Introduction. Jose Francis 1 Biswajit Dubashi. Adithan Chandrasekaran 3

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1 Med Oncol (2015)32:213 DOI /s ORIGINAL PAPER Influence of Sokal, Hasford, EUTOS scores and pharmacogenetic factors on the complete cytogenetic response at 1 year in chronic myeloid leukemia patients treated with imatinib Jose Francis 1 Biswajit Dubashi 2 Rajan Sundaram 1 Suresh Chandra Pradhan 1 Adithan Chandrasekaran 3 Received: 23 June 2015 / Accepted: 29 June 2015 Springer Science+Business Media New York 2015 Abstract Imatinib mesylate is currently considered the first-line treatment for chronic myeloid leukemia (CML). Sokal, Hasford and EUTOS are the three major risk categorization scores available for CML patients. The present study aimed to explore the influence of three risk score, genetic polymorphisms of ABCB1, OCT1, ABCG2 and trough level concentration on complete cytogenetic response at 1 year and overall survival. The mean time period of follow-up was months, and the overall survival was 94.6 %. The Sokal score (P 0.014), Hasford score (P 0.016) and MDR1 C3435T (P 0.001) tend to influence the overall survival in the patients. The patients who had better overall survival had early complete cytogenetic response (P ). The ABCG2 C421A was the covariate which had correlation with the complete cytogenetic response. A perceptive approach incorporating pharmacogenetic evaluation with major risk categorization score at the initial stage will help in ensuring better treatment success in CML patients treated with imatinib. Keywords Imatinib mesylate Cytogenetic response Overall survival Pharmacogenetics & Biswajit Dubashi drbiswajitdm@gmail.com Department of Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry , India Department of Medical Oncology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry , India Department of Clinical Pharmacology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry , India Introduction The introduction of imatinib has transfigured the treatment of chronic myeloid leukemia (CML) [1, 2]. The initial trials have shown a good overall survival rate in imatinib-treated patients [3, 4]. The timely monitoring of clinical response has significant importance in assuring treatment accomplishment. As the treatment with imatinib endures for lifetime of the patients and with the advent of newer tyrosine kinases as options for imatinib failure, the need for surrogate markers to predict response and survival at an early stage is warranted. Various risk evaluation score metrics are available for categorization of patients with CML, namely Sokal, Hasford and EUTOS scores [5 7]. These three risk scores are different from one another in many aspects and have been widely studied with respect to the prognostic implications and disease response in patients treated with imatinib [8 10]. Apart from the various risk score metrics, systemic exposure of imatinib mesylate is another factor which correlates with the treatment response [11, 12]. The measurement of steady-state trough level concentration (Cmin.ss) is of importance in predicting the response to treatment, apart from identifying adherence to treatment. Therapeutic drug monitoring (TDM) is considerably merited in improvising the management of CML patients on imatinib [13, 14]. A number of genetic polymorphisms associated with drug transporter genes involved in imatinib transport across the cells have been studied with relation to response of imatinib treatment [15, 16]. Genetic polymorphisms in drug transporter genes have been associated with drug-induced adverse effects and response predictions for many anticancer agents. The drug transporter genes ABCB1, OCT1, ABCG2 are the ones widely studied for

2 213 Page 2 of 6 Med Oncol (2015)32:213 exploring the pharmacogenetic relationship with imatinib response [17, 18]. Considering the above-mentioned details, the primary aim of the study was to identify the potential influence of three prognostic scores, genetic polymorphisms of ABCB1, OCT1, ABCG2 and trough level concentration with the complete cytogenetic response (CCyR) at 1 year of treatment. Further, these variables were correlated with the overall survival in CML patients. Methods Patient cohort The patients who were identified as CML-Chronic phase (presence of Philadelphia chromosome) at Regional Cancer Centre, JIPMER, Puducherry, and on T. Imatinib 400 mg od were included in the study. The study was approved by the Institute ethics committee, and written informed consent was obtained from all patients. The patient response details were recorded during the treatment period as per the latest European Leukemia Network (ELN) recommendations. The baseline demographics were recorded, and the three scores were calculated at the initiation of the treatment. For the analysis, the lower- and intermediate-risk groups of Sokal and Hasford scores were combined as mentioned in the ELN recommendations [19]. Trough level concentration and genotype assessment The samples for trough level concentration assessment of imatinib at steady state (after minimum period of 7 days) in CML patients were collected between 21 and 27 h after last drug administration [11]. The quantification of imatinib in plasma was determined using high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS; Waters, Milford, MA, USA) [20]. The LC-MS/MS method involved a simple protein precipitation method using acetonitrile. The DNA of the patients for genotyping was extracted from peripheral blood by phenol chloroform method. Various SNPs, namely ABCB C[T (rs ), ABCB G[T/A (rs ); ABCG2 421 C[A (rs ); OCT C[T (rs ), OCT A[G (rs628031), OCT C[A (rs622342), were selected for the study. The genotyping was carried out on real-time thermocycler (ABI Prism 7300, Foster city, CA, USA) using validated real-time TaqMan SNP genotyping probes (Applied Biosystems, Foster City, CA, USA). All samples were analyzed in duplicates, and negative controls were included to ensure authenticity of the results. Statistical analysis All statistical tests were done using SPSS version 19.0 (IBM PASW Statistics; 19.0). The overall survival was estimated using Kaplan Meier method followed by logrank test to explore the influence of various covariates on the survival functions. The influence of various factors over the response was evaluated using logistic regression analyses, initially by bivariate analysis followed by univariate analysis. Bootstrapping was performed for the covariates which were found to have significant association with the response. The comparison between patients with overall survival and complete cytogenetic response was performed using Fisher s exact test. P value of \0.05 was considered statistically significant. Results Patient demographics A total number of 111 CML-chronic phase patients and on stable dose (400 mg) were included in the study. The baseline patient characteristics are illustrated in Table 1. Table 1 Patient baseline and response characteristics Patient characteristics (n = 111) Values Gender male/female (n) 72/39 Age median (range) 40 (18 74) years Body weight mean ± SD 54.2 ± 12.3 kg Body surface area (mean) 1.54 m 2 Complete cytogenetic response Yes 83 No 23 Not available 5 Sokal score category Low risk 36 Intermediate risk 51 High risk 24 Hasford score category Low risk 59 Intermediate risk 26 High risk 13 Not available 13 EUTOS score category Low risk 56 High risk 42 Not available 13

3 Med Oncol (2015)32:213 Page 3 of 6213 Association of various factors with complete cytogenetic response The complete cytogenetic response data at 1 year were available for 106 patients. The percentage of CCyR was 78.3 %. The influence of various demographic characteristics, prognostic scores, pharmacogenetic factors and trough level concentration on CCyR was analyzed using binary logistic regression initially with all covariates followed by univariate analysis and is presented in Table 2. The ABCG2 421 C[A polymorphism was found to influence the CCyR of the patient in the univariate analysis which was verified by simulation using bootstrap method (n = 1000). The bootstrap results confirmed the association of ABCG2C421A with CCyR with P value (95 % Confidence interval limits ). Fig. 1 The overall survival of 111 CML patients in the present study Overall survival data The mean time period of follow-up was months, and the overall survival was 94.6 % (Fig. 1). The influence of covariates on overall survival was analyzed using log-rank test. The Sokal score (P 0.014), Hasford score (P 0.016) and MDR1 C3435T (P 0.001) tend to influence the overall survival in the patients and is presented in Figs. 2, 3 and 4. The low- and intermediate-risk groups (54.3 months) as per Sokal classification have a better mean survival compared to the higher-risk group (39.0 months). Similarly, the Hasford classification also showed a better overall survival for low-risk group (53.5 months) with respect to high-risk group (25.0 months). The demographic characteristics and Cmin.ss did not influence the overall survival in the study cohort. The MDR1 C3435T was the only pharmacogenetic factor which showed an association with the overall Fig. 2 The influence of Sokal score on overall survival in the study cohort Table 2 Association of various factors over the CCyR at 1 year Factors P value Odds ratio 95 % confidence interval Lower Upper Age Gender Body weight Sokal score Hasford score EUTOS score ABCG2 421 C[A OCT C[T OCT A[G OCT C[A ABCB C[T ABCB G[T/A Cmin.ss Bold value indicates statistical significance (P = 0.035)

4 213 Page 4 of 6 Med Oncol (2015)32:213 Fig. 3 The influence of Hasford score on overall survival in the study cohort Fig. 4 The influence of MDR1 C3435T on overall survival in the study cohort survival. The T allele patients have better overall survival (54.8 months) compared to patients with C allele (43.9 months). Further, comparison was performed between the groups who had overall survival with complete cytogenetic response at 1 year which showed a significant relation (P value ). Discussion The treatment of CML with imatinib endures almost for the entire life span of the patients, and this demands the development of biomarkers for predicting the survival and response at an early stage of the patients. There exists the need for reliable prognostic scoring system for risk categorization along with biomarkers to predict response for the successful therapy with imatinib in CML. Various attempts have been made to validate the superiority of the available three scores [8 10, 21]. Many pharmacogenetic factors and circulating imatinib drug concentration have been widely studied for exploring its relation with therapeutic response, so as to elucidate a potential biomarker [17, 18, 22]. The present study compared the influence of three scoring systems, pharmacogenetic factor and trough level imatinib concentration at steady state with the achievement of complete cytogenetic response in the patients. The drug transporter gene ABCG2 421 C[A genetic polymorphism showed a significant association (P 0.035) with CCyR at 1 year. The importance of ABCG2 drug efflux gene with respect to imatinib transport is widely been studied. The ABCG2 genetic polymorphism has proven to result in varied imatinib concentration in human body. Thus, ABCG2 421 C[A will be a good candidate gene to further explore its importance for predicting the response in CML patients. However, the three risk evaluation scores, trough level concentration and other pharmacogenetic factors did not influence the response. The overall survival rate was 94.6 % over a mean follow-up period of months in the present study which is comparable with the previously published results [3, 4]. The Sokal and Hasford scoring systems were significantly associated with the overall survival in the study cohort (P and 0.016, respectively). As the EUTOS score was basically developed to predict the likelihood of achieving response [7], these results does not demonstrate the superiority of Sokal and Hasford score over the EUTOS score. However, all the three scoring systems were not associated with the CCyR at 1 year in the present study. Apart from Sokal score and Hasford scores, MDR1 C3435T polymorphism was another variable which showed a correlation (P 0.001) with the overall survival. The MDR T genotype patients had a better overall survival compared to the MDR C genotype patients, which demonstrates the protective effect of 3435T mutation. The association of MDR1 genotypes with response has been depicted earlier [23]. A study by Kim et al. [24] has shown a contradictory result that TT genotype had a lower overall survival, but was not confirmed in the multivariate analysis. The ABCG2 421 C[A which showed an association with complete cytogenetic response did not influence the overall survival in the present study. However, the results from the present study need to be validated in a larger patient population. The steady-state trough level concentration measurement was another covariate of interest which has been widely studied in relation to imatinib therapy. A recent study to fill up the dearth of evidence regarding implementation of routine TDM for imatinib puts forward the suggestion that it will be of use in patients who are susceptible for dose adjustment [25]. But in the present study, there was no influence of Cmin.ss with

5 Med Oncol (2015)32:213 Page 5 of 6213 respect to overall survival and response. Further comparison between patients who had overall survival with complete cytogenetic response at 1 year revealed a significant association (P ). This result demonstrates that achieving an early cytogenetic response is crucial for better overall survival of the patients. The achieving of complete cytogenetic response at 1 year can be considered as a predictor for overall survival and also for switching to second-generation TKIs for improving the response. Conclusion To conclude, the Sokal and Hasford scores not the EUTOS showed an influence over the overall survival in the present study. The ABCG2 C421A polymorphism was found to correlate with the CCyR at the end of 1 year. The patients who had complete cytogenetic response at 1 year had better overall survival in the study cohort. An astute approach integrating pharmacogenetics evaluation along with risk score categorization at the initial stage will help in ensuring better treatment success in CML patients treated with imatinib. Acknowledgments The study was supported by a Grant received from Science and Engineering Research Board, Government of India (SERB-SB/FT/LS-147/2012). Compliance with Ethical Standards Conflict of interest References None declared. 1. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. N Engl J Med. 2001;344: Lakshmaiah KC, Bhise R, Purohit S, et al. Chronic myeloid leukemia in children and adolescents: results of treatment with imatinib mesylate. Leuk Lymphoma. 2012;53: Deininger M, O Brien S, Guilhot F, et al. 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