LDH, or immunophenotype was not predictive for outcome. Multivariate analysis could not be done because of a large degree of colinearity between the six MRPs. Kaplan-Meier curves and log-rank tests analyzed the effect of MRPs on relapse-free survival. The median MRP expression was used as cutoff point for high and low MRP expression. Table 4A provides the data for relapse-free survival in all patients with low versus high MRP expression. It shows that patients with high expression of all MRPs, except MRP4, had a shorter relapse-free survival than patients with low MRP expression (MRP1, P = 0.005; MRP2, P = 0.008; MRP3, P = 0.001; MRP5, P = 0.016; MRP6, P = 0.037). The same effect of high MRP expression could be observed on overall survival (Table 4B). In Fig. 2, the relapse-free survival curves for the various MRPs are depicted. When childhood patients were analyzed alone, high MRP3 expression levels lead to shorter relapse-free survival (5.3 versus 4.9 years, P = 0.033). For adults, MRP2 and MRP6 influenced relapse-free survival (MRP2, 10.7 versus 5.8 years, P = 0.049; MRP6, 11.2 versus 3.3 years, P = 0.008). Because a few children and adults were treated according to a different regimen, we corrected for treatment protocol. The additional adjustments for protocol did not change the results of the survival analyses.



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  		Fig. 1. ROC curves predicting relapse for the different MRPs in children and adults. A, ROC curves for MRP1, MRP2, and MRP3. B, ROC curves for MRP5, MRP6, and the cluster of MRP1, MRP2, MRP3, MRP5, and MRP6.

 

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  		Table 3. MRP expression and area under the ROC curve for children and adults

 

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  		Table 4. Effect of high MRP expression on relapse-free survival (A) and overall survival (B) in children and adults

 


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  		Fig. 2. Relapse-free survival in children and adults from the various MRPs subdivided into two groups of low (L) and high (H) MRP expression. N, number; O, observed events. A, MRP1; B, MRP2; C, MRP3; D, MRP4; E, MRP5; F, MRP6.

 
In summary, ALL patients who will eventually relapse can be recognized at diagnosis by high expression levels of MRP1, MRP2, MRP3, MRP5, and MRP6 in leukemic cells. This expression profile is comparable in children and adults but is more often present in adults.

Multidrug resistance–associated protein expression and clinical features. To investigate whether high MRP expression is an independent prognostic factor, the association with clinical features, including age, gender, WBC count, LDH, immunophenotype, and chromosomal aberrations [t(9;22), t(12;21), t(4;11), or 11q23 rearrangements], was determined. MRP1 expression was higher in T-lineage ALL compared with B-lineage ALL (including pro-B, pre-B, and common ALL; median MRP1 expression, 1.00 versus 0.66, P = 0.031). In contrast, MRP4 was expressed at a lower level in T-lineage ALL in comparison with B-lineage ALL (median MRP4 expression, 0.48 versus 0.83, P = 0.007). For MRP2, MRP3, MRP5, and MRP6, no association between immunophenotype and MRP expression was observed.

No effect of gender was found on the expression of MRP genes in children and adults. Age correlated positively with MRP1 expression. Higher initial WBC counts were associated with a lower expression of MRP3, MRP4, and MRP6. High LDH levels were correlated to a low expression of MRP3 and MRP6. The Spearman correlation coefficients for age, initial WBC counts, and LDH were very small (r = 0.23-0.27).

There were few ALL patients with chromosomal aberrations. Nevertheless, we analyzed whether these patients differed in their MRP expression pattern. Five adults and one child had a BCR/ABL translocation. No difference in MRP expression was observed in these patients compared with patients without the translocation. The translocation t(4;11) was present in three adults and two children, and these patients showed a higher MRP2 and MRP4 expression (P = 0.027 and P = 0.033, respectively) than patients without the translocation. The favorable translocation t(12;21) was found in only two children and no obvious differences were observed between the MRP expression of patients with and without the translocation. Hyperdiploidy (more than 50 chromosomes) of leukemic blasts, which has an incidence of 15% to 20% in childhood ALL, was present in 11 children and was not associated with a high or low expression of MRP genes.


   Discussion
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 Discussion
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This is the first study describing the association between MRP1 to MRP6 expression and clinical outcome in childhood and adult ALL. In children, high expression of MRP1, MRP3, and MRP5 is associated with unfavorable outcome whereas MRP2 and MRP6 can predict relapse in adults. In addition, adults showed a higher expression of MRP1, MRP2, and MRP3 compared with children. Interestingly, this difference disappeared when children and adults were categorized on the basis of clinical outcome. Relapsed patients showed a higher expression of all MRP genes, except MRP4, than patients who remained in complete remission. These results suggest that the prognosis of childhood ALL is comparable with adult ALL if childhood leukemic cells have intrinsic cellular properties similar to adult leukemic cells. This supports the studies of Greaves (3, 4) who suggested that the transforming events in adult and childhood ALL occur in general at a different maturation level in the hematopoietic stem cell compartment. In adult ALL, the transformation is considered to occur in the multipotent stem cell whereas childhood ALL originates from the more committed lymphoid progenitor cell. The level of the transforming event is not strictly connected to a certain age group because there is an overlapping spectrum of the course of the disease between children and adults with ALL.

The association between maturation stage and ABC transporters has already been observed in normal hematopoiesis. The expression and functional activity of P-glycoprotein and breast cancer resistance protein were found to be higher in normal hematopoietic stem cells with an immature phenotype (27–29). In human normal bone marrow cells, a higher MRP expression was observed compared with ALL cells. In contrast, healthy normal B and T lymphocytes were found to have a lower MRP expression than ALL cells (11).

Thus far, only a few studies have addressed the role of a number of MRPs in ALL. No association between expression levels and clinical outcome was observed for MRP1 and MRP2 in childhood ALL in these studies (7, 8, 10, 11). For MRP3, children with high expression showed lower overall survival rates than patients with low MRP expression, which is in agreement with our study (11). The discrepancy with our study for MRP1 and MRP2 might be caused by the limited number of studied childhood ALL patients with an unfavorable outcome in their study. Because we selected more children with an unfavorable outcome, our observations about the prognostic significance of MRP expression may possibly not be generalized to the usual ALL population. However, we found no differences in important prognostic factors, such as WBC counts, age, and unfavorable chromosomal translocations, between the studied patients and the patients that did not participate in our study. In contrast to former studies, we also investigated adult patients who showed the unfavorable MRP profile more often. By including more patients with a relapse in our study population, we were able to describe and detect a possible influence of MRP expression on prognosis. In AML, several studies on MRP1 expression have been reported (30–32), of which the majority did not observe an association between MRP1 expression and clinical outcome. For MRP2, MRP3, and MRP5, one study reported no up-regulation in relapse (31) whereas Steinbach et al. (11) found an association between MRP3 (and possibly MRP2) and a worse prognosis.

We determined the mRNA expression of six MRPs. The assessment of functional MRPs could not be done in our stored patient samples. However, no functional assays for MRP4, MRP5, and MRP6 are available for leukemic cells.

A remarkable finding was that five of the six studied MRPs have a higher expression in relapsed ALL patients. Why MRP4 is different in this regard remains unclear. MRP4 transports drugs comparable with those of MRP5, including methotrexate and 6-mercaptopurine. The level of expression of MRP4 and MRP5 cannot be compared in the ALL samples. Possibly, a compensation mechanism exists that down-regulates MRP4 when MRP5 is already functionally active. To further investigate this, functional assays for MRP4 and MRP5 are required.

The profile with high expression of multiple MRPs indicates that it will be of limited value to counteract the effects of a single MRP with regard to drug resistance. A more general approach is required, especially because the different MRPs have strongly overlapping substrate specificities and are capable of transporting many different chemotherapeutic drugs. Thus far, no clinical trials in leukemia are known that make use of specific MRP inhibitors. This study underscores the importance of the design of a more general MRP inhibitor that can modulate all MRP transporters simultaneously.

Recently, the results of microarray analysis were reported in patients with childhood ALL in relation to drug sensitivity and clinical outcome (33). The gene expression patterns were investigated in relation to in vitro cellular drug resistance to prednisone, vincristine, asparaginase, and daunorubicin. In this analysis, no differences were found in the expression of genes known to play a role in drug resistance, including MDR1 and MRP1. The discrepancy with our study might be related to the fact that the selection of genes was based on the drug sensitivity assay, which is another approach than clinical end points, such as relapse-free survival as used in the present study. Moreover, MRPs are not involved in the transport of prednisone or asparaginase and will subsequently not be recognized as an unfavorable prognostic factor.

In summary, the present study shows that a subset of ALL patients with high MRP expression has an unfavorable prognosis independently of age. Early recognition of a profile with high MRP expression could identify patients with an increased risk for relapse that could benefit from treatment adaptations based on this knowledge.


   Footnotes
 
Grant support: Foundation of Pediatric Oncology Groningen grant SKOG 99-01 (S.L.A. Plasschaert).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5/19/05; revised 9/13/05; accepted 9/23/05.


   References
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