Tumor Biol. DOI 10.1007/s13277-016-4908-2
ORIGINAL ARTICLE
DPYD gene polymorphisms are associated with risk and chemotherapy prognosis in pediatric patients with acute lymphoblastic leukemia Xiao-Qiang Zhao 1,2 & Wei-Jie Cao 1 & Hai-Ping Yang 2 & Xue-Wen Yang 2 & Ping Tang 1 & Ling Sun 1 & Xing Gao 1
Received: 3 November 2015 / Accepted: 22 January 2016 # International Society of Oncology and BioMarkers (ISOBM) 2016
Abstract We aimed to investigate the association between dihydropyrimidine dehydrogenase (DPYD) gene polymorphisms and the risk of pediatric acute lymphoblastic leukemia (ALL) and its prognosis after chemotherapy. A total of 147 pediatric ALL patients diagnosed by our hospital between January 2011 and December 2014 were included in the case group, and 102 healthy people who received a physical examination during the same time frame in our hospital were included in the control group. DNA sequencing was applied for site determination and genotyping of the DPYD 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms. The genotype and allele frequencies of the two groups were compared. A significant difference was found in the comparison of the mutant gene and allele frequencies of the 85T > C polymorphism between the case and control groups (P < 0.05). The CT and CC genotypes in the 85T > C polymorphism were associated with the risk of the disease (OR = 1.592, 95 % CI = 1.010–2.509), suggesting that the recessive gene (85C) was more likely to lead to the occurrence of ALL compared with the dominant gene (85T) (P < 0.05). Patients carrying the C allele of the 85T > C polymorphism presented higher damage of their liver functions and higher
infection rates compared with patients carrying the non-C allele (P < 0.05). A higher proportion of liver function damage and a higher infection rate were found in patients with the GA genotype in the IVS14 + 1G > A polymorphism compared with the GG genotype (P < 0.05). The complete remission (CR) rate in patients with the GG genotype in the IVS14 + 1G > A polymorphism was higher than in patients with the GA genotype (P = 0.020). After 5-fluorouracil/calcium folinate (5-FU/CF)-based chemotherapy, the event-free survival (EFS) rate of patients with the TT genotype was higher than patients with the CT and CC genotypes (P < 0.05). Our results revealed that the C allele of the 85T > C polymorphism might be associated with susceptibility to pediatric ALL. Patients carrying the C allele may have an increased risk of ALL. Thus, the 85T > C polymorphism may be a predictor of CR for pediatric ALL patients. Keywords DPYD gene polymorphism . Acute lymphoblastic leukemia . 5-Fluorouracil . 85T > C . 2194G > A . 1156G > T . VS14 + 1G > A
Introduction * Ling Sun [email protected] * Xing Gao [email protected]
1
Department of Hematology, First Affiliated Hospital to Zhengzhou University, Jianshe East Road, No. 1, Two Seven District, Zhengzhou 450052, Henan, People’s Republic of China
2
Department of Hematology, First Affiliated Hospital of Henan University of Science and Technology, Luoyang 471003, Henan, People’s Republic of China
Similar to cancer in general, acute lymphoblastic leukemia (ALL) most likely arises from interactions between exogenous and endogenous exposures, genetic (inherited) susceptibility, and chance [1]. Approximately 6000 new ALL cases with a ratio of 1.3:1 of males to females are estimated to be diagnosed annually in the USA. These cases primarily include children, and approximately 60% of cases are found in people aged A (52 %) is regarded as the most common mutation and has been the most well-studied [9]. Dihydropyrimidine dehydrogenase (DPD, EC1) is encoded by DPYD and is deemed the rate-controlling enzyme for endogenous pyrimidine and fluoropyrimidine catabolism, which is responsible for the elimination of approximately 80 % of administered 5-fluorouracil (5-FU) [10]. 5-FU is a chemotherapeutic that can lead to severe toxic side effects, including hematologic toxicities and death, in advanced or adjuvant settings due to deficiencies in the catabolic pathway [11]. As previously reported, DPD deficiency can result in lifethreatening complications that are correlated with more than 40 sequence variations in the DPYD gene [12]. Many studies have focused on DPYD gene polymorphisms and the toxicity of 5-FU in many types of cancer, including colorectal cancer, gastrointestinal (GI) malignancy, and breast cancer. However, few studies have concentrated on the effects of the gene polymorphisms themselves on the cancer [11, 13]. In this study, we investigated the four different loci (85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A) of the DPYD gene in 147 ALL patients who received surgical treatment and 5-FUbased combined chemotherapy to clarify the relationship between the DPYD gene polymorphisms and ALL.
Materials and methods
their legal guardians. This study complied with the guidelines and principles of the Declaration of Helsinki [14]. Study subjects A total of 147 patients (79 males and 68 females aged 2– 12 years) diagnosed with ALL in the department of hematology in the First Affiliated Hospital to Zhengzhou University between January 2011 and December 2014 were included in the case group. The 147 ALL patients were divided into two types according to the immunophenotyping of the European Group for the Immunological Characterization of Leukemia (EGIL): 122 patients with B cell acute lymphoblastic leukemia (B-ALL) and 25 patients with T cell acute lymphoblastic leukemia (T-ALL) [15]. The enrollment of all patients was based on the diagnostic criteria of the second edition of the Diagnosis and treatment of blood diseases [16]. ALL was diagnosed according to the following basic diagnostic criteria: (1) patients with symptoms of fever, paleness, fatigue, and bleeding accompanied by infiltration into many organs, including the liver, spleen, and lymph nodes; (2) decreased hemachrome, red blood cell counts, and blood cells, whereas the white cell count presented an increase, normal level, or a decrease and its classification included several lymphoblasts and prolymphocytes or no existing lymphoblasts and prolymphocytes; and (3) most of the nuclear cells (primarily lymphocyte hyperplasia) in the bone marrow smear showed obvious or extreme hyperplasia with a minority of nuclear cells presenting as hypoplastic marrow (lymphoblast and prolymphocyte ≥ 30 %). A total of 102 age-matched healthy volunteers (47 males and 55 females aged 2–14 years) who were not biologically related to the 147 ALL patients and received a physical examination in our hospital in the same time frame were enrolled in the control group. Patients were not enrolled if they were >14 years of age; had other severe heart, lung and brain diseases; severe damage of the liver and kidney functions; or other diseases. DNA extraction A fasting peripheral blood (5 mL) sample was collected from both the case and control group patients in the morning in tubes containing ethylenediaminetetraacetic acid (EDTA). The Blood Genome DNA Extraction Kit (Takara Biotechnology [Dalian] co., LTD) was used to extract genomic DNA from the peripheral white blood cells. The DNA density was adjusted to 100 ng/μL, and the samples were stored in −20 °C freezers prior to the experiments.
Ethics statement Primer design This study was approved by the Ethical Committee of the First Affiliated Hospital to Zhengzhou University. Written informed consent was obtained from all study subjects and/or
Premier 5.0 was used to design primers, which were synthesized by Sangon (Shanghai, China). The principle used for
Tumor Biol.
primer design was as follows: (1) the amplified fragment was in the range from 300 to 600 bp; (2) the distance between the two ends and the exon was approximately 100 bp, and the downstream sequencing primers could not have more than 7 single base tandem repeats or short tandem repeats (STRs); (3) the primer length comprised 18–27 basic groups, with an optimum length of 20 bp; and (4) the Tm value was controlled between 58 and 63 °C, with an optimum Tm value of 60 °C. A Tm value of 70 °C was set for the fragment with a GC volume > 70 %. The primer sequences are presented in Table 1.
Polymerase chain reaction The PCR reaction mixture had a total volume of 30 μL and contained the following components: DNA template (0.2 μL), 10 × buffer (3.0 μL) (containing magnesium ion), 1.2 mL of the dNTP mixture (2.5 mmol/L), 1.6 μL of the primers (0.8 μL of the upstream primer and 0.8 μL of the downstream primer), 0.5 μL of TaKaRa Taq (5 U/μL), and 20.2 μL of DEPC-treated water. The PCR amplification was performed based on the touchdown procedure: pre-denaturing (94 °C for 45 s), annealing for 45 s (the annealing temperatures of 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A were 66, 58, 58, and 56.5 °C, respectively), elongation at 72 °C for 45 s, and a final elongation for 10 min (Table 1).
Electrophoresis detection and sequencing The PCR products were analyzed by 1.5 % agarose gel electrophoresis. PCR products with specific amplification fragments were used for sequencing. The Beckman Coulter CEQS000 genetic analysis system and CEQTM DTCS Quick Start Kit (Beckman Coulter) were used for sequencing from both the forward and reverse directions.
Table 1 Primer sequences of DYPD gene polymorphism 85T > C
2194G > A 1156G > T IVS14 + 1G > A
Therapeutic strategy 5-Fluorouracil/calcium folinate (5-FU/CF)-based chemotherapy was administered to the 147 ALL patients. On the first day, the patients received a 3-h intravenous drip with 85 mg/ m2 oxaliplatin (Jiangsu Hengrui Medicine Co., Ltd), a 2-h intravenous drip with 200 mg/m2 calcium folinate, intravenous injection of 400 mg/m2 5-FU, and a 22-h intravenous drip of 600 mg/m2 5-FU with a continuous micro-pump. The next day, the patients received a 2-h intravenous drip with 200 mg/m2 calcium folinate, intravenous injection with 400 mg/m2 5-FU, and a 22-h intravenous drip of 600 mg/m2 5-FU with a continuous micro-pump. A 14-day course of treatment was selected. All patients received at least two courses (average 4–6 courses). Evaluation of adverse drug reactions The difference in the clinical efficiency of the two groups was recorded, and the adverse reactions of patients after treatment were also evaluated in our study. Toxic reactions to the anticancer drugs were divided into complete remission (CR), partial remission, and non-remission based on the Acute and Subacute Toxicity Grading Criteria of Anticancer Drugs (WHO) [17]: (1) CR: no clinical symptoms and signs caused by the infiltration of leukemia cells, platelets ≥ 100 × 109/L, hemoglobin (Hb) ≥ 100 g/L in males and ≥ 90 g/L in females, absolute value of neutrophil granulocytes ≥ 1.5 × 109/L, no leukemia cells found in the peripheral blood, primitive lymphocytes and naive lymphocytes ≤5 %, and red cell and megakaryocytic series presented normal; (2) partial remission: primitive lymphocyte and naive lymphocytes 5–20 % or one of the clinical symptoms or the hemogram did not reach the standard of CR; and (3) non-remission: all three items (myelogram, hemogram, and clinical symptoms) did not reach the standard of complete remission.
Primer sequences
Annealing temperature (°C)
Annealing time (s)
Circle
F: 5′-CCTGGCTTTAAATCCTCGA ACA-3′ R:5′-GTTTTTCTCGTTAGAAACGTCTT ATCC-3′ F: 5′-TGCATTTTTCTGGGATGTGA-3′ R: 5′-GGGATCATAAAGGGCACAAA-3′ F:5′-TAGATGGAACTTGCTAAGGA-3′ R:5′-GAACTGAACCAAAGGCACTG-3′
60
45
30
58
45
40
58
45
45
56.5
45
30
F: 5′-AAAATGTGAGAAGGGACCTCA3′ R: 5′-ATGCATCAGCAAAGCAACTG-3′
DPYD dihydropyrimidine dehydrogenase gene, F forward, R reverse
Tumor Biol.
Follow-up After treatment with surgery and chemotherapy, patient data were collected via telephone, outpatient visits, petition letter, and reviewing case information after the approval of the family members. The deadline for follow-up was December 30, 2014, with a follow-up time of 0.5–36 months (follow-up rate, 100%). The change in the patient’s condition was evaluated regularly through follow-up information obtained for the evaluation of the therapeutic regimen and event-free survival (EFS) rate. The EFS refers to the proportion of the patients free of recurrence and metastasis from the operation date to the final follow-up. Statistical analysis The SPSS 19.0 (IBM Corporation, Somers, NY, USA) software was used for the statistical analysis. Hardy-Weinberg’s equilibrium was applied to detect the representative enrolled patients. Categorical data were measured by the χ2 test and presented as a ratio or percentage. Continuous data were presented as the mean ± standard deviation (SD) and tested by Student’s t test. One-way analysis of variance (ANOVA) was conducted to compare continuous data among multiple groups. The Kaplan-Meier method was applied to analyze the EFS rate. P values 0.05). Comparison of the proportion of abnormal chromogenes between the case and control groups demonstrated no significant differences (case group 25.85 % and control group 20.59 %) (P > 0.05). However, significant differences were found between the case and control groups in the comparisons of white blood cell counts (WBC), red blood cell counts (RBC), hemoglobin concentrations, and platelet counts (all P < 0.01). Genotyping of DPYD gene polymorphisms Specific PCR amplifications were conducted for the DPYD loci (85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A); then, the PCR products were analyzed using 1.5 % agarose gel electrophoresis. Sequencing was applied for specific electrophoresis bands (Fig. 1). Figure 1 showed that the 85T > C polymorphism had three genotypes (TT, CT, and CC), the 2194G > A polymorphism had two genotypes (GG and GA),
the 1156G > T polymorphism had three genotypes (GG, GT, and TT) and the IVS14 + 1G > A polymorphism had the two same genotypes as the 2194G > A polymorphism. DPYD genotype and allele frequencies For the DPYD 85T > C polymorphism, 1 case in the ALL patients (7.48 %) was mutant (CC genotype), 49 cases (33.33 %) were heterozygous (CT genotype), and 87 cases (59.18 %) were wild type (TT genotype). In the control group, the mutant (CC genotype), heterozygous (CT genotype), and wild-type (TT genotype) genotypes were represented by one case (0.98 %), 32 cases (31.37 %), and 69 cases (67.75 %), respectively. A significant difference was detected between the mutant frequencies (CC genotype) in the 85T > C polymorphism between the case and control groups (P < 0.05). An association was found between the C genic mutation in the 85T > C polymorphism and the risk of ALL (case 23.81 % and control 16.67 %) (OR = 1.592, 95 % CI = 1.010–2.509). Thus, the recessive gene (85C) is more likely to lead to the occurrence of ALL than the dominant gene (85T) (P < 0.05). In the 2194G > A polymorphism, 16 cases in ALL patients (10.88 %) were heterozygous (GA genotype) and 131 (89.12 %) were wild type (GG genotype). In the control group, 9 cases (8.82 %) were heterozygous (GA genotype) and 93 cases (91.18 %) were wild type (GG genotype). For the 1156G > T polymorphism, 2 cases in the ALL patients (1.36 %) were mutant (TT genotype), 43 cases (29.25 %) were heterozygous (GT genotype), and 102 cases (69.39 %) were wild type (GG genotype), whereas in the control group, one case was mutant (TT genotype), 25 cases (24.51 %) were heterozygous (GT genotype), and 76 cases (74.51 %) were wild type (GG genotype). In the IVS14 + 1G > A polymorphism, 2 cases in the ALL patients (8.16%) were heterozygous (GA genotype) and 135 cases (91.84%) were wild type (GG genotype), whereas in the control group, 6 cases (5.88 %) were heterozygous (GA genotype) and 96 cases (94.12 %) were wild type (GG genotype). Comparisons of the genotype and allele frequencies of the 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms between the case and control groups showed no existing significant differences, and no obvious associations were found between these genotypes and allele frequencies and the risk of ALL or toxicity (all P > 0.05) (Table 3). Association between DPYD gene polymorphisms and adverse reactions after chemotherapy For the DPYD 85T > C polymorphism, the proportion of liver function damage and the infection rate was higher in patients with the CT and CC genotypes (liver function damage 15 % and infection rate 20 %) compared with patients with the TT genotype (liver function damage 3.45 % and infection rate
Tumor Biol. Table 2 Baseline characteristics of case and control groups
Group
Case group
Control group
n Total
Percentage
147
n
P value
Percentage
102
Gender Male Female Age ≥6 A polymorphism)
compared with the GG genotype (liver function damage 6.67 % and infection rate 11.11 %) (P < 0.05). No significant differences in the liver function damage and infection rates were observed in the different genotypes of the 2194G > A
A
B
T C T G T G T T C
T C TGNGT T C
1
2
TC TGCGT TC
T GG CG T T A C
3
A 85T>C 1) TT; 2) CT; 3) CC
C
GT G T G A A T T
1
GT G T N A A T T
2
C 1156G>T 1) GG; 2) GT
T GG CN T T A C
2
1
B 2194G>A 1) GG; 2) GA
GT G T T A A T T
3
D
CA A CG T A A G
CA A CN T A A G
2
1
D IVS14+1 G>A 1) GG; 2) GA
Tumor Biol. Table 3 Distributions of genotype and allele frequencies of DPYD 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms [n(%)]
Genotypes
χ2
P value
OR(95 % CI)
69(67.75) 32(31.37)
0.487
0.485
Ref 0.823(0.477 ∼ 1.422)
5.908
0.015
0.115(0.014 ∼ 69.270)
Case group
Control group
(n = 147)
(n = 102)
87(59.18) 49(33.33)
85T > C TT CT CC
11(7.48)
1(0.98)
CT + CC T
60(40.82) 223(75.19)
33(32.35) 170(83.33)
C 2194G > A
71(23.81)
34(16.67)
GG
131(89.12)
93(91.18)
GA G
16(10.88) 278(94.56)
Ref 1.592(1.010 ∼ 2.509)
4.053
0.044
9(8.82) 195(95.59)
0.283
0.595
0.792(0.336 ∼ 1.871) Ref
16(5.44)
9(4.41)
0.268
0.605
1.247(0.540 ∼ 2.880)
GG
102(69.39)
76(74.51)
GT TT
43(29.25) 2(1.36)
25(24.51) 1(0.98)
0.715 0.106
0.398 0.745
0.780(0.439 ∼ 1.388) 0.671(0.060 ∼ 7.542)
GT + TT G T
45(30.61) 247(84.01) 47(15.99)
26(25.49) 177(86.76) 27(13.24)
0.721
0.396
Ref 1.247(0.748 ∼ 2.080)
135(91.84) 12(8.16) 282(95.92) 12(4.08)
96(94.12) 6(5.88) 198(97.06) 6(2.94)
0.467
0.494
0.45
0.503
A 1156G > T
Ref
Ref
IVS14 + 1G > A GG GA G A
Ref 0.703(0.255 ∼ 1.939) Ref 1.404(0.518 ∼ 3.805)
Ref reference, CI confidence interval, DPYD dihydropyrimidine dehydrogenase gene
and 1156G > T polymorphisms (both P > 0.05). Moreover, no significant differences were found in mucosal lesions, gastrointestinal reactions, and cytopenia between the different genotypes of the 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms (all P > 0.05) (Table 4).
Association between DPYD gene polymorphisms and the CR rate of ALL patients In the DPYD 85T > C polymorphism, a significant difference was found in the CR rate for the ALL patients between the CC and TT genotypes (P = 0.005); moreover, the CC (63.64 %) and CT (87.76 %) genotypes had lower CR rates compared with the TT (91.95 %) genotype. In the IVS14 + 1G > A polymorphism, the CR rate in patients with the GG (89.63 %) genotype was significantly higher than the CR rate in patients with the GA (66.67 %) genotype (P = 0.020). No significant differences were found between the different genotypes of the 2194G > A (GA 81.25 % and GG 89.31 %) and 1156G > T polymorphisms (TT 100 %, GT 86.05 %, and GG 89.22 %) (all P > 0.05) (Table 5).
Association between DPYD gene polymorphisms and the EFS of ALL patients After surgical treatment combined with 5-FU/CF-based chemotherapy, the EFS of the TT genotype was significantly higher than the EFS of the CT and CC genotypes (85 % in the CT and CC genotypes and 95 % in the TT genotype; P < 0.05). However, no significant differences were found between the EFS rates in the different genotypes of the 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms (all P > 0.05) (Table 6 and Fig. 2a–d).
Discussion Our present study was conducted to investigate the association between DPYD gene polymorphisms and the risk of pediatric ALL and its prognosis after chemotherapy. Our results demonstrated that the C allele of the 85T > C polymorphism might be associated with susceptibility to pediatric ALL. Thus, patients carrying the C
Tumor Biol. Table 4
Association between DPYD gene polymorphism and adverse reaction after chemotherapy [n(%)]
Genotypes
Number
Mucosal lesion
Gastrointestinal reaction
Liver function damage
Renal function damage
Cytopenia
Infection
87
14(16.09)
23(26.44)
3(3.45)
2(2.30)
19(21.84)
7(8.05)
60
10(16.67) 0.009
17(28.33) 0.064
9(15.00) 6.321
6(10.00) 4.093
14(23.33) 0.046
12(20.00) 4.509
0.926
0.800
0.012
0.043
0.831
0.034
22(16.79) 2(12.50) 0.192
35(26.72) 5(31.25) 0.148
11(8.40) 1(6.25) 0.088
7(5.34) 1(6.25) 0.023
29(22.14) 4(25.00) 0.067
16(12.21) 3(18.75) 0.541
0.661
0.701
0.767
0.880
0.796
0.462
16(15.69) 8(17.78) 0.100 0.752
27(26.47) 13(28.89) 0.092 0.761
8(7.84) 4(8.89) 0.046 0.831
6(5.88) 2(4.44) 0.126 0.723
22(20.59) 12(26.67) 0.663 0.416
14(13.73) 5(11.11) 0.190 0.663
85T > C TT CT + CC χ2 P value 2194G > A GG GA
131 16
χ2 P value 1156G > T GG GT + TT
102 45
χ2 P value IVS14 + 1G > A GG
135
22(16.30)
37(27.41)
9(6.67)
7(5.19)
30(22.22)
15(11.11)
GA χ2 P value
12
2(16.67) 0.001 0.974
3(25.00) 0.032 0.858
3(25.00) 4.941 0.026
1(8.33) 0.212 0.645
3(25.00) 0.221 0.825
4(33.33) 4.702 0.03
DPYD dihydropyrimidine dehydrogenase gene
allele may have an increased risk of ALL, and the 85T > C polymorphism may be a predictor of CR for pediatric ALL patients. Table 5 Association between DPYD gene polymorphism and CR rate in ALL patients Genotypes
Number (%)
CR (%)
P value
85T > C TT
87 (59.18)
80 (91.95)
–
49 (33.33) 11 (7.48)
43 (87.76) 7 (63.64)*
0.424 0.005
131 (89.12) 16 (10.88)
117 (89.31) 13 (81.25)
– 0.341
102 (69.39) 43 (29.25) 2 (1.36)
91 (89.22) 37 (86.05) 2 (100)
– 0.588 0.623
135 (91.84) 12 (8.16)
121 (89.63) 8 (66.67)#
– 0.02
CT CC 2194G > A GG GA 1156G > T GG GT TT IVS14 + 1G > A GG GA
To date, high inter- and intraindividual variations have been found in DPD levels. This mutability may influence patients receiving 5-FU treatment with regard to toxicity, resistance, and efficacy [18]. Some factors were reported to play important roles in the dysregulation of DPYD, including genetic and epigenetic regulations such as promoter hypermethylation and variation in transcription factor expression [19]. Moreover, previous investigations into the molecular Table 6 rate
Effect of DPYD gene polymorphism on event-free survival
Genotypes
36 months EFS (%)
χ2
P value
95.00 85.00
5.615
0.021
91.60 87.50
0.269
0.604
90.20 93.33
0.389
0.533
91.11 91.67
0.022
0.881
DPYD dihydropyrimidine dehydrogenase gene, ALL acute lymphoblastic leukemia, CR complete remission
85T > C TT CT + CC 2194G > A GG GA 1156G > T GG GT + TT IVS14 + 1G > A GG GA
*P < 0.05, compared with TT genotype; # P < 0.05, compared with GG genotype
DPYD dihydropyrimidine dehydrogenase gene, EFS event-free survival
Tumor Biol.
A
B
100
85T T
EPS(%)
EPS(%)
100
2194GA 2194GG
85CT+CC
95 90
95
90
85
80
85
0
20
40
month
60
C
80
1156GG 1156GT+TT
100
95
90
85
0
20
40
month
mechanisms underlying DPD deficiency indicated that genetic alterations in DPYD, including exon skipping, deletion, and missense mutations, could lead to changes in the mRNA and amino acid sequences and thus result in a DPD-deficient phenotype and a deficiency in DPD activity [20]. Furthermore, a study conducted by Zhang et al. suggested that a significant proportion of the observed severe toxicities might be due to DPYD variants [21]. Genetic variations in DPYD can also result in an enzyme deficiency state, which in turn may lead to severe toxicity or other side effects via imbalance of the nucleotide pool, such as DNA or RNA damage [22]. Several genetic variations have been shown to play crucial roles in DPYD enzyme activity, including 85T > C and IVS14 + 1G > A [23, 24]. Moreover, the DPYD gene plays an important role in the occurrence and development of some neoplastic hematologic disorders. These findings provided a research foundation for investigations into the response to treatment with chemicals. Our study investigated the four DPYD gene loci (85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms) and revealed that the 85T > C polymorphism might be associated with susceptibility to pediatric ALL. Indeed, patients carrying the C allele may have an increased risk for ALL. Our study found that the CC and CT genotypes had lower CR rates compared with the TT genotype in the 85T > C polymorphism; additionally, the altered gene loci had lower CR rates. Historically, patients with inherited DPD deficiency are prone to severe toxic and even lethal effects of 5-FU treatment, which underlines the importance of this enzyme in 5FU metabolism [25]. Previous evidence demonstrated that the genetic variations in DPYD led to enzyme deficiency [18].
60
80
0
20
40
60
80
month
D
EPS(%)
EPS(%)
Fig. 2 Event-free survival (EFS) rate of the different genotypes in the 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms (DPYD dihydropyrimidine dehydrogenase gene; a EFS rate of the genotypes in the 85T > C polymorphism; b EFS rate of the genotypes in the 2194G > A polymorphism; c EFS rate of the genotypes in the 1156G > T polymorphism; d EFS rate of the genotypes in the IVS14 + 1G > A polymorphism)
IVS14GG IVS14GA
100
95
90
85
0
15
30
45
60
month
Moreover, 50 % of the normal DPD activity level in cancer patients was suggested to be sufficient to activate the development of severe 5-FU toxicity. This finding clearly established that the inactivation of one DPYD allele could result in a decrease in DPD expression sufficient to induce 5-FU toxicity, thereby providing a possible genetic basis for this toxicity [26]. A study conducted by Ichikawa et al. demonstrated that a higher DPD mRNA level, which was shown to be a good marker for DPD activity, resulted in a negative prognosis and poorer survival in colorectal carcinoma patients receiving chemotherapy regimens containing 5-FU [27, 28]. However, previous evidence also demonstrated that DPD deficiency rather than a range of enzyme activities produced by multiple variants was a single factor that was responsible for the majority of enzyme deficiencies [29]. Our study demonstrated that patients carrying the TT genotype presented a higher EFS rate compared with patients carrying the 85C genotype in the 85T > C polymorphism, suggesting that the 85T > C polymorphism might be a predictor of the CR for pediatric ALL patients. 5-FU is one of the most common chemotherapeutics and is influenced by DPD activity; thus, the efficacy and toxic reaction were also affected by the changes in 5-FU metabolism [30]. Pharmacokinetics proved that a partial or complete lack of DPD activity caused by genetic variations could reduce the ability to metabolize and clear 5-FU in vivo. The half-life was significantly prolonged, the decomposition was decreased, and the synthesis was increased, resulting in enhanced cell toxicity that contributed to a relatively low prognosis [31, 32]. To summarize, our study provided evidence that the C allele of the 85T > C polymorphism was associated with
Tumor Biol.
susceptibility to pediatric ALL. Thus, patients carrying the C allele may have an increased risk of ALL and the 85T > C polymorphism may be a predictor of CR for pediatric ALL patients. Clinically, the 85T > C polymorphism can be used as a guide for individualized treatment and the decision to utilize 5-FU in ALL patients. Nevertheless, one limitation of this study is the relatively small sample size, which needs to be addressed in a future study. Acknowledgments The authors are grateful to those who gave valuable advice on this paper. Compliance with ethical standards This study was approved by the Ethical Committee of the First Affiliated Hospital to Zhengzhou University. Written informed consent was obtained from all study subjects and/or their legal guardians. This study complied with the guidelines and principles of the Declaration of Helsinki. Conflicts of interest None
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ORIGINAL ARTICLE
DPYD gene polymorphisms are associated with risk and chemotherapy prognosis in pediatric patients with acute lymphoblastic leukemia Xiao-Qiang Zhao 1,2 & Wei-Jie Cao 1 & Hai-Ping Yang 2 & Xue-Wen Yang 2 & Ping Tang 1 & Ling Sun 1 & Xing Gao 1
Received: 3 November 2015 / Accepted: 22 January 2016 # International Society of Oncology and BioMarkers (ISOBM) 2016
Abstract We aimed to investigate the association between dihydropyrimidine dehydrogenase (DPYD) gene polymorphisms and the risk of pediatric acute lymphoblastic leukemia (ALL) and its prognosis after chemotherapy. A total of 147 pediatric ALL patients diagnosed by our hospital between January 2011 and December 2014 were included in the case group, and 102 healthy people who received a physical examination during the same time frame in our hospital were included in the control group. DNA sequencing was applied for site determination and genotyping of the DPYD 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms. The genotype and allele frequencies of the two groups were compared. A significant difference was found in the comparison of the mutant gene and allele frequencies of the 85T > C polymorphism between the case and control groups (P < 0.05). The CT and CC genotypes in the 85T > C polymorphism were associated with the risk of the disease (OR = 1.592, 95 % CI = 1.010–2.509), suggesting that the recessive gene (85C) was more likely to lead to the occurrence of ALL compared with the dominant gene (85T) (P < 0.05). Patients carrying the C allele of the 85T > C polymorphism presented higher damage of their liver functions and higher
infection rates compared with patients carrying the non-C allele (P < 0.05). A higher proportion of liver function damage and a higher infection rate were found in patients with the GA genotype in the IVS14 + 1G > A polymorphism compared with the GG genotype (P < 0.05). The complete remission (CR) rate in patients with the GG genotype in the IVS14 + 1G > A polymorphism was higher than in patients with the GA genotype (P = 0.020). After 5-fluorouracil/calcium folinate (5-FU/CF)-based chemotherapy, the event-free survival (EFS) rate of patients with the TT genotype was higher than patients with the CT and CC genotypes (P < 0.05). Our results revealed that the C allele of the 85T > C polymorphism might be associated with susceptibility to pediatric ALL. Patients carrying the C allele may have an increased risk of ALL. Thus, the 85T > C polymorphism may be a predictor of CR for pediatric ALL patients. Keywords DPYD gene polymorphism . Acute lymphoblastic leukemia . 5-Fluorouracil . 85T > C . 2194G > A . 1156G > T . VS14 + 1G > A
Introduction * Ling Sun [email protected] * Xing Gao [email protected]
1
Department of Hematology, First Affiliated Hospital to Zhengzhou University, Jianshe East Road, No. 1, Two Seven District, Zhengzhou 450052, Henan, People’s Republic of China
2
Department of Hematology, First Affiliated Hospital of Henan University of Science and Technology, Luoyang 471003, Henan, People’s Republic of China
Similar to cancer in general, acute lymphoblastic leukemia (ALL) most likely arises from interactions between exogenous and endogenous exposures, genetic (inherited) susceptibility, and chance [1]. Approximately 6000 new ALL cases with a ratio of 1.3:1 of males to females are estimated to be diagnosed annually in the USA. These cases primarily include children, and approximately 60% of cases are found in people aged A (52 %) is regarded as the most common mutation and has been the most well-studied [9]. Dihydropyrimidine dehydrogenase (DPD, EC1) is encoded by DPYD and is deemed the rate-controlling enzyme for endogenous pyrimidine and fluoropyrimidine catabolism, which is responsible for the elimination of approximately 80 % of administered 5-fluorouracil (5-FU) [10]. 5-FU is a chemotherapeutic that can lead to severe toxic side effects, including hematologic toxicities and death, in advanced or adjuvant settings due to deficiencies in the catabolic pathway [11]. As previously reported, DPD deficiency can result in lifethreatening complications that are correlated with more than 40 sequence variations in the DPYD gene [12]. Many studies have focused on DPYD gene polymorphisms and the toxicity of 5-FU in many types of cancer, including colorectal cancer, gastrointestinal (GI) malignancy, and breast cancer. However, few studies have concentrated on the effects of the gene polymorphisms themselves on the cancer [11, 13]. In this study, we investigated the four different loci (85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A) of the DPYD gene in 147 ALL patients who received surgical treatment and 5-FUbased combined chemotherapy to clarify the relationship between the DPYD gene polymorphisms and ALL.
Materials and methods
their legal guardians. This study complied with the guidelines and principles of the Declaration of Helsinki [14]. Study subjects A total of 147 patients (79 males and 68 females aged 2– 12 years) diagnosed with ALL in the department of hematology in the First Affiliated Hospital to Zhengzhou University between January 2011 and December 2014 were included in the case group. The 147 ALL patients were divided into two types according to the immunophenotyping of the European Group for the Immunological Characterization of Leukemia (EGIL): 122 patients with B cell acute lymphoblastic leukemia (B-ALL) and 25 patients with T cell acute lymphoblastic leukemia (T-ALL) [15]. The enrollment of all patients was based on the diagnostic criteria of the second edition of the Diagnosis and treatment of blood diseases [16]. ALL was diagnosed according to the following basic diagnostic criteria: (1) patients with symptoms of fever, paleness, fatigue, and bleeding accompanied by infiltration into many organs, including the liver, spleen, and lymph nodes; (2) decreased hemachrome, red blood cell counts, and blood cells, whereas the white cell count presented an increase, normal level, or a decrease and its classification included several lymphoblasts and prolymphocytes or no existing lymphoblasts and prolymphocytes; and (3) most of the nuclear cells (primarily lymphocyte hyperplasia) in the bone marrow smear showed obvious or extreme hyperplasia with a minority of nuclear cells presenting as hypoplastic marrow (lymphoblast and prolymphocyte ≥ 30 %). A total of 102 age-matched healthy volunteers (47 males and 55 females aged 2–14 years) who were not biologically related to the 147 ALL patients and received a physical examination in our hospital in the same time frame were enrolled in the control group. Patients were not enrolled if they were >14 years of age; had other severe heart, lung and brain diseases; severe damage of the liver and kidney functions; or other diseases. DNA extraction A fasting peripheral blood (5 mL) sample was collected from both the case and control group patients in the morning in tubes containing ethylenediaminetetraacetic acid (EDTA). The Blood Genome DNA Extraction Kit (Takara Biotechnology [Dalian] co., LTD) was used to extract genomic DNA from the peripheral white blood cells. The DNA density was adjusted to 100 ng/μL, and the samples were stored in −20 °C freezers prior to the experiments.
Ethics statement Primer design This study was approved by the Ethical Committee of the First Affiliated Hospital to Zhengzhou University. Written informed consent was obtained from all study subjects and/or
Premier 5.0 was used to design primers, which were synthesized by Sangon (Shanghai, China). The principle used for
Tumor Biol.
primer design was as follows: (1) the amplified fragment was in the range from 300 to 600 bp; (2) the distance between the two ends and the exon was approximately 100 bp, and the downstream sequencing primers could not have more than 7 single base tandem repeats or short tandem repeats (STRs); (3) the primer length comprised 18–27 basic groups, with an optimum length of 20 bp; and (4) the Tm value was controlled between 58 and 63 °C, with an optimum Tm value of 60 °C. A Tm value of 70 °C was set for the fragment with a GC volume > 70 %. The primer sequences are presented in Table 1.
Polymerase chain reaction The PCR reaction mixture had a total volume of 30 μL and contained the following components: DNA template (0.2 μL), 10 × buffer (3.0 μL) (containing magnesium ion), 1.2 mL of the dNTP mixture (2.5 mmol/L), 1.6 μL of the primers (0.8 μL of the upstream primer and 0.8 μL of the downstream primer), 0.5 μL of TaKaRa Taq (5 U/μL), and 20.2 μL of DEPC-treated water. The PCR amplification was performed based on the touchdown procedure: pre-denaturing (94 °C for 45 s), annealing for 45 s (the annealing temperatures of 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A were 66, 58, 58, and 56.5 °C, respectively), elongation at 72 °C for 45 s, and a final elongation for 10 min (Table 1).
Electrophoresis detection and sequencing The PCR products were analyzed by 1.5 % agarose gel electrophoresis. PCR products with specific amplification fragments were used for sequencing. The Beckman Coulter CEQS000 genetic analysis system and CEQTM DTCS Quick Start Kit (Beckman Coulter) were used for sequencing from both the forward and reverse directions.
Table 1 Primer sequences of DYPD gene polymorphism 85T > C
2194G > A 1156G > T IVS14 + 1G > A
Therapeutic strategy 5-Fluorouracil/calcium folinate (5-FU/CF)-based chemotherapy was administered to the 147 ALL patients. On the first day, the patients received a 3-h intravenous drip with 85 mg/ m2 oxaliplatin (Jiangsu Hengrui Medicine Co., Ltd), a 2-h intravenous drip with 200 mg/m2 calcium folinate, intravenous injection of 400 mg/m2 5-FU, and a 22-h intravenous drip of 600 mg/m2 5-FU with a continuous micro-pump. The next day, the patients received a 2-h intravenous drip with 200 mg/m2 calcium folinate, intravenous injection with 400 mg/m2 5-FU, and a 22-h intravenous drip of 600 mg/m2 5-FU with a continuous micro-pump. A 14-day course of treatment was selected. All patients received at least two courses (average 4–6 courses). Evaluation of adverse drug reactions The difference in the clinical efficiency of the two groups was recorded, and the adverse reactions of patients after treatment were also evaluated in our study. Toxic reactions to the anticancer drugs were divided into complete remission (CR), partial remission, and non-remission based on the Acute and Subacute Toxicity Grading Criteria of Anticancer Drugs (WHO) [17]: (1) CR: no clinical symptoms and signs caused by the infiltration of leukemia cells, platelets ≥ 100 × 109/L, hemoglobin (Hb) ≥ 100 g/L in males and ≥ 90 g/L in females, absolute value of neutrophil granulocytes ≥ 1.5 × 109/L, no leukemia cells found in the peripheral blood, primitive lymphocytes and naive lymphocytes ≤5 %, and red cell and megakaryocytic series presented normal; (2) partial remission: primitive lymphocyte and naive lymphocytes 5–20 % or one of the clinical symptoms or the hemogram did not reach the standard of CR; and (3) non-remission: all three items (myelogram, hemogram, and clinical symptoms) did not reach the standard of complete remission.
Primer sequences
Annealing temperature (°C)
Annealing time (s)
Circle
F: 5′-CCTGGCTTTAAATCCTCGA ACA-3′ R:5′-GTTTTTCTCGTTAGAAACGTCTT ATCC-3′ F: 5′-TGCATTTTTCTGGGATGTGA-3′ R: 5′-GGGATCATAAAGGGCACAAA-3′ F:5′-TAGATGGAACTTGCTAAGGA-3′ R:5′-GAACTGAACCAAAGGCACTG-3′
60
45
30
58
45
40
58
45
45
56.5
45
30
F: 5′-AAAATGTGAGAAGGGACCTCA3′ R: 5′-ATGCATCAGCAAAGCAACTG-3′
DPYD dihydropyrimidine dehydrogenase gene, F forward, R reverse
Tumor Biol.
Follow-up After treatment with surgery and chemotherapy, patient data were collected via telephone, outpatient visits, petition letter, and reviewing case information after the approval of the family members. The deadline for follow-up was December 30, 2014, with a follow-up time of 0.5–36 months (follow-up rate, 100%). The change in the patient’s condition was evaluated regularly through follow-up information obtained for the evaluation of the therapeutic regimen and event-free survival (EFS) rate. The EFS refers to the proportion of the patients free of recurrence and metastasis from the operation date to the final follow-up. Statistical analysis The SPSS 19.0 (IBM Corporation, Somers, NY, USA) software was used for the statistical analysis. Hardy-Weinberg’s equilibrium was applied to detect the representative enrolled patients. Categorical data were measured by the χ2 test and presented as a ratio or percentage. Continuous data were presented as the mean ± standard deviation (SD) and tested by Student’s t test. One-way analysis of variance (ANOVA) was conducted to compare continuous data among multiple groups. The Kaplan-Meier method was applied to analyze the EFS rate. P values 0.05). Comparison of the proportion of abnormal chromogenes between the case and control groups demonstrated no significant differences (case group 25.85 % and control group 20.59 %) (P > 0.05). However, significant differences were found between the case and control groups in the comparisons of white blood cell counts (WBC), red blood cell counts (RBC), hemoglobin concentrations, and platelet counts (all P < 0.01). Genotyping of DPYD gene polymorphisms Specific PCR amplifications were conducted for the DPYD loci (85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A); then, the PCR products were analyzed using 1.5 % agarose gel electrophoresis. Sequencing was applied for specific electrophoresis bands (Fig. 1). Figure 1 showed that the 85T > C polymorphism had three genotypes (TT, CT, and CC), the 2194G > A polymorphism had two genotypes (GG and GA),
the 1156G > T polymorphism had three genotypes (GG, GT, and TT) and the IVS14 + 1G > A polymorphism had the two same genotypes as the 2194G > A polymorphism. DPYD genotype and allele frequencies For the DPYD 85T > C polymorphism, 1 case in the ALL patients (7.48 %) was mutant (CC genotype), 49 cases (33.33 %) were heterozygous (CT genotype), and 87 cases (59.18 %) were wild type (TT genotype). In the control group, the mutant (CC genotype), heterozygous (CT genotype), and wild-type (TT genotype) genotypes were represented by one case (0.98 %), 32 cases (31.37 %), and 69 cases (67.75 %), respectively. A significant difference was detected between the mutant frequencies (CC genotype) in the 85T > C polymorphism between the case and control groups (P < 0.05). An association was found between the C genic mutation in the 85T > C polymorphism and the risk of ALL (case 23.81 % and control 16.67 %) (OR = 1.592, 95 % CI = 1.010–2.509). Thus, the recessive gene (85C) is more likely to lead to the occurrence of ALL than the dominant gene (85T) (P < 0.05). In the 2194G > A polymorphism, 16 cases in ALL patients (10.88 %) were heterozygous (GA genotype) and 131 (89.12 %) were wild type (GG genotype). In the control group, 9 cases (8.82 %) were heterozygous (GA genotype) and 93 cases (91.18 %) were wild type (GG genotype). For the 1156G > T polymorphism, 2 cases in the ALL patients (1.36 %) were mutant (TT genotype), 43 cases (29.25 %) were heterozygous (GT genotype), and 102 cases (69.39 %) were wild type (GG genotype), whereas in the control group, one case was mutant (TT genotype), 25 cases (24.51 %) were heterozygous (GT genotype), and 76 cases (74.51 %) were wild type (GG genotype). In the IVS14 + 1G > A polymorphism, 2 cases in the ALL patients (8.16%) were heterozygous (GA genotype) and 135 cases (91.84%) were wild type (GG genotype), whereas in the control group, 6 cases (5.88 %) were heterozygous (GA genotype) and 96 cases (94.12 %) were wild type (GG genotype). Comparisons of the genotype and allele frequencies of the 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms between the case and control groups showed no existing significant differences, and no obvious associations were found between these genotypes and allele frequencies and the risk of ALL or toxicity (all P > 0.05) (Table 3). Association between DPYD gene polymorphisms and adverse reactions after chemotherapy For the DPYD 85T > C polymorphism, the proportion of liver function damage and the infection rate was higher in patients with the CT and CC genotypes (liver function damage 15 % and infection rate 20 %) compared with patients with the TT genotype (liver function damage 3.45 % and infection rate
Tumor Biol. Table 2 Baseline characteristics of case and control groups
Group
Case group
Control group
n Total
Percentage
147
n
P value
Percentage
102
Gender Male Female Age ≥6 A polymorphism)
compared with the GG genotype (liver function damage 6.67 % and infection rate 11.11 %) (P < 0.05). No significant differences in the liver function damage and infection rates were observed in the different genotypes of the 2194G > A
A
B
T C T G T G T T C
T C TGNGT T C
1
2
TC TGCGT TC
T GG CG T T A C
3
A 85T>C 1) TT; 2) CT; 3) CC
C
GT G T G A A T T
1
GT G T N A A T T
2
C 1156G>T 1) GG; 2) GT
T GG CN T T A C
2
1
B 2194G>A 1) GG; 2) GA
GT G T T A A T T
3
D
CA A CG T A A G
CA A CN T A A G
2
1
D IVS14+1 G>A 1) GG; 2) GA
Tumor Biol. Table 3 Distributions of genotype and allele frequencies of DPYD 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms [n(%)]
Genotypes
χ2
P value
OR(95 % CI)
69(67.75) 32(31.37)
0.487
0.485
Ref 0.823(0.477 ∼ 1.422)
5.908
0.015
0.115(0.014 ∼ 69.270)
Case group
Control group
(n = 147)
(n = 102)
87(59.18) 49(33.33)
85T > C TT CT CC
11(7.48)
1(0.98)
CT + CC T
60(40.82) 223(75.19)
33(32.35) 170(83.33)
C 2194G > A
71(23.81)
34(16.67)
GG
131(89.12)
93(91.18)
GA G
16(10.88) 278(94.56)
Ref 1.592(1.010 ∼ 2.509)
4.053
0.044
9(8.82) 195(95.59)
0.283
0.595
0.792(0.336 ∼ 1.871) Ref
16(5.44)
9(4.41)
0.268
0.605
1.247(0.540 ∼ 2.880)
GG
102(69.39)
76(74.51)
GT TT
43(29.25) 2(1.36)
25(24.51) 1(0.98)
0.715 0.106
0.398 0.745
0.780(0.439 ∼ 1.388) 0.671(0.060 ∼ 7.542)
GT + TT G T
45(30.61) 247(84.01) 47(15.99)
26(25.49) 177(86.76) 27(13.24)
0.721
0.396
Ref 1.247(0.748 ∼ 2.080)
135(91.84) 12(8.16) 282(95.92) 12(4.08)
96(94.12) 6(5.88) 198(97.06) 6(2.94)
0.467
0.494
0.45
0.503
A 1156G > T
Ref
Ref
IVS14 + 1G > A GG GA G A
Ref 0.703(0.255 ∼ 1.939) Ref 1.404(0.518 ∼ 3.805)
Ref reference, CI confidence interval, DPYD dihydropyrimidine dehydrogenase gene
and 1156G > T polymorphisms (both P > 0.05). Moreover, no significant differences were found in mucosal lesions, gastrointestinal reactions, and cytopenia between the different genotypes of the 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms (all P > 0.05) (Table 4).
Association between DPYD gene polymorphisms and the CR rate of ALL patients In the DPYD 85T > C polymorphism, a significant difference was found in the CR rate for the ALL patients between the CC and TT genotypes (P = 0.005); moreover, the CC (63.64 %) and CT (87.76 %) genotypes had lower CR rates compared with the TT (91.95 %) genotype. In the IVS14 + 1G > A polymorphism, the CR rate in patients with the GG (89.63 %) genotype was significantly higher than the CR rate in patients with the GA (66.67 %) genotype (P = 0.020). No significant differences were found between the different genotypes of the 2194G > A (GA 81.25 % and GG 89.31 %) and 1156G > T polymorphisms (TT 100 %, GT 86.05 %, and GG 89.22 %) (all P > 0.05) (Table 5).
Association between DPYD gene polymorphisms and the EFS of ALL patients After surgical treatment combined with 5-FU/CF-based chemotherapy, the EFS of the TT genotype was significantly higher than the EFS of the CT and CC genotypes (85 % in the CT and CC genotypes and 95 % in the TT genotype; P < 0.05). However, no significant differences were found between the EFS rates in the different genotypes of the 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms (all P > 0.05) (Table 6 and Fig. 2a–d).
Discussion Our present study was conducted to investigate the association between DPYD gene polymorphisms and the risk of pediatric ALL and its prognosis after chemotherapy. Our results demonstrated that the C allele of the 85T > C polymorphism might be associated with susceptibility to pediatric ALL. Thus, patients carrying the C
Tumor Biol. Table 4
Association between DPYD gene polymorphism and adverse reaction after chemotherapy [n(%)]
Genotypes
Number
Mucosal lesion
Gastrointestinal reaction
Liver function damage
Renal function damage
Cytopenia
Infection
87
14(16.09)
23(26.44)
3(3.45)
2(2.30)
19(21.84)
7(8.05)
60
10(16.67) 0.009
17(28.33) 0.064
9(15.00) 6.321
6(10.00) 4.093
14(23.33) 0.046
12(20.00) 4.509
0.926
0.800
0.012
0.043
0.831
0.034
22(16.79) 2(12.50) 0.192
35(26.72) 5(31.25) 0.148
11(8.40) 1(6.25) 0.088
7(5.34) 1(6.25) 0.023
29(22.14) 4(25.00) 0.067
16(12.21) 3(18.75) 0.541
0.661
0.701
0.767
0.880
0.796
0.462
16(15.69) 8(17.78) 0.100 0.752
27(26.47) 13(28.89) 0.092 0.761
8(7.84) 4(8.89) 0.046 0.831
6(5.88) 2(4.44) 0.126 0.723
22(20.59) 12(26.67) 0.663 0.416
14(13.73) 5(11.11) 0.190 0.663
85T > C TT CT + CC χ2 P value 2194G > A GG GA
131 16
χ2 P value 1156G > T GG GT + TT
102 45
χ2 P value IVS14 + 1G > A GG
135
22(16.30)
37(27.41)
9(6.67)
7(5.19)
30(22.22)
15(11.11)
GA χ2 P value
12
2(16.67) 0.001 0.974
3(25.00) 0.032 0.858
3(25.00) 4.941 0.026
1(8.33) 0.212 0.645
3(25.00) 0.221 0.825
4(33.33) 4.702 0.03
DPYD dihydropyrimidine dehydrogenase gene
allele may have an increased risk of ALL, and the 85T > C polymorphism may be a predictor of CR for pediatric ALL patients. Table 5 Association between DPYD gene polymorphism and CR rate in ALL patients Genotypes
Number (%)
CR (%)
P value
85T > C TT
87 (59.18)
80 (91.95)
–
49 (33.33) 11 (7.48)
43 (87.76) 7 (63.64)*
0.424 0.005
131 (89.12) 16 (10.88)
117 (89.31) 13 (81.25)
– 0.341
102 (69.39) 43 (29.25) 2 (1.36)
91 (89.22) 37 (86.05) 2 (100)
– 0.588 0.623
135 (91.84) 12 (8.16)
121 (89.63) 8 (66.67)#
– 0.02
CT CC 2194G > A GG GA 1156G > T GG GT TT IVS14 + 1G > A GG GA
To date, high inter- and intraindividual variations have been found in DPD levels. This mutability may influence patients receiving 5-FU treatment with regard to toxicity, resistance, and efficacy [18]. Some factors were reported to play important roles in the dysregulation of DPYD, including genetic and epigenetic regulations such as promoter hypermethylation and variation in transcription factor expression [19]. Moreover, previous investigations into the molecular Table 6 rate
Effect of DPYD gene polymorphism on event-free survival
Genotypes
36 months EFS (%)
χ2
P value
95.00 85.00
5.615
0.021
91.60 87.50
0.269
0.604
90.20 93.33
0.389
0.533
91.11 91.67
0.022
0.881
DPYD dihydropyrimidine dehydrogenase gene, ALL acute lymphoblastic leukemia, CR complete remission
85T > C TT CT + CC 2194G > A GG GA 1156G > T GG GT + TT IVS14 + 1G > A GG GA
*P < 0.05, compared with TT genotype; # P < 0.05, compared with GG genotype
DPYD dihydropyrimidine dehydrogenase gene, EFS event-free survival
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A
B
100
85T T
EPS(%)
EPS(%)
100
2194GA 2194GG
85CT+CC
95 90
95
90
85
80
85
0
20
40
month
60
C
80
1156GG 1156GT+TT
100
95
90
85
0
20
40
month
mechanisms underlying DPD deficiency indicated that genetic alterations in DPYD, including exon skipping, deletion, and missense mutations, could lead to changes in the mRNA and amino acid sequences and thus result in a DPD-deficient phenotype and a deficiency in DPD activity [20]. Furthermore, a study conducted by Zhang et al. suggested that a significant proportion of the observed severe toxicities might be due to DPYD variants [21]. Genetic variations in DPYD can also result in an enzyme deficiency state, which in turn may lead to severe toxicity or other side effects via imbalance of the nucleotide pool, such as DNA or RNA damage [22]. Several genetic variations have been shown to play crucial roles in DPYD enzyme activity, including 85T > C and IVS14 + 1G > A [23, 24]. Moreover, the DPYD gene plays an important role in the occurrence and development of some neoplastic hematologic disorders. These findings provided a research foundation for investigations into the response to treatment with chemicals. Our study investigated the four DPYD gene loci (85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms) and revealed that the 85T > C polymorphism might be associated with susceptibility to pediatric ALL. Indeed, patients carrying the C allele may have an increased risk for ALL. Our study found that the CC and CT genotypes had lower CR rates compared with the TT genotype in the 85T > C polymorphism; additionally, the altered gene loci had lower CR rates. Historically, patients with inherited DPD deficiency are prone to severe toxic and even lethal effects of 5-FU treatment, which underlines the importance of this enzyme in 5FU metabolism [25]. Previous evidence demonstrated that the genetic variations in DPYD led to enzyme deficiency [18].
60
80
0
20
40
60
80
month
D
EPS(%)
EPS(%)
Fig. 2 Event-free survival (EFS) rate of the different genotypes in the 85T > C, 2194G > A, 1156G > T, and IVS14 + 1G > A polymorphisms (DPYD dihydropyrimidine dehydrogenase gene; a EFS rate of the genotypes in the 85T > C polymorphism; b EFS rate of the genotypes in the 2194G > A polymorphism; c EFS rate of the genotypes in the 1156G > T polymorphism; d EFS rate of the genotypes in the IVS14 + 1G > A polymorphism)
IVS14GG IVS14GA
100
95
90
85
0
15
30
45
60
month
Moreover, 50 % of the normal DPD activity level in cancer patients was suggested to be sufficient to activate the development of severe 5-FU toxicity. This finding clearly established that the inactivation of one DPYD allele could result in a decrease in DPD expression sufficient to induce 5-FU toxicity, thereby providing a possible genetic basis for this toxicity [26]. A study conducted by Ichikawa et al. demonstrated that a higher DPD mRNA level, which was shown to be a good marker for DPD activity, resulted in a negative prognosis and poorer survival in colorectal carcinoma patients receiving chemotherapy regimens containing 5-FU [27, 28]. However, previous evidence also demonstrated that DPD deficiency rather than a range of enzyme activities produced by multiple variants was a single factor that was responsible for the majority of enzyme deficiencies [29]. Our study demonstrated that patients carrying the TT genotype presented a higher EFS rate compared with patients carrying the 85C genotype in the 85T > C polymorphism, suggesting that the 85T > C polymorphism might be a predictor of the CR for pediatric ALL patients. 5-FU is one of the most common chemotherapeutics and is influenced by DPD activity; thus, the efficacy and toxic reaction were also affected by the changes in 5-FU metabolism [30]. Pharmacokinetics proved that a partial or complete lack of DPD activity caused by genetic variations could reduce the ability to metabolize and clear 5-FU in vivo. The half-life was significantly prolonged, the decomposition was decreased, and the synthesis was increased, resulting in enhanced cell toxicity that contributed to a relatively low prognosis [31, 32]. To summarize, our study provided evidence that the C allele of the 85T > C polymorphism was associated with
Tumor Biol.
susceptibility to pediatric ALL. Thus, patients carrying the C allele may have an increased risk of ALL and the 85T > C polymorphism may be a predictor of CR for pediatric ALL patients. Clinically, the 85T > C polymorphism can be used as a guide for individualized treatment and the decision to utilize 5-FU in ALL patients. Nevertheless, one limitation of this study is the relatively small sample size, which needs to be addressed in a future study. Acknowledgments The authors are grateful to those who gave valuable advice on this paper. Compliance with ethical standards This study was approved by the Ethical Committee of the First Affiliated Hospital to Zhengzhou University. Written informed consent was obtained from all study subjects and/or their legal guardians. This study complied with the guidelines and principles of the Declaration of Helsinki. Conflicts of interest None
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