ITPA:c.94C>A and NUDT15:c.415C>T Polymorphisms and Their Relation to Mercaptopurine-Related Myelotoxicity in Childhood Leukemia in Thailand

Figures & data

Figure 1 Study flow diagram.

Figure 2 Gel electrophoresis of 328-bp PCR products of ITPA:c.94C>A variant (A) before and (B) after NspI restriction enzyme digestion and 269-bp PCR products of NUDT15:c.415C>T variant (C) before and (D) after TaaI restriction enzyme digestion, respectively (lane 1–4, M-100 bp marker). The 328-bp PCR products of ITPA:c.94C>A variant were digested by NspI and yielded 238-bp and 90-bp fragments as shown in lane 1 and 3; (B) suggesting the heterozygous for ITPA:c.94C>A variant. The 269 bp-PCR products of NUDT15:c.415C>T variant were digested by TaaI and yielded 142-bp and 127-bp fragments as shown in lane 2 and 4; (D) indicating the heterozygous for NUDT15:c.415C>T variant.

Table 1 Patient Demographic Data

Patients (n=99)	N (%)
Age at diagnosis (years)
Mean±SD	5.1±4.4
Median (min–max)	4.5 (0.3–18.0)
Gender
Male	55 (56)
Female	44 (44)
Diagnosis
Pre-B ALL	87 (88)
T-ALL	10 (10)
MPAL	1 (1)
APL	1 (1)
CNS status
CNS-1	91 (92)
CNS-2	6 (6)
CNS-3	2 (2)
Ploidy
Normal	95 (96)
Hyperdiploidy	4 (4)
Risk stratification for ALL (n=97)
Standard	47 (48)
High	42 (43)
Very high	8 (8)
Disease status
Remission	83 (84)
Relapse	16 (16)

Note: Data are presented as mean±SD and median (range) for continuous variables and number (%) for categorical variables.

Abbreviations: ALL, acute lymphoblastic leukemia; ANC, absolute neutrophil count; APL, acute promyelocytic leukemia; CNS, central nervous system; MPAL, mixed phenotype acute leukemia.

Table 2 Prevalence of ITPA:c.94C>A and NUDT15:c.415C>T Polymorphisms

Genetic Polymorphisms	N (%)
ITPA:c.94C>A
Wild type	65 (66)
Heterozygous genotype	34 (34)
NUDT15:c.415C>T
Wild type	82 (83)
Heterozygous genotype	17 (17)
Both ITPA:c.94C>A and NUDT15:c.415C>T heterozygous genotypes
Yes	4 (4)
No	95 (96)

Note: Data are presented as number (%) for categorical variables.

Abbreviations: ITPA, inosine triphosphate pyrophosphohydrolase; NUDT15, nucleoside diphosphate linked moiety X-type motif 15.

Table 3 Characteristics of Patients with Acute Leukemia According to ITPA:c.94C>A and NUDT15:c.415C>T Genotypes

	ITPA:c.94C>A				NUDT15:c.415C>T					ITPA:c.94C>A and NUDT15:c.415C>T
	WT		GT		p-value	WT		GT		p-value	WT		GT
	N	%	N	%		N	%	N	%		N	%	N	%
Age at diagnosis (years)	6.4±4.3		5.3±4.1		0.241	6.2±4.3		5.4±3.8		0.437	6.1±4.3		5.0±2.9
Gender
Male	32	58	23	42	0.080	49	89	6	11	0.065	54	98	1	2
Female	33	75	11	25		33	75	11	25		41	93	3	7
Diagnosis
Pre-B ALL	56	64	31	36	0.659	71	82	16	18	0.900	83	95	4	5
T-ALL	7	70	3	30		9	90	1	10		10	100	0	0
MPAL	1	100	0	0		1	100	0	0		1	100	0	0
APL	1	100	0	0		1	100	0	0		1	100	0	0
CNS status
CNS-1	59	65	32	35	0.569	75	82	16	18	0.801	87	96	4	4
CNS-2	5	83	1	17		5	83	1	17		6	100	0	0
CNS-3	1	50	1	50		2	100	0	0		2	100	0	0
Ploidy
Normal	62	65	33	35	0.688	79	83	16	17	0.672	91	96	4	4
Hyperdiploidy	3	75	1	25		3	75	1	25		4	100	0	0
Risk stratification for ALL (N=97)
Standard	28	60	19	40	0.286	38	81	9	19	0.360	44	94	3	6
High	31	74	11	26/50		37	88	5	12		42	100	0	0
Very high	4	50	4			5	63	3	37		7	88	1	12
Disease status
Remission	54	65	29	35	0.776	68	82	15	18	0.588	79	95	4	5
Relapse	11	69	5	31		14	88	2	12		16	100	0	0

Notes: Data are presented as mean±SD for continuous variables and number (%) for categorical variables; comparison between two independent data sets was analyzed using Fisher’s exact test for categorical data and Mann–Whitney U-test for continuous data; p-value<0.05 was considered as statistically significant.

Abbreviations: ALL, acute lymphoblastic leukemia; ANC, absolute neutrophil count; APL, acute promyelocytic leukemia; CNS, central nervous system; GT, genetic polymorphism; ITPA, inosine triphosphate pyrophosphohydrolase; NUDT15, nucleoside diphosphate linked moiety X-type motif 15; MPAL, mixed phenotype acute leukemia; WT, wild type.

Figure 3 Associations between genetic variants and various blood counts as well as 6-MP administrative dosing among patients with acute leukemia undergoing 6-MP containing regimens. (A) ANC, (B) platelet count, (C) hemoglobin, and (D) daily 6-MP administrative dosing among patients with ITPA:c.94C>A polymorphism versus those with ITPA wide type. (E) ANC, (F) platelet count, (G) hemoglobin, and (H) daily 6-MP administrative dosing among patients with NUDT15:c.415C>T polymorphism versus those with NUDT15 wide type.

Notes: Data in the graph are shown as dot plots. One dot plot represents a mean blood count or daily 6-MP administrative dosing on each cycle of treatment during the maintenance phase of leukemia treatment protocols. The two independent data sets were analyzed using independent sample t-test and Mann–Whitney U-test; p-value<0.05 was considered as statistically significant.

Abbreviations: ANC, absolute neutrophil count; ITPA, inosine triphosphate pyrophosphohydrolase; NUDT15, nucleoside diphosphate linked moiety X-type motif 15; 6-MP, mercaptopurine.

Table 4 Effect of ITPA:c.94C>A and NUDT15:c.415C>T Polymorphisms and 6-MP Dosing on ANC

ANC	Coefficient	p-value	95% CI
			Lower	Upper
Model 1
ITPA:c.94C>A	11.90	0.948	−343.11	366.92
6-MP dose	−8.40	0.062	−17.22	0.42
Model 2
NUDT15:c.415C>T	−463.81	0.004*	−778.53	−149.09
6-MP dose	−9.29	0.038*	−18.05	−0.52
Model 3
ITPA:c.94C>A and NUDT15:c.415C>T	−527.56	0.046*	−1045.65	−9.48
6-MP dose	−8.41	0.061	−17.19	0.38

Notes: Longitudinal data from time-dependent variables were analyzed using multivariate GEE to examine the associations between ITPA and NUDT15 polymorphisms as well as 6-MP administrative dosing and ANC measured monthly during the maintenance phase of acute leukemia protocols; *p-value<0.05 was considered as statistically significant.

Abbreviations: ANC, absolute neutrophil count; ITPA, inosine triphosphate pyrophosphohydrolase; NUDT15, nucleoside diphosphate linked moiety X-type motif 15; 6-MP, mercaptopurine.

Table 5 Overall Frequency of Myelotoxicity

CBC (N=1635)	Genetic Polymorphisms
	ITPA:c.94C>A		NUDT15:c.415C>T		ITPA:c.94C>A and NUDT15:c.415C>T
	Heterozygous	Wild type	Heterozygous	Wild type	Yes	No
	N=549	N=1086	N=261	N=1374	N=59	N=1576
ANC (cells/mm^3)
<500	23	65	25	63	3	85
500–999	90	146	47	189	14	222
1,000–1,500	101	207	62	246	14	294
Hemoglobin (g/dL)
<8.0	4	21	5	11	1	15
8.0–9.9	60	111	34	137	4	167
10–12	285	484	149	620	38	731
Platelets (cells/mm^3)
<25,000	3	3	0	6	0	6
25,000–49,999	2	8	3	7	1	9
50,000–74,999	3	8	0	11	0	11
75,000–150,000	42	100	19	123	7	135

Abbreviations: ANC, absolute neutrophil count; CBC, complete blood count; ITPA, inosine triphosphate pyrophosphohydrolase; NUDT15, nucleoside diphosphate linked moiety X-type motif 15.