ORIGINAL ARTICLE. Key Words: Glutathione-S-transferase, Acute lymphocytic leukemia, polymorphic enzymes

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1 ORIGINAL ARTICLE Genetic Polymorphim of Glutathione-S-Tranferae P1, T1 and M1 in Pediatric with Acute Lymphocytic Leukemia in a Philippine Tertiary Hopital Maria Melanie Liberty B. Alcauin 1, Pamela D. Fajardo 1, Catherine Lynn T. Silao 1,2, Amy Goleta-Dy 1, Eufroina A. Melendre 1, Eva Maria C. Cutiongco-dela Paz 1,2, Carmencita David-Padilla 1,2 1 Department of Pediatric, College of Medicine and Philippine General Hopital, Univerity of the Philippine Manila; 2 Intitute of Human Genetic, National Intitute of Health, Univerity of the Philippine Manila Abtract Introduction. Glutathione S-tranferae (GST) are major detoxifying enzyme that modify uceptibility in cancer including acute lymphocytic leukemia (ALL). Thi paper determine the frequency of GST polymorphim (M1, T1, P1) in Filipino ALL patient and control ubject and compare the frequencie between the two group. Method. Pediatric ALL patient at the UP-PGH Medical Center een from January to June 2007 were enrolled. Age and ex matched ubject without ALL from the UP-PGH Outpatient Department were included a control. Genomic DNA wa extracted from peripheral blood of each ubject. GSTM1 and T1 polymorphim were determined uing polymerae chain reaction (PCR) while retriction fragment length polymorphim (RFLP) analyi wa employed for the determination of GSTP1 polymorphim. Matched Odd Ratio wa ued to compare the genomic frequencie of control and ALL patient. Reult. The preence of GSTT1 and GSTM1 polymorphim howed a trend toward protection from having ALL, with OR 0.59 (95% CI: ) and OR 0.86 (95% CI: ), repectively. Having the GSTP1 polymorphim wa hown to be a rik factor [OR 1.7 (95% CI: )]. Concluion. Difference in GST polymorphim frequencie were noted between the control group and ALL patient. GSTT1 and GSTM1 polymorphim appear protective while having the GSTP1 polymorphim confer increaed rik for ALL. Key Word: Glutathione-S-tranferae, Acute lymphocytic leukemia, polymorphic enzyme Introduction Reearch on cancer i now geared toward identifying hot factor that modify uceptibility and treatment outcome. Numerou tudie have been undertaken in the earch for gene that may increae or decreae an individual rik of Correponding Author: Catherine Lynn T. Silao, MD, PhD Intitute of Human Genetic, National Intitute of Health Philippine, Univerity of the Philippine Manila 625 Pedro Gil Street, Ermita, Manila 1000, Philippine Telephone: cltilao@pot.upm.edu.ph uceptibility of having cancer. Much focu i given to gene encoding for detoxifying enzyme that interact with variou carcinogen and gene that play a role in the prevention of carcinogenei. Glutathione-S-tranferae are major detoxifying enzyme that provide critical defene from variou chemical and environmental carcinogen. They are a multigene family of phae II detoxification enzyme that catalyze the conjugation of both endogenou and exogenou electrophile with the non-protein thiol glutathione. The reulting conjugate i more water oluble and in mot intance, le toxic. Thi reult in protection againt toxicity and chemical carcinogenei, epecially during the initiation phae. 1 Six clae of GST have been identified with four (α, µ, σ, π) being well characterized and known to be polymorphic. The polymorphim that have been mot extenively evaluated a biomarker of cancer rik are GSTM1, GSTT1, and GSTP1. Mutation in ubtype P1, T1, and M1 polymorphim have been implicated a rik factor in a number of malignancie - colorectal carcinoma, 2,3 gatric carcinoma, 3,4 lung cancer, 5,6,7 bladder carcinoma, 5 hamartoma, 7 gallbladder carcinoma, 8 eophageal carcinoma, 9 and cervical carcinoma, 10 among other. In mot tudie, polymorphim were analyzed in aociation with known environmental rik, like moking. Hematologic malignancie were included in imilar tudie with evidence pointing to aociation between acute lymphocytic leukemia (ALL) and GST polymorphim genotype. 11,12 Acute lymphocytic leukemia (ALL) i the mot common form of malignancy in childhood. ALL i more common in male than in female with a noted peak of incidence between age 2 and 5 year. Three to four out of 100,000 white children are affected. In the United State, it account for approximately three quarter of diagnoed cae of leukemia annually. 13 Local data on the incidence of ALL i not available. However, in the Pediatric Hematology Section of the Philippine General Hopital, an average of 22 new cae are een yearly. Race wa long recognized a an important factor in the rik aement of ALL with thi malignancy being more common in the white population. Racial difference in 22 ACTA MEDICA PHILIPPINA

2 Frequency of GST polymorphim in ALL uceptibility and even treatment outcome have been hown to be preent Survival variability by race and ethnicity wa alo demontrated with ALL children from white and Aian/Pacific Ilander population having better urvival rate compared to black, Hipanic, and American Indian/ Alakan native. 16,17 To our knowledge, thi i the firt tudy comparing the frequency and poible aociation between common GST polymorphim and ALL in a Filipino population. The author alo decribe the demographic feature of pediatric ALL patient in the UP-PGH Medical Center. Thi paper may provide additional baeline data to the currently few tudie 20,21 on the frequency of GST polymorphim in the Filipino population. It will alo help future reearch that attempt to elucidate the difference in ALL uceptibility and urvival variability among race with level of detoxifying enzyme a a parameter. Method Subject, 18 year of age and below, diagnoed with ALL through morphology and/or flow cytometry and undergoing treatment at the Department of Pediatric in the Univerity of the Philippine-Philippine General Hopital (UP-PGH) Medical Center from January 2007 to June 2007 were included in the tudy population. Age- and exmatched ubject without chronic illne, malignancy nor ign of a hematologic diorder, een at the UP-PGH Out Department, were included a control. Demographic characterization of ALL patient wa done by chart review and follow- up interview. Informed conent wa obtained from both patient and control or their guardian before the tudy. All ample were collected in accordance with local intitutional review board or medical ethic committee guideline. Genotyping for GSTM1, GSTP1, GSTT1 Genomic DNA wa extracted uing the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) from peripheral blood of both patient and control. A 220-bp fragment from GSTM1 wa PCR-amplified uing the primer F: 5 - GAA CTC CCT GAA AAG CTA AAG C- 3 and R: 5 - GTT GGG CTC AAA TAT ACG GTG G - 3. A 312-bp fragment from the CYP1A1 gene wa co-amplified a internal control uing the primer CYP1A1_f: 5 - GAA CTG CCA CTT CAG CTG TCT-3 and CYP1A1_r: 5 - CAG CTG CAT TTG GAA GTG CTC - 3. A 442-bp fragment from GSTP1 wa PCR-amplified uing the primer GSTP1_f: 5 -GTA GTT TGC CCA AGG TCA AG - 3 and GSTP1_r: 5 - AGC CAC CTG AGG GGT AAG - 3. The reulting PCR product wa digeted with 2.5U Al26wI for 4h at 37 C. A 480-bp fragment from GSTT1 wa PCR-amplified uing the primer F: 5 - TTC CTT ACT GGT CCT CAC ATC TC - 3 and R: 5 - TCA CCG GAT CAT GGC CAG CA - 3. A 312-bp fragment from the CYP1A1 gene wa co-amplified a internal control uing F: 5 - GAA CTG CCA CTT CAG CTG TCT-3 and R: 5 - CAG CTG CAT TTG GAA GTG CTC-3. Each PCR reaction mixture contained: ng genomic DNA, 1X PCR Buffer, 2mM MgCl2, 200 um DNTP, 0.01 um of each primer, and 0.75 U Taq Polymerae. The amplification profile i a follow: initial denaturation (5 min, 94 C), 35 cycle of denaturation (1 min, 94 C), annealing (50, 65 C), and extenion (1 min, 72 C), followed by final extenion (5 min, 72 C). All PCR product and diget were reolved in a 2% agaroe gel. Statitical analyi Frequencie of polymorphim of ALL patient and control were tatitically done uing the Matched-Odd Ratio formula. Reult From January 2007 to June 2007, 50 patient with ALL were een for follow-up conult or for chemotherapy in the Hematology-Oncology Outpatient Clinic of the Department of Pediatric, Univerity of the Philippine-Philippine General Hopital. There wa a 1.6:1 male preponderance oberved with 80% of patient falling in the 1-9 year age group. Of the 26 patient with ALL who had immunophenotying done, 84% had B lineage. Four of 10 who had cytogenetic tudie performed had chromoomal abnormalitie. The majority of the patient had no known family hitory of malignancy. Pallor wa the mot frequent preenting ymptom (Table 1). The GSTT1, GSTP1 and GSTM1 gene polymorphim are hown in Figure 1-3. The Ile/Ile GSTP1 polymorphim i the mot frequent genotype in ALL patient (52%). The control group frequency i 42%. The heterozygou Ile/Val Table 1. Characteritic of the tudy population of 50 children with ALL Gender Male 31 Female 19 Age at Diagnoi <1 Year Year Year 10 Phenotype/Lineage T-Lineage 4 B-Lineage 22 Unpecified 24 Cytogenetic Normal 6 Abnormal 4 Not Done 40 Family Hitory of Malignancy Preent 7 Abent 43 Preenting Sign/Symptom Pallor 18 Bleeding and Pallor 10 Bleeding 8 Fever 9 lymphadenopathy 3 Bone Pain 2 ACTA MEDICA PHILIPPINA 23

3 Frequency of GST polymorphim in ALL genotype ha imilar frequencie in both cae and control group. The Val/Val genotype i more frequently een in the control group (16% v. the 2% in ALL group). The GSTM1 null genotype i more frequently oberved in both control and cae. The GSTT1 wild type polymorphim i more frequently oberved in the control group than the null genotype but the revere i true for the ALL group (Table 2). 500 bp 400 bp 300 bp M bp 312 bp M bp 500 bp 400 bp 442 bp Figure 3. GSTT1 gene polymorphim. M i the molecular weight marker; lane 1 i the negative control. Lane 2 i the normal control. Lane 4, 5, 7, 8, 11, 13 repreent the wild type allele a hown by the preence of the 480 bp band. Lane 3, 6, 9, 10, 12 repreent the null allele. The 312 bp band i the CYP1A1 exon 7 which erve a the internal control. Figure 1A. Undigeted 442 bp PCR product of the GSTP1 gene of ALL patient (lane 3-15) and normal control (lane 16-24). M repreent the 100 bp marker. Lane 1, blank. Lane 2, repreent a heterozygote control. 400 bp 300 bp 200 bp 100 bp M bp 329 bp 216 bp 113 bp Figure 1B. Confirmation of the GSTP1 polymorphim in ALL patient (lane 3-15) and normal control (lane 16-24) by Al26wI digetion of amplified genomic DNA. Lane 2 repreent the undigeted amplicon. Lane 6, 7, 9, 16-18, and 20 how the wild type allele (329, 113 &/or 107 bp). Lane 3, 5, 11-13, 15, 19, how the heterozygou mutant genotype (329, 216, 113 &/or 107 bp) while lane 4, 8, and 14 how the homozygou mutant genotype (216, 113 &/or 107 bp). M, 100 bp ladder; Lane 1 Blank. Table 2. Ditribution of GSTT1, GSTM1 and GSTP1 genotype in ALL cae (n = 50) and control (n = 50) GENOTYPE ALL cae (%) (%) TOTAL GSTP1 Ile/Ile 26 (52) 21 (42) 47 Ile/Val 23 (46) 21 (42) 44 Val/Val 1 (2) 8 (16) 9 GSTM1 Preent 16 (32) 18 (36) 34 Null 34 (68) 32 (64) 66 GSTT1 Preent 23 (46) 30 (60) 53 Null 27 (54) 20 (40) 47 Matched Odd Ratio howed that a trend toward protection from having ALL with the preence of the common GSTT1 and GSTM1 wild polymorphim with OR 0.59(95% CI: ) and OR 0.86 (95% CI: ), repectively. Having the common GSTP1 polymorphim, however, wa hown to be a rik factor to having ALL with OR 1.7 (95% CI: ) (Table 3). M Table 3. Matched Odd Ratio for GSTT1, GSTM1, and GSTP1 400 bp 300 bp 200 bp 100 bp 312 bp 220 bp Figure 2. GSTM1 gene polymorphim. M i the 100 bp molecular weight marker. Lane 1 i the negative control. Lane 2, 6 and 7 repreent the wild type allele a hown by the preence of the 220 bp band. Lane 3-5 repreent the null allele. The 312 bp band i the CYP1A1 exon 7 which erve a the internal control for the PCR run. a. GSTM b. GSTT c. GSTP1 Other *other- heterozygou Ile/Val/and Val/Val Dicuion The demographic characteritic of the ALL patient in thi tudy follow the uual characteritic reported in the literature, namely: male preponderance with a male to female ratio of 1.5:1; the majority diagnoed within 24 ACTA MEDICA PHILIPPINA

4 Frequency of GST polymorphim in ALL the 1-9 age group; and predominant B-cell lineage on phenotyping. 13,14 Immunophenotying and cytogenetic/ chromoomal analyi were done only in a minority of the patient, primarily due to unavailability of fund. Both of thee parameter are routinely done in developed countrie. Thee are ued for better rik aement and prognotication. Thee alo help in tailoring chemotherapeutic protocol to be implemented on a pecific patient. In our ALL patient, morphologic claification wa ued for rik aement and prognotication when immunophenotyping wa not available. Much interet ha been generated with the poibility of uing detoxifying enzyme a biomarker for rik aement of variou malignancie. The ultimate goal of invetigation uch a thi tudy i to improve cure rate or treatment outcome by tailoring treatment baed on one genetic makeup. 22 With thoe patient having the genotype aociated with high rik given more intenive therapy. Previou tudie have hown that there i marked geographical and ethnic variation in the ditribution of gene for polymorphic GST. 24 Report on genomic frequencie for thee enzyme are available for black and Caucaian population with a few tudie reported on Japanee, Chinee and Indian population, and even fewer tudie on the Malay race. The previou two local tudie on GST polymorphim in cancer were done on adult population and involved olid tumor. 20,21 Thi i the firt tudy to involve a Filipino pediatric population with a hematologic malignancy. The frequencie of GSTM1 and GSTT1 null genotype in thi tudy are noticeably different and higher than previou report. 7, 8, 12, 14, 15, 20, 21, 23 The frequencie of null genotype are even higher than thoe reported by Davi et al for the white population in which ALL occurred at relatively high incidence. The frequencie are imilar to the Japanee genotype reported by Imanihi et al, alo for GSTM1 and GSTT1. GSTP1 genotype are imilar overall with thoe reported on the Chinee population 23 and on the Filipino population by a local tudy on colorectal cancer. 21 Surpriingly, the local tudy on an adult population 21 howed much lower frequencie, with GSTM1 null at 26.2% for patient with colorectal cancer and 34% for the control. Likewie with GSTT1 null genotype at 31.8% for the cancer cae and 9%for the control. Thee difference need to be validated in a bigger tudy population. Thi tudy report a trend toward protection from having ALL with preence of the common GSTT1 and GSTM1 polymorphim and, hence, an increaed rik with GSTM1 and GSTT1 null genotype. Although thi trend ha been demontrated in mot olid tumor in the adult population, conflicting reult were reported by previou tudie on ALL. Krajinovic et al 25 reported an increae rik of ALL with GSTM1 null genotype but not with the GSTT1 null genotype. In a report by Davie et al, 12 however, no aociation wa noted for both null genotype. The GSTP1 common polymorphim wa hown in thi tudy to be a rik factor for having ALL, contrary to another report by Krajinovic et al. 26 A pointed out by Modal et al, 27 the difference in the aociation or lack of them in the previou tudie may be explained by other factor like the heterogeneity in environmental toxin expoure and the effect of other detoxifying gene. Difference in the frequencie of GST polymorphim were noted between the control and patient with ALL. Thi tudy report a trend toward protection from having ALL with preence of the common GSTT1 and GSTM1 polymorphim while the common GSTP1 polymorphim apparently increae rik for ALL. Thi obervation i baed on a limited number of ubject and need to be confirmed in a larger population tudy. Acknowledgment The author acknowledge the invaluable technical aitance of Moie Anthony Arana, Chritian Eric Abaya, Karen Hernandez and Daffodil Canon. Thi tudy wa funded by the Intitute of Human Genetic. 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