Abstract
Background The reduced folate carrier (RFC1) is a major methotrexate transporter whose impaired function was recognized as a frequent mechanism of antifolate resistance. Recently, a G80A polymorphism has been described in the RFC1. This study evaluated the effect of the G80A polymorphism in the RFC1 gene on survival and risk of relapse of acute lymphoblastic leukemia.
Methods and Results Seventy patients with acute lymphoblastic leukemia were genotyped by polymerase chain reaction restriction fragment length polymorphism method. An association between the polymorphism and risk of relapse was found (P < 0.05). Patients with the G/A genotype have 3.97 (95% confidence interval, 1.12–14.06) and carriers of the A/A genotype have 7.84 (95% confidence interval, 1.66–37.10) higher chance of a relapse. Other variables such as age and leukocyte count were associated (P < 0.05) with the risk of relapse of disease. Individuals with G/A or A/A genotypes had poorer survival (log-rank test, P = < 0.05).
Conclusions These data suggest a role of the polymorphism G80A in the risk of relapse and the mortality risk in patients with acute lymphoblastic leukemia from the State of Guerrero, Mexico.
In Mexico, acute leukemia is considered a problem of public health; it represents the fourth position of mortality of all neoplastic malignancies in children younger than 15 years. Date from 1996 to 2000 show a mortality rate of 63.7 per million children, one of the highest rates reported in the world.1In 2005, leukemia was the second cause of mortality in the State of Guerrero in children younger than 15 years.2An antineoplastic agent used as treatment for patients with acute lymphoblastic leukemia (ALL) is the methotrexate (MTX), which was introduced 5 decades ago to clinical oncology. The reduced folate carrier gene (RFC1) is a major MTX transporter whose impaired function was recognized as a frequent mechanism of antifolate resistance.3Recently, the G80A polymorphism, which replaces His by Arg at position 27 of the RFC1 protein, was identified. Children with ALL with the A variant had worse outcome than patients with the G/G genotype.4In the present study, we have retrospectively analyzed the association between the genetic polymorphisms G80A and both survival and risk of relapse in childhood ALL in a Mexican population.
Study Population
The patients (n = 70) with ALL were included in this study of the pediatric oncology service of the State Cancer Institute from the south of Mexico (Acapulco, Guerrero) between August 2005 and August 2010, diagnosed with ALL through bone marrow aspirate based on French-American-British morphological criteria, cytochemical staining properties, and immunophenotyping of blast cells. The diagnosis of ALL was further subclassified into T-lineage (CD3+, CD7+, plus CD2+ or CD5+, or both) or B-lineage (CD22+, CD19+, HLA-DR+, and CD10±). Multiagent chemotherapeutic protocols used were 96091, 96092, or CIE-10:C9.1.0 of the Cancer Institute from Guerrero State and previously described.5,6
Children with low-risk ALL received a triple intrathecal therapy (MTX, cytarabine, hydrocortisone [HDC]) during the induction phase: MTX, 8 mg/m2 for 1 to 2 years, 10 mg/m2 for 2 to 3 years, 12 mg/m2 for 3 to 8 years, and 15 mg/m2 after 8 years; cytarabine, 16 mg/m2 for 1 to 2 years, 20 mg/m2 for 2 to 3 years, 24 mg/m2 for 3 to 8 years, and 30 mg/m2 after 8 years; HDC, 8 mg/m2 for 1 to 2 years, 10 mg/m2 for 2 to 3 years, 12 mg/m2 for 3 to 8 years, and 15 mg/m2 after 8 years at days 1, 22, 29, and 36. During the consolidation phase, the patients received 2 g/m2 per 24 hours of MTX at weeks 7, 10, 13, 16, 19, and 22; and 48 hours after, they received folinic acid rescue, 15 mg/m2 every 3 hour for 9 times; and during maintenance therapy, they received 50 mg/m2 of MTX weekly until week 130. Children with high-risk ALL received a triple intrathecal therapy (MTX, cytarabine, HDC) during the induction phase: MTX (8–15 mg), cytarabine (16–30 mg), HDC (8–15 mg), depending on age, at days 1, 15, 29, 45, and 59. During the consolidation phase, they received 5 g/m2 of MTX at days 8, 22, 36, and 50 and triple intrathecal therapy at days 8, 22, 33, and 50, with leucovorin rescue, 15 mg/m2, after 48 hours. During the maintenance phase, they received 25 mg/m2 MTX weekly5,6until week 130.
The bone marrow samples of patients or blood samples used in this study were part of the samples taken for clinical diagnostic tests in the hospital; informed consent was obtained. The study and the informed consent procedure were approved by the Institutional Review Board of the Cancer Institute. Complete remission was defined by less than 5% blast cells in the bone marrow and normalization of peripheral blood counts at 4 weeks after starting induction therapy. Relapse was defined as the reappearance of more than 20% blast cells in the marrow or the presence of localized leukemic infiltrates at any site after completion of induction chemotherapy. Worse outcome was defined as a lack of response to induction therapy, a relapse after achieving complete remission, or death due to any cause. Risk classification (standard risk: 1–9 years of age and presenting white blood cell [WBC] count of <50,000/mm3; high risk: <1 and >9 years of age and WBC count >50,000/mm3). Patients’ characteristics and clinical prognostic factors at diagnosis (sex, age, WBC count, and risk group) are given in Table 1.
Genotyping
The G80A polymorphism was detected using the reported polymerase chain reaction primers by Dervieux et al.7in 2004: forward primer (5′-AGTGTCACCTTCGTCCCCTC-3′) and reverse primer (5′-CTCCCGCGTGAAGTTCTT-3′) and using previously established protocols.8The polymerase chain reaction products (230 bp) were digested with 3 units of the HhaI enzyme (New England Biolabs, Berverly, MA). Individuals with the A/A genotype presented 2 fragments (162 and 68 bp), individuals with the G/A genotype presented 4 fragments (162, 125, 68, and 37 bp), and those with the G/G genotype 3 fragments (126, 68, and 37 bp).
Statistical Analysis
Univariate logistic regression analysis for the association with the risk of relapse of ALL was tested first for G80A genetic polymorphism, sex, and other clinical characteristics, and those factors were included in a second analysis, the multivariate logistic analysis. The log-rank test and Kaplan-Meier curves were used to analyze the effect of the G80A genetic polymorphism, and relapse of ALL on overall survival. Overall survival (OS) was defined as the time between surgery and either death or the time of the last follow-up. P < 0.05 was considered statistically significant. All statistical analyses were done using SPSS software, version 15.0 (SPSS, Chicago, IL) and STATA software, version 9.2 (StataCorp, College Station, TX).
RESULTS AND DISCUSSION
The 70 patients with ALL had ages ranging from 1.0 to 18 years (mean ± SD age, 7.65 ± 4.67 years). There were 45 males (64.29%) and 25 females (35.71%). Eighteen patients (25.71%) were in the age group of 1 to 9 years (standard risk). Fifty-two patients (72.29%) were younger than 1 year old and older than 9 years (high risk) at the time of initial diagnosis; 68.57% had a relapse of ALL. White blood cell count and characteristics of immunophenotype are described in Table 1.
This report is the first to describe the contribution of polymorphism in RFC1, to the effects of both survival and risk of relapse in childhood ALL. A significant difference in the frequency of RFC1 genotypes was observed between children with and without relapse (Tables 1, 2). Carriers of the RFC1 A variant had a higher risk for events than those with the G/G genotype (odds ratio [OR], = 6.92; 95% confidence interval, 1.58–31.38; P = 0.012). Similarly, the Kaplan-Meier analysis showed that carriers of the A variant had the worst ALL outcomes (P = 0.026; Fig. 1D). In the multivariate analysis, OR estimates for patients with RFC1 A/A genotype retained their significance (OR, 7.84; 95% confidence interval, 1.66–37.10; P = 0.009) in the presence of other prognostic factors, which also influenced ALL outcome (age, WBC, and risk classes; Table 2). The Kaplan-Meier analysis of overall survival curves showed significant results of relapse of ALL and G80A genetic polymorphism. We found no significant associations between the sex and overall survival (log-rank test, P = 0.79; Fig. 1A). The different overall survival was evident between the individuals with and without relapse of ALL (log-rank test, P = <0.001; Fig. 1B).
We found patients who did not respond to chemotherapy with MTX of which 9 (45.00%) of 20 patients were carriers of the G/G genotype, 17 (85.00%) of 20 patients were carriers of the G/A genotype, and 22 (73.00%) of 30 patients were carriers of the A/A genotype (P = 0.007, Fisher exact test). Moreover, the patients were evaluable for toxicity. The incidence of renal, hepatic, and mucous membrane toxicities during induction or consolidation phase was mainly found in the patients with the risk genotype (A/A), a result similar to that reported by Laverdiere et al., in children carriers of the A/A genotype, who had worse response than patients with the G/G genotype (P = 0.04). Evidence is shown here of the correlation between the genotypes, the polymorphism G80A in the RFC1 gene, and lack of response to MTX. Most of the A/A patients experienced lack of response to MTX more frequently.
The association between RFC1 polymorphism and ALL outcome suggests that this variant might contribute to the estimation of ALL prognosis. Our results are in line with those previously reported by Laverdièr et al., showing an association between the A/A genotype and a reduced survival in the patients with ALL.4However, the genotypic frequencies reported in this study differ (P < 0.001) from those reported by Laverdièr et al. in French Canadian origin population with ALL, where the G/A genotype (48.0%) was the most frequently reported, followed by the G/G genotype (29.9%) and the A/A genotype (22.1%). The differences found in the distribution of the risk genotype (A/A) in the French Canadian population compared to the Mexican population, can be due to differences in genetic background between populations. The population from the State of Guerrero in Mexico has 22.0% African genetic background according to the report by the INMEGEN in 2007.9,10Morin et al.11suggested that patients with A/A genotype of RFC1 tended to have higher folate polyglutamate levels in blood cells. Recent evidence suggests that patients with the RFC1 A/A genotype responded to therapy better than those with the G/G and G/A genotypes.7Other studies have explored variations in the RFC1 expression in leukemic cells and demonstrated that low expression of RFC1 is related to a poor outcome in childhood leukemia.12Jansen et al.,13suggested that higher messenger RNA levels was related to the RFC1 A variant.
Therefore, additional studies are needed to explain the underlying mechanism linking RFC1 polymorphism and ALL outcome. A prospective study assessing intracellular MTX levels and RFC1 substrate binding affinities in patients with and without RFC1 A variant is under way in our laboratory. It would also be important to assess the relative impact of RFC1 polymorphism on ALL outcome with regard to other variants relevant to MTX response.
ACKNOWLEDGMENTS
The authors thank their patients and the patients’ parents for their collaboration; and Ana Bertha Rivera Ramírez and Marco A. Terán Porcayo of the State Cancer Institute for their contributions of biologic material and facilitating access to clinical data. The authors also thank the National Council of Science and Technology (CONACYT, Mexico) for a fellowship awarded to Adriana Contreras Quiroz (August 2007 to July 2009).