DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukemia patients with NUDT15 variant genotypes

Hee Young Ju; Ji Won Lee; Hee Won Cho; Ju Kyung Hyun; Youngeun Ma; Eun Sang Yi; Keon Hee Yoo; Ki Woong Sung; Rihwa Choi; Hong Hoe Koo; Soo-Youn Lee

doi:10.1371/journal.pone.0245667

. 2021 Jan 22;16(1):e0245667. doi: 10.1371/journal.pone.0245667

DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukemia patients with NUDT15 variant genotypes

Hee Young Ju ¹, Ji Won Lee ¹, Hee Won Cho ¹, Ju Kyung Hyun ¹, Youngeun Ma ², Eun Sang Yi ³, Keon Hee Yoo ¹, Ki Woong Sung ¹, Rihwa Choi ^4,⁵, Hong Hoe Koo ^1,^*, Soo-Youn Lee ^5,^*

Editor: A M Abd El-Aty⁶

PMCID: PMC7822258 PMID: 33481917

Abstract

Background

Large inter-individual variations in drug metabolism pose a challenge in determining 6-mercaptopurine (6MP) doses. As the last product of 6MP metabolism, DNA-thioguanine nucleotide (DNA-TGN) could reflect the efficacy of 6MP, especially in patients harboring variants in the 6MP metabolism pathway. The aim of this study was to investigate the clinical significance of DNA-TGN monitoring in Korean pediatric acute lymphoblastic leukemia (ALL) patients, focusing on the NUDT15 genotype.

Methods

The subjects of this study were patients who underwent ALL treatment with 6MP. Tests for the NUDT15 and TPMT genotypes were performed, and prospective DNA-TGN and erythrocyte TGN samples were collected after two weeks or more of 6MP treatment. DNA-TGN was quantified using the liquid chromatography-tandem mass spectrometry method.

Results

A total of 471 DNA-TGN measurements in 71 patients were analyzed, which ranged from 1.0 to 903.1 fmol thioguanine/μg DNA. The 6MP intensity demonstrated a significant relationship with DNA-TGN concentration (P<0.001). Patients harboring NUDT15 variants were treated with a lower dose of 6MP (P<0.001); however, there was no significant difference in DNA-TGN concentration when compared to patients carrying wild-type NUDT15 (P = 0.261). These patients also presented higher variation in DNA-TGN levels (P = 0.002) and DNA-TGN/6MP intensity (P = 0.019) compared to patients carrying wild-type NUDT15. DNA-TGN concentration did not show a significant correlation with WBC count (P = 0.093).

Conclusions

Patients harboring NUDT15 variants demonstrated similar DNA-TGN concentrations even at low doses of 6MP and showed high variability in DNA-TGN. Particularly in patients with NUDT15 variants who need a reduced 6MP dose, DNA-TGN could be applied as a useful marker to monitor the therapeutic effect of 6MP.

Introduction

Mercaptopurine is one of the key drugs used for maintenance treatment of childhood acute lymphoblastic leukemia (ALL). However, some patients experience toxicity during mercaptopurine treatment, such as severe infections related to bone marrow suppression, in addition to technical issues, such as the need for dose reduction or frequent treatment interruptions. Mercaptopurine is known to have narrow therapeutic indices and it is also known that frequent dose changes or discontinuation of mercaptopurine is associated with increased relapse of ALL [1]. Maintaining an appropriate concentration of thiopurine metabolites is important for preventing relapse or the occurrence of toxic events after ALL treatment.

If accurate metabolite measurements can be used to adjust the dose of 6-mercaptopurine (6MP), ALL patients, especially those with a variety of metabolic variations, can expect better treatment outcomes. To date, the degree of cytopenia and liver function have been used to adjust the dose of 6MP during ALL treatment. However, the metabolism of 6MP varies greatly, and the dosage may vary by up to 10 fold between patients; this uncertainty makes it difficult to successfully carry out therapeutic adjustments using these classical methods [2]. Moreover, it is known that varying 6MP doses and 6MP drug interruptions are related to varying thioguanine nucleotide (TGN) levels and an increase in relapse rate [1]. Other studies have evaluated the use of erythrocyte TGN (RBC TGN) as an indicator of 6MP metabolism, but these studies have found that RBC TGN is not a robust prognostic marker for relapse [3]. Recently, DNA-incorporated thioguanine nucleotides (DNA-TGNs) have been proposed as the primary mediators of 6-mercaptopurine cytotoxicity [4]. In addition, a large-scale prospective study by the Nordic Society of Pediatric Hematology and Oncology (NOPHO) group demonstrated that the concentration of DNA-TGN is negatively correlated with relapse [5].

Several genes have been linked to the metabolism of 6MP used in the treatment of ALL, including TPMT and NUDT15 [6]. Variants of these two genes have been linked to an increased risk of leukopenia during 6MP treatment [7, 8]. Patients carrying variant TPMT or NUDT15 alleles, which exhibit decreased activity compared to the wild type, experience increased toxicity even at low doses of 6MP. Particularly, NUDT15 variants have been shown to be more prevalent in Asian populations than in other ethnic groups [9–11]. As a nucleotide diphosphatase, NUDT15 is known to inactivate thiopurine metabolites by converting TGTP to TGMP and therefore negatively regulates the cytotoxic effects of this class of drugs [12]. It can then be assumed that in individuals harboring NUDT15 variants, even low doses of 6MP would produce relatively high DNA-TGN concentrations, thereby allowing for effective treatment.

To date, three papers have been published on DNA-TGN levels, covering patients with NUDT15 variant genotypes [13–15]. These studies have reported that patients with the NUDT15 variant genotype show higher DNA-TGN levels and toxicity when 6MP is administered at regular doses. In contrast, the present study focused on DNA-TGN in patients with NUDT15 or TPMT variant genotypes who were treated with reduced doses of 6MP. In addition, in this study, since repeated blood tests were performed in patients continuously receiving 6MP, changes in DNA-TGN over time could be observed.

The aim of this study was to investigate the pattern of DNA-TGN when 6MP is administered at reduced doses in patients harboring NUDT15 variants (dynamically adjusted based on the degree of cytopenia). It was designed to evaluate whether DNA-TGN concentration differs based on the genotype of NUDT15, and whether DNA-TGN levels reflect treatment-related toxicity during ALL treatment. In addition, the variation of DNA-TGN during maintenance treatment with 6MP was explored.

Methods

1. Study patients

In this study, the participants were recruited from patients undergoing 6MP-based treatment for ALL or lymphoma at the Department of Pediatrics, Samsung Medical Center, Seoul, Republic of Korea. The enrollment period was from January 2018 to February 2020, and patients with newly diagnosed as well as relapsed ALL or lymphoma were included in the study. Patients who were not expected to receive 6MP treatment and those who did not wish to participate in the study were excluded. The number of participants in the study was not set in advance because of the exploratory study design of DNA-TGN in patients with an existing treatment schedule.

The present study protocol was reviewed and approved by the Institutional Review Board of Samsung Medical Center (approval No. 2017-11-161). Informed consent was obtained from all subjects when they were enrolled. Written consent was obtained from the parents of minors and from patients for adults.

2. Treatment

Patients were treated in accordance with the Children’s Oncology Group (COG) regimen-based treatment. Regardless of their risk group, patients who received 6MP for two or more weeks were enrolled in this study. The initial dose for 6MP maintenance therapy was determined based on the lowest 6MP dose that did not result in cytopenia (described in previous treatment cycles, including consolidation, interim maintenance, or delayed intensification). The 6MP dosage was rounded up to the nearest available dose for administration, and the dosage was gradually increased upon identifying that 6MP was tolerable. The intended final dose of 6MP for patients harboring wild-type NUDT15 and TPMT was set at 50 mg/m²/day. 6MP intensity was defined as the ratio between the prescribed daily 6MP dose (mg/m²/day) and the standard daily dose (50 mg/m²/day).

3. Sample collection and analysis

Prospective metabolite samples (DNA-TGN, RBC TGN, methylmercaptopurine nucleotides (MMPN)) were acquired simultaneously after continuous administration of 6MP for at least 14 days after initiation of constant-dosing 6MP treatment: after 2 weeks for the protocol in which patients took 6MP for 2 weeks; after 2 and 4 weeks for the protocol in which patients took 6MP for 4 weeks; after 2, 4, and 8 weeks for the protocol in which patients took 6MP for 8 weeks; and after 2 and 4 weeks of the 1^st cycle of maintenance treatment. Blood sampling for DNA-TGN was performed along with routine sampling to monitor complete blood cell count and chemistry tests during chemotherapy. If the 6MP dose was changed or if the treatment was stopped, additional samples were collected. DNA- and RBC- TGN concentrations and MMPN were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The DNA-TGN test was performed with isotope-labeled TG-d3 and guanine-d3 as internal standards, as previously described. Chromatographic etheno-TG peaks were normalized using etheno-G by calculating TG responses as etheno-TG area/etheno-G area (DNA-TG = [etheno-TG response/etheno-G response]/[etheno-TG-d3 response/etheno-G-d3 response]) [16, 17]. In this study, blood samples were collected prospectively; however, the DNA-TGN test was not performed in real time. Therefore, the dose of 6MP was adjusted according to white blood cell (WBC) count, aspartate aminotransferase (AST), alanine transaminase (ALT), but not DNA-TGN values. Genotyping of NUDT15 and TPMT was performed before the start of the 6MP maintenance therapy via direct sequencing. Direct Sanger sequencing of exons 1 and 3 of NUDT15 and direct sequencing of exons 3 to 10 of TPMT were performed after PCR [18, 19]. Since NUDT15 *1/*2 and *3/*6 genotypes were not distinguished technically by this method, *1/*2 represents *1/*2 or *3/*6 [18].

4. Data collection

Clinical data were collected upon diagnosis and included 6MP dose, treatment interruption, relapse, survival, and treatment-related toxicity. Laboratory results were collected for WBC, absolute neutrophil count (ANC), platelet count, AST, ALT, and total bilirubin.

5. Statistical analysis

Descriptive analyses were conducted for age, sex, diagnosis, NUDT15, TPMT genotype, 6MP dose, DNA-TGN, and RBC TGN. Fisher’s exact test was performed to determine differences in sex, diagnosis, and relapse status according to genotypes. Correlation analysis was performed to determine the correlation of DNA-TGN by 6MP dose and WBC count by DNA-TGN. Fisher’s exact test was used to determine the differences in the rate of toxicity in the NUDT15 variant and wild type groups. For the association of DNA-TGN by 6MP dose and hematologic toxicity, analyses were conducted only on the samples collected during maintenance treatment to rule out the effects of other drugs. The Kruskal-Wallis test was performed to compare the differences in DNA-TGN according to sex, diagnosis, and relapse state. In addition, the same test was used to compare the differences in DNA-TGN, 6MP intensity, and DNA-TGN/6MP intensity according to genotypes. To determine the difference in DNA-TGN/6MP intensity according to genotype, normalization of DNA-TGN/6MP intensity was done. To evaluate the distribution of DNA-TGN, DNA-TGN/6MP intensity and RBC TGN values between and within patients, coefficients of variation (CV) were calculated. Linear mixed model analysis was performed to compare the pattern of change between NUDT15 variant and wild type groups. When performing CV and linear mixed model analyses to determine the degree of variability, only the values collected during the maintenance treatment of patients from which were taken DNA-TGN samples more than three times during the maintenance period were used.

All statistical analyses were performed using SPSS v25 (SPSS Inc., Chicago, IL, USA), and all graphical representations were prepared using Prism 7 (Graph Pad Software, San Diego, CA, USA). All statistical tests used two-sided probability, and significance was set at P <0.05.

Results

1. Patients and samples

A total of 72 patients were enrolled in this study, but one patient was excluded from the analysis due to a lack of clinical information. Of the 71 patients in this study, 47 were male and 24 were female; 19 patients harbored an NUDT15 variant and 3 patients harbored a TPMT variant. None of the patients harbored both genetic variants. Detailed clinical information is presented in Table 1.

Table 1. Participant and DNA-TGN sample characteristics.

Characteristic	Total Patients^* (N = 71)	NUDT15 WT^†, TPMT WT (N = 49)	NUDT15 variant, TPMT WT (N = 19)	NUDT15 WT, TPMT variant (N = 3)	P value
Sex
Male	47	35	11	1	0.235
Female	24	14	8	2
Median age	6.9	6.5	8.5	8.9	0.441
(range)	(1.2–23.3)	(1.2–21.2)	(2.4–23.3)	(4.9–11.3)	0.441
Diagnosis
B-ALL^‡	59	41	15	3	0.522
T-ALL	7	5	2	0
B-NHL^§	1	1	0	0
T-NHL	4	2	2	0
Previous relapse
Yes	68	48	18	2	0.078
No	3	1	1	1
Variant genotypes			NUDT15	TPMT	N/A
			^1/^2, n = 8	^1/^3, n = 3
			^1/^3, n = 4
			^1/^5, n = 3
			^1/^6, n = 3
			^2/^3, n = 1
On-therapy	48	35	11	2	0.564
Off-therapy	23	14	8	1
Relapse after enrolment	1	1	0	0	N/A
Death	0	0	0	0	N/A
Number of DNA-TGN samples taken	471	291	167	13	0.858
Mean number of DNA-TGN samples for each patient	6.6	5.9	8.8	4.3	N/A
Mean number of DNA-TGN samples for each patient	(range, 1–21)	(range, 1–16)	(range, 1–21)	(range, 1–10)	N/A
Timepoint at which DNA-TGN sample was collected
Consolidation	20	16	4	0	0.153
Interim maintenance	86	60	25	1
Delayed intensification	6	2	4	0
Maintenance	359	213	134	12
Median 6MP intensity during maintenance treatment	0.33 (range, 0.01–2.3)	0.4 (range, 0.07–2.3)	0.22 (range, 0.02–1.43)	0.03 (range, 0.01–0.52)	<0.001

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*There was no patient harboring both NUDT15 and TPMT variants;

^†WT, wild type;

^‡ALL, acute lymphoblastic leukemia;

^§NHL, non-Hodgkin lymphoma.

A total of 472 DNA-TGN samples were collected. As one sample was collected during the treatment interruption period, only 471 samples were used for analysis. The mean number of DNA-TGN samples collected per patient was 6.6 (range, 1–21). A total of 359 measurements were performed during the maintenance therapy, with 112 measurements taken before the initiation of the maintenance therapy. DNA-TGN levels from whole measurements ranged from 1.0 to 903.1 fmol TG/μg DNA (fmol thioguanine/μg DNA), with a median level of 119.0 fmol TG/μg DNA.

The median follow-up time was 18.1 months (range, 3.2–27.3 months). At the time of the last data collection, 23 patients were off therapy, and 48 patients were on therapy. There were no deaths during this study, but one patient exhibited a relapse of ALL. This patient was sent for DNA-TGN evaluation during the consolidation treatment, and relapse was confirmed immediately after that. The treatment protocol was changed, and hematopoietic stem cell transplantation was performed. Relapse was not observed in patients who underwent ALL maintenance treatment.

When comparing the distribution of DNA-TGN, male (vs. female, P = 0.001) and Pre-B ALL (vs. Pre-T ALL, lymphoma, P<0.001) showed higher levels of DNA-TGN. However, when comparing DNA-TGN/6MP intensity, there was no significant difference according to sex (P = 0.18), disease (P = 0.061), or previous relapse (P = 0.675).

2. DNA-TGN results stratified based on the NUDT15 status

The distribution of DNA-TGN levels was stratified based on the genotype of NUDT15 and TPMT (Fig 1A). There was a significant positive correlation between DNA-TGN levels and 6MP dosage in all patients (P<0.001). When examining the distribution of DNA-TGN according to the 6MP dose by the subgroup of NUDT15 variants, NUDT15 *1/*2, *1/*3, and *1/*5, showed higher DNA-TGN/6MP intensity than that of wild-type patients. However, there was no statistically significant difference between NUDT15 *1/*6 or TPMT variant genotype patients and wild-type patients, but the level was lower in one DNA-TGN test performed on the NUDT15 *2/*3 genotype patient (Fig 1B).

Patients with NUDT15 variant genotypes were treated with significantly lower 6MP intensities when compared to patients with the wild-type NUDT15 genotype (P<0.001). (Fig 2A) However, there was no significant difference in the DNA-TGN levels between patients with NUDT15 variant genotypes, TPMT variant genotypes, and both wild types (P = 0.261) (Fig 2B). The ratio of DNA-TGN/6MP intensity was significantly higher in patients harboring NUDT15 variants than that in patients harboring the wild-type NUDT15 (P<0.001); however, no difference was observed between patients harboring TPMT variants and the wild type (P = 0.323) (Fig 2C).

Fig 2 — (A) 6MP intensity (B) DNA-TGN (C) DNA-TGN/6MP intensity (WT, wild type; v, variant).

The median DNA-TGN level during the maintenance period in patients harboring both NUDT15 and TMPT wild-type genes was 126.2 fmol TG/μg DNA. In patients harboring NUDT15 variants, 73.8% of the 6MP dose was needed to reach the median DNA-TGN level compared to patients harboring wild-type NUDT15.

3. Toxicity during treatment

Of the 63 patients who underwent maintenance therapy, eight patients manifested leukopenia (WBC < 1,500/μL) at the time of DNA-TGN sampling. Among these patients, four harbored NUDT15 variants (two *1/*2, one *1/*5, one *1/*6 genotype), and no patient with a TPMT variant experienced cytopenia. The DNA-TGN concentrations during these leukopenia episodes ranged from 27.8 to 504.8 fmol TG/μg DNA. Three of the patients developed leukopenia before 12 weeks of treatment, and their 6MP intensities were 0.17, 0.12, and 0.08, respectively. However, in the five cases of leukopenia that occurred after 12 weeks of 6MP-based maintenance therapy, the 6MP intensity ranged from 0.22 to 0.98 (median 0.4).

Three DNA-TGN samples were collected when treatment was discontinued for reasons other than cytopenia (AST or ALT elevation in two cases, fever with viral infection in one case). More detailed information on the measurements associated with cytopenia or toxicities resulting from treatment interruptions are described in Table 2.

Table 2. Cases with toxicity related to treatment.

Patient	NUDT15	TPMT	Time of Sample	6MP intensity^*	MTX intensity^†	DNA-TGN (fmol TG/μg DNA)	RBC TGN (μmol/L)	MMPN^‡ (μmol/L)	WBC (/μL)	ANC (/μL)	Reason of interruption
Cytopenia
1	^1/^1	^1/^1	10^th cycle	0.98	1.01	504.8	364.7	35039.8	1280	540	-
2	^1/^1	^1/^1	2^nd cycle	0.40	0.97	63.8	200	1150.3	1260	200	-
3	^1/^1	^1/^1	3^rd cycle	0.22	1.03	149.1	385.7	469.3	780	390	-
4	^1/^1	^1/^1	3^rd cycle	0.28	0.89	107.6	261.9	610.5	1440	310	-
5	^1/^5	^1/^1	7^th cycle	0.47	1.04	235.9	313.2	1057.1	1440	910	-
6	^1/^2	^1/^1	1^st cycle (2 weeks from start)	0.17	0.69	68.7	65.9	1080.5	1410	940	-
7	^1/^2	^1/^1	1^st cycle (6 weeks from start)	0.12	0.61	62.0	70.4	154.6	770	270	-
8	^1/^6	^1/^1	1^st cycle (10 weeks from start)	0.08	0.98	27.8	51.5	85.4	1040	320	-
Treatment interruption for reasons other than cytopenia
9	^1/^1	^1/^1	3^rd cycle	0.28	0.94	90.0	236.3	791.9	2420	1310	AST^§ 1040
10	^1/^1	^1/^1	2^nd cycle	0.47	0.82	80.5	208.9	2064.9	2400	830	AST^§ 1145
11	^1/^2	^1/^1	4^th cycle	0.18	0.97	74.8	94.5	413.2	5210	4120	Fever with viral infection

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*6MP intensity, 6MP dose (mg/m²/day) / 50 mg/m²/day;

^†MTX intensity, Methotrexate dose (mg/m²/week) / 20 (mg/m²/week);

^‡MMPN, methylmercaptopurine nucleotides;

^§AST, aspartate aminotransferase.

When these patients were excluded, the WBC counts for all the other patients were within the reference range. Within the total 359 measurements gathered during maintenance treatment, WBC count did not exhibit a significant correlation with the DNA-TGN level (P = 0.093) or RBC TGN (P = 0.425). When analyzing separately the patients harboring NUDT15 variants, the DNA-TGN level also showed no association with WBC (P = 0.87). Among the 17 measurements with the highest DNA-TGN scores of 400 or more, cytopenia occurred in only one case.

4. Inter-individual variability

To evaluate the degree of change in DNA-TGN within each patient, the CV for DNA-TGN was calculated for each patient and its distribution was examined. CV was checked in 51 patients who provided at least three or more DNA-TGN samples during the maintenance period. Among the three patients with TPMT variant genotypes, only one was tested for DNA-TGN multiple times; therefore, patients with TPMT variant genotypes were not included in the CV analysis. The CV for DNA-TGN ranged from 13.9% to 76.1% in patients harboring wild-type NUDT15, with a median value of 32.7%. In patients carrying NUDT15 variants, the CV ranged from 29.5% to 104% and the median value was 51.4%, which were significantly higher than those in patients harboring wild-type NUDT15 (P = 0.001). The CV for RBC TGN ranged from 29.5% to 143.6% (median 53.5%) in patients harboring NUDT15 variants, and 13.7% to 76.1% in patients harboring wild-type NUDT15 (median 36.2%) (P = 0.009). As this large variation in DNA-TGN and RBC TGN may have been due to frequent changes in the 6MP dose in the NUDT15 variant group, evaluation of the CV in DNA-TGN/6MP intensity was also performed to eliminate the effect of dose variation. The CV for the DNA-TGN/6MP intensity ratios ranged from 23.8% to 90.5% (median 45.4%) in patients harboring the NUDT15 variants, which was significantly higher (P = 0.019) than that in patients harboring wild-type NUDT15 (5.6% to 71.8%, median 32.3%).

5. Changes in 6MP dose during the maintenance treatment

Changes in the 6MP dose were analyzed in patients who underwent maintenance therapy for more than three months. In total, 34 patients with both wild-type NUDT15 and TPMT, 11 patients harboring NUDT15 variants, and two patients harboring TPMT variants had undergone maintenance therapy for 3 months or longer. The median starting doses of 6MP were 21.43 mg/m², 9.83 mg/m² and 0.89 mg/m², respectively (P<0.001).

The last 6MP dose was compared between the groups based on the assumption that the last 6MP dose was the highest tolerable dose. There were significant differences in the last doses of 6MP between the 3 groups (median dose of 28.8 vs. 13.5 vs. 17.7 mg/m² in the wild type, NUDT15 variant, and TPMT variant groups, respectively; P = 0.037) (Fig 3A). Five patients took a higher dose of 6MP than the intended dose at the end of the treatment due to a high WBC count.

The change in 6MP intensity during maintenance therapy was plotted based on NUDT15 status (Fig 3B). In addition, the change in DNA-TGN/6MP intensity during the maintenance therapy was plotted against NUDT15 status (Fig 3C), showing high variability in patients harboring the NUDT15 variants. However, the linear mixed model analysis did not reveal any significant differences in the pattern of DNA-TGN concentration with respect to time, between patients harboring wild-type NUDT15 and variants (P = 0.238).

Discussion

As the last product of 6MP metabolism, DNA-TGN could be used as a marker for evaluating the efficacy of 6MP, especially in patients harboring pharmacogenetic variants of genes coding for proteins involved in the 6MP metabolism pathway. In patients harboring NUDT15 variants, DNA-TGN accumulates, leading to treatment-related toxicity. Following the publication of the first report linking NUDT15 polymorphisms to thiopurine-induced leukopenia in a Korean study on patients with inflammatory bowel disease [7], a high incidence of NUDT15 polymorphism has been reported in East Asian populations. In addition, treatment-related toxicity was reported to be high in ALL patients harboring NUDT15 polymorphisms [10, 20, 21].

A preclinical study using NUDT15 knockout mice showed that a reduction in the dose of 6MP resulted in decreased treatment-related toxicity without attenuating treatment outcomes. In the study, DNA-TGN levels in NUDT15 knockout mice (which corresponds to NUDT15 homozygous variant) were similar to those in NUDT15 wild-type mice treated with a normal dose of 6MP [14]. However, tolerable doses of 6MP have been shown to vary considerably in patients with heterozygous variant for NUDT15 as compared to those with homozygous variant for NUDT15. The Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines suggest initiating 6MP treatment at a reduced dose in patients with a NUDT15 variant genotype, and then adjusting the 6MP dose based on the observed degree of myelosuppression [22]. However, while this dosing method can prevent toxicity, it is difficult to predict the treatment effects.

In this study, DNA-TGN samples were serially collected from patients with ALL receiving 6MP treatment in a prospective manner and were then subjected to a variety of in-depth analyses.

First, the correlation between DNA-TGN and 6MP intensity was examined based on NUDT15 status. Patients harboring NUDT15 variants exhibited higher DNA-TGN/6MP intensity ratios, consistent with the findings of a previous report [13]. Three previously published papers have demonstrated that higher DNA-TGN concentrations coincided with a higher incidence of cytopenia in patients harboring NUDT15 variants when the patients received the standard dose of 6MP [13–15]. In contrast, in this study, patients harboring the NUDT15 variants began their treatment of 6MP at a reduced dose, and it was estimated that an equivalent median DNA-TGN concentration could be achieved using 73.8% of the 6MP dose used in patients harboring wild-type NUDT15. This result suggests that in patients harboring NUDT15 variants, thiopurine dose-reduction strategies reduce toxicity while not reducing the therapeutic effect.

Second, the distribution and changing patterns in DNA-TGN concentrations were evaluated based on the genotypes of NUDT15. The results demonstrate that DNA-TGN and RBC TGN tend to be more variable in patients with the NUDT15 variant genotype. As frequent 6MP dose changes in patients with the NUDT15 variant genotype could cause some changes in the DNA-TGN and RBC TGN values, variation in DNA-TGN/6MP intensity was also evaluated. DNA-TGN/6MP intensity ratios were also more variable in patients harboring NUDT15 variants than those in patients harboring wild-type NUDT15. A previous COG study reported a high risk of relapse in ALL patients with large RBC TGN variability [1]. In that study, variability in drug dosing and treatment interruptions (possibly to maintain ANC within a certain range) were thought to be associated with varying levels of TGN. Patients harboring NUDT15 variants who begin treatment of 6MP at a reduced dosage and are then gradually acclimatized to increasing doses over time, also show large variations in DNA-TGN and RBC TGN levels. This suggests that while adjusting 6MP dose during treatment, frequent measurement of metabolites such as DNA-TGN should be performed to improve overall therapeutic efficacy.

Thiopurine is known to have a delayed cytotoxic effect. At least two or more S phases of cell cycles are needed for 6TGN to incorporate into DNA, and this 6TGN-substituted DNA replicates and presents mismatches, eventually leading to cell death [4]. Considering the timing of the toxicity onset, it is helpful to test DNA-TGN after 2 weeks or more, when the dose of 6MP is changed, or when toxicity occurs, rather than immediately after starting 6MP.

Third, the DNA-TGN levels in relation to the clinical observations were evaluated. The results of this study did not show any correlation between DNA-TGN or RBC TGN and WBC counts. This may be because the treatment was interrupted in only a small number of recruited patients due to the development of cytopenia. In this study, the DNA-TGN test was performed at the beginning of the maintenance period in many cases. During this period, 6MP was administered at a low dose, and therefore, only a few cases presented cytopenia. Other factors that may cause hematologic toxicity—simultaneously administered medications and infection—should also be considered in the interpretation of results. Since methotrexate (MTX) is continuously administered in addition to 6MP during maintenance treatment, treatment-related toxicity can be caused by both drugs. However, in this study, MTX was not included in the analysis because it was considered that there would be little variation in toxicity due to MTX, as the dose of MTX was kept near constant in each patient. Previous studies have reported that there is a difference in 6MP tolerance according to sex [23], but this study did not show a difference in DNA-TGN values compared to 6MP intensity according to sex.

The appropriate target therapeutic range of DNA-TGN concentration is not yet known. To determine the target therapeutic range of DNA-TGN, long-term follow-up with variable 6MP doses would be needed to determine the relationship between DNA-TGN and clinical outcomes, including relapse and toxicity. In addition to the few studies already performed regarding DNA-TGN, this study can help establish the standard for therapeutic dose adjustment of 6MP based on DNA-TGN.

Study strength and limitations

This appears to be the first study to analyze prospective serial DNA-TGN samples in a large number of patients—including those, which carry NUDT15 variant genotypes—treated with reduced doses of 6MP. In addition, this is the first study to report detailed clinical data for these patients. However, this study has several limitations. The overall rate of cytopenia was low due to the strategy of starting 6MP treatment at a low dose and increasing the dose in patients that tolerated it well. Therefore, it was difficult to estimate the exact concentration of DNA-TGN at which toxicity occurs. In addition, as the follow-up period was short, it was impossible to evaluate the relationship of clinical outcomes including relapse, survival, and long-term complications, with DNA-TGN level. Since drug metabolism may differ in relapsed patients, it would be better to investigate relapsed patients separately if the number of patients is sufficient [24].

Conclusion

In conclusion, this study demonstrated that DNA-TGN positively correlates with the dose of 6MP, and that DNA-TGN concentration can be maintained at levels similar to those observed in patients carrying wild-type NUDT15—even at lower doses of 6MP—in patients harboring NUDT15 variants. In addition, DNA-TGN concentrations showed high variability in patients harboring NUDT15 variants, which suggests the need for close DNA-TGN monitoring to provide more finely tuned treatment. This study was able to demonstrate the efficacy of DNA-TGN concentration in monitoring the treatment effects of 6MP, particularly in patients carrying NUDT15 variant genotypes who need a reduced 6MP dose. Further prospective studies are needed to determine whether treatment outcomes can be improved by employing DNA-TGN values to titrate 6MP doses in patients harboring wild-type and variant NUDT15.

Supporting information

S1 Dataset

(XLSX)

Click here for additional data file.^{(48.3KB, xlsx)}

S1 Appendix. Detailed description of study methods.

(DOCX)

Click here for additional data file.^{(28.7KB, docx)}

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This study was supported by a grant from the Korean Foundation for Cancer Research (KFCR-2017-D-1). The funder provided financial support for the study, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the authors are articulated in the ‘author contributions’ section. All authors have no potential conflicts of interest to declare.

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PLoS One. doi: 10.1371/journal.pone.0245667.r001

Decision Letter 0

A M Abd El-Aty

20 Oct 2020

PONE-D-20-27853

DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukaemia patients with NUDT15 variant genotypes

PLOS ONE

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**********

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5. Review Comments to the Author

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Reviewer #1: The article investigated the amount of thioguanine incorporated into DNA (DNA-TGN) of mononuclear cells in patients receiving maintenance chemotherapy for acute lymphoblastic leukemia, and emphasized the importance of DNA-TGN monitoring in assessing the toxicity and efficacy of 6-mercaptopurine among those harboring NUDT15 variants.

The large parts of results are already reported in the previous reports, but they found high variability in DNA-TGN in patients with NUDT15 variant genotypes, which possibly lead to negative impacts on the outcome of leukemia.

The author concluded that DNA-TGN monitoring may be superior to the combination of the genomic diagnosis of NUDT15 genomic variants and 6MP dosing based on the results of genomic analysis for NUDT15 and TPMT.

The conclusion is not completely proved with the results of this study.

For example, is the amount of DNA-TGN at one point sometime after 6MP-administration enough to assess the 6MP efficacy? How about is time x DNA-TGN level during maintenance therapy?

In the study, the authors identified 4 different heterozygotes and 1 compound heterozygote, but no homozygote, as well as the wild type genotype occupying the majority of the study subjects.

The readers may want to know the relationship between the variant genotypes and the results of 6MP-metabolites.

Do authors think that the results of DNA-TGN can be reflected to 6MP dosing at clinic immediately?

It takes at least two weeks after initiating 6MP administration. If so, it seems not practical.

Some minor comments are indicated below.

1. Patients with relapsed ALL are included in the study subjects.

Some relapsed patients may have received maintenance therapy with 6MP previously.

The episode could affect 6MP metabolism in somatic cells as well as leukemia cells.

2. Please clearly describe the definition of 6MP intensity. Is that the ration of each tolerable dose for 50mg/m2/day or a cumulative dose administrated before DNA-TGN measurement for a dosage calculated by 50mg/m2/day x administration days?

3. What is reasons for sex difference among the subjects?

How is the age distribution or the ratio of T-cell type malignancy?

Can authors have some discussion about sex difference in 6MP-tolerance or sensitivity?

4. One of purposes of this study was described as “to evaluate whether DNA-TGN can reflect the clinical changes during ALL treatment”. Median follow-up among the subjects was only 18.1 months. It is too short to evaluate clinical impact of the markers related to 6MP metabolism. If the study can include some surrogate markers such as change of MRD, this study could have more suggestive for clinical practice.

5. Is there any relationship between the average or minimum WBC count and DNA-TGN in patients with NUDT15 variants?

6. In Figure 1, a few patients with NUDT15 genetic variants showed more than 1.0 of 6MP intensity. What does it mean?

Reviewer #2: congratulations on this interesting work titled " DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukaemia patients with NUDT15 variant genotypes"

however, some issues in this manuscript need to be addressed before it is made available in the public domain.

Major

1) Difficult to follow the language

1) line 74: Inherited genetic variations in the anti-leukemic drug metabolism have been recognized as the risk factors for ALL relapse—may be authors meant toxicity as we know that we have sufficient literature on the evidence related to the incidence of

Citations for higher frequencies in Asians whether authors meant all Asians or specific populations within Asians- Japanese, Chinese, Indians, any other reports? Or public database where these frequencies are found

The study s main objective is to investigate the toxicity and introduction is focused on relapse?

Main research question seems association of DNA TGN levels with that of NUDT15 variants and consequent toxicities—one main problem with the methodology is that all the pati8ents were started with low dose which is however is an advantage for the investigation as they can compare the DNA TGN levels at low doses in patients with NUDT15 variants and without the variants.

More details on the treatment can be provided in supplementary material.

Instead of adjusting the tGN-DNA levels with 6MP intensity which is a ratio of 6MP dose given to that of standard dose. Authors should directly adjust the levels with the dose administered (normalization). Ideally higher TGN-DNA adjusted to the dose would be

LC-MS method validation is not described. Whether external validation QC samples or interday and intraday CV, as the study period is 2 years. – sometime the observed variability could be incorporated by the analytical method used, differences in the storage periods etc. Which sample was used how it was collected? not clear for the reader the main analyte measured to be associated with genotypes.

The ratio of DNA TGN/ TGN or DNA-TGN adjusted t that of 6MP dose ca be compared between the genotype groups using parametric or non-parametric tests depending upon the distribution of the data

Relapse and death are phenotypes that are too early to be evaluated during the study period.

Whether the time point of sample collection was associated with that of the levels ? as I see that authors must compare within each phase rather comparing distribution across all phases. For e.g. comparison within consolidation phase ? seems there is quite difference in the time points used for sample collection, why not standardizing the time point for sample collection.

Whether authors have used ROC analysis to define cutoffs for the DNA TGN levels associated with that of toxicity?

For inter individual variability, since authors have multiple readings for DNA TGN why not using repeated measured ANOVA (if data is normal distributed) or non-parametric tests (nonparametric marginal model) to compare the levels between genotype groups?

It was not clear if the authors investigated only specific varinats in NUDt15 or sequenced the whole?, if only looked at specific variants then variability may be explained by the presence or absence of other variants ?

Conclusion is not supported by the findings in the study. In fact authors showed the relation of DNA TGN levels and NUDt15 that correlated with that of 6MP dosing , because similar TGN DNA levels obtained using the low doses in variant carriers of NUDT15. IN this case, as authors warrant may be close monitoring of the individuals based on NUDT15 genotype ? for e.g. higher variability in levels seen in the variant carriers, so may be NUDt15 variant carriers need close monitoring of the levels and dose adjustments. However, variants were associated with that of 6MP dosing.

minor and when analysis is modified may be need ot modify the following .

Figure 1 is not easily readable. I would simply take the dose adjusted TGN DNA levels and compare between the groups based on genotypes. And then see the proportion of cytopenia based on ROC curve for these adjusted DNA TGN levels.

Fig 3A—the representation can be mentioned as the dose ratio i.e. last dose to the initial dose, and then compare the dose adjustment between the groups based on genotypes, that can give clear idea whether there was dose reduction or dose increment occurred within each group. —on parametric comparisons can be made as it is likely that the distribution would be non-normal

Fig 3B 6MP intensity can be shown as mean with SD bars on each cycle of maintenance-

Fig 3C also same as Fig 3B , Fig 3C is not readable and representation must be changed

Reviewer #3: Statistical Comments:

The statistical analysis part was collectively written, results are shown in Table(1),

with P-values where you have Three groups, all t-test, U-test are only for two groups

please clear that

In addition where is the results for Mixed Model

Please Pinpoint the statistical methods used for each P-value shown, so the

results will be more informative.

Reviewer #4: The authors reported a prospective study of serial collection of DNA-TGN in 72 patients including their NUDT15 genotypes and found that patients with NUDT15 variant showed higher DNA-TGN variability. The results are informative, however, there are few comments listed below to improve clarity.

Major comments

1. Table 1

There are eight patients genotyped as NUDT15 *1/*2 in this study, however, ref 14 had mentioned that the genotyping methods you used could not distinguish NUDT15 *1/*2 from *3/*6. Did the NUDT15 diplotypes for those patients had been confirmed by other methods?

2. Table 1

The statistical significance test (Chi-square test) used in table 1 was not suitable due to more than 20% of expected counts less than 5.

3. page 12, line 232-239

Please revise figure 2 by orders of these results you mentioned in manuscript and label figures (2A, 2B, 2C) after each results.

4. Line 257

Please correct the range of DNA-TGN.

5. line 259-260

Please correct the patient number and the range of DNA-TGN.

6. Table 2

Please add the unit used for measurements including DNA-TGN, RBC-TGN, MMPN, WBC and ANC. Also add measurement method for MMPN in “sample collection and analysis” section since MMPN had been mentioned here.

7. Discussion line 403-407

Please make more discussion and cite references to support your proposal since that the outcomes were not correlate with DNA-TGN level in this study.

8. Clinically, the NUDT15 genotyping will be done before the administration of 6MP. DNA-TGA will be available after the patients take the 6MP. The clinicans might adjust the dose of 6MP by white cell counts and ANC. Might you comment about this?

Minor comments

Pleases add p-value in figure 2 and figure 3A

**********

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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PLoS One. 2021 Jan 22;16(1):e0245667. doi: 10.1371/journal.pone.0245667.r002

Author response to Decision Letter 0

12 Nov 2020

< Response to editors and reviewers >

First of all, thank you for reviewing this paper in detail and giving valuable advices. We have made our best efforts to revise the manuscript, and we hope that it has been answered properly. Please let us know if there is lacking points. We will make additional corrections. We hope that the revised manuscript could be suitable for publication in PLOS ONE.

(Please look at the separate file named "DNATGN_response to reviewers_last", as the text written on the web are easily scattered and figures/tables are not visible. )

=====================================================================================================

< Editors’ comments >

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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- We have checked that this paper meets the style standards suggested by PLOS ONE.

Upon resubmission, please provide the following:

•The name of the colleague or the details of the professional service that edited your manuscript

�- We have got the professional editing service by Editage.

•A copy of your manuscript showing your changes by either highlighting them or using track changes (uploaded as a *supporting information* file)

�- We made a “DNATGN_revised manuscript with track changes_last” file.

•A clean copy of the edited manuscript (uploaded as the new *manuscript* file)

- We also made a “DNATGN_revised manuscript” file.

3. You indicated that you had ethical approval and informed consent for your study.

- Thank you for your comment. We additionally described the information in the methods section.

- Consent was obtained from parents for minors and from patients for adults. (line 152-153)

4. Please provide a sample size and power calculation in the Methods, or discuss the reasons for not performing one before study initiation.

- Thank you for your comment. We discussed the reason for not performing sample size calculation in the methods section.

- The number of participants in the study was not set in advance because it was an exploratory study of DNA-TGN in patients treated according to the existing treatment schedule. (line 147-149)

5. In your Methods section, please provide additional information about the participant recruitment method and the demographic details of your participants.

Please ensure you have provided sufficient details to replicate the analyses such as:

a) a description of any inclusion/exclusion criteria that were applied to participant recruitment,

� - We appreciate for your comments. We described the content as follows;

� - Patients who were not expected to receive 6MP treatment and those who did not wish to participate in the study were excluded from the study. (line 145-147)

b) the methods used to collect patient samples.

� - We appreciate for your comments. We described the content as follows;

- Prospective metabolite samples (DNA-TGN, erythrocyte 6-thioguanine nucleotides (RBC TGN), methylmercaptopurine nucleotides (MMPN)) were acquired simultaneously after administration of 6MP for at least 14 days after initiation of constant-dosing 6-MP treatment—at 2 weeks for the protocol taking 6MP for 2 weeks, at 2 and 4 weeks for the protocol taking 6MP for 4 weeks, at 2 and 4 and 8 weeks for the protocol taking 6MP for 8 weeks, and at 2 and 4 weeks of 1st cycle of maintenance treatment. The blood sampling for DNA-TGN was done along with routine samplings to monitor complete blood cell count and chemistry tests during chemotherapy. (line 168-175)

6. Please ensure your methods are described in sufficient detail for others to replicate the study. Specifically, please provide a brief summary of the methods involved in LC-MS and genotyping.

�- We have further described the contents you mentioned as follows.

�- DNA- and RBC- TGN concentrations and MMPN were measured using liquid chromatography-tandem mass spectrometry (LC-MS). DNA-TGN test was done with isotope-labeled TG-d3 and guanine-d3 as internal standards as previously described. Chromatographic etheno-TG peaks were normalized using etheno-G by calculating TG responses as etheno-TG area/etheno-G area (DNA-TG=[etheno-TG response/etheno-G response]/[etheno-TG-d3 response/etheno-G-d3 response]).[12,13] (line 176-181)

Genotyping of NUDT15 and TPMT was performed before the 6MP maintenance therapy by direct sequencing. Direct Sanger sequencing of exons 1 and 3 of NUDT15, and direct sequencing of exon 2 to 9 of TPMT were performed after PCR. [14,15]. (line 185-187)

�- Thank you for your comment. I validated the ORCID ID following the comment.

8. Thank you for stating the following in the Financial Disclosure section:

We note that one or more of the authors are employed by a commercial company: Green Cross Laboratories

� - The author who works at the Green Cross Laboratory previously worked at Samsung Medical Center with other authors. The Korean Foundation for Cancer Research, which provided funding for this study, is a completely separate institution from the workplace where the author above works. The funders did not participate in the design or implementation of the study, and only provided the research fund.

�- We also updated the author roles of the online submission form.

Please also include the following statement within your amended Funding Statement.

� -We additionally described following statement at the funding statement part.

� -The funder provided financial support for the study, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the authors are articulated in the ‘author contributions’ section. (line 502-505)

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

� - The commercial affiliation of Choi R, one of the co-authors, did not play a role in this study.

�- In the current state of revision, it is confirmed that all authors of this study have no conflict of interest.

We additionally described the following statement in the “Competing Interests Statement” part.

�- All authors have no potential conﬂicts of interest to declare. There is an author who belong to a commercial affiliation, but the commercial affiliation does not alter our adherence to PLOS ONE policies on sharing data and materials. (line 498-500)

c. Please include both an updated Funding Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf.

� - We included both an updated Funding Statement and Competing Interests Statement in the cover letter.

Editor's comments:

� - Thank you for your kind comments. We stated the novel point of this study additionally in the introduction part.

� - To date, two papers have been published on DNA-TGN measured in patients with NUDT15 variant genotype. Previous studies have reported higher DNA-TGN level and toxicity in patients with the NUDT15 mutant genotype when 6MP is given in regular dose. In contrast, our study focused on DNA-TGN in patients with NUDT15 or TPMT variant genotypes, who were treated with reduced doses of 6MP. In addition, in this study, since repeated blood tests were performed in patients continuously receiving 6MP, changes in DNA-TGN over time could be observed. (line 127-133)

� - Also, we modified the last paragraph of introduction to make the meaning clearer.

�- In this study, we investigated the pattern of DNA-TGN in patients harbouring the NUDT15 variants when 6MP was administered at reduced doses (dynamically adjusted based on degree of cytopenia). This study was designed to evaluate whether the DNA-TGN concentration differ based on genotype of NUDT15, and whether DNA-TGN can reflect the treatment-related toxicity during ALL treatment. In addition, we sought to determine the pattern of change in DNA-TGN during maintenance treatment including 6MP. (line 134-139)

======================================================================

< Reviewers’ comments >

For example, is the amount of DNA-TGN at one point sometime after 6MP-administration enough to assess the 6MP efficacy? How about is time x DNA-TGN level during maintenance therapy?

�- Thank you for the kind advice. It would be ideal to access the time x DNA-TGN level as the reviewer suggested. But it was difficult to analyze the time x DNA-TGN level because there was no accurate record of the actual time taken and the time the DNA TGN was collected. However, it was assumed that there would be a certain level of regularity, as most patients took the drug on an empty stomach before bedtime (9-10p) and collected the blood sample at morning before clinic. In the future, while conducting prospective research, we will try to study on the advice given.

In the study, the authors identified 4 different heterozygotes and 1 compound heterozygote, but no homozygote, as well as the wild type genotype occupying the majority of the study subjects. The readers may want to know the relationship between the variant genotypes and the results of 6MP-metabolites.

�- Thank you for the important point. We additionally analyzed 6MP metabolite results according to variant genotypes, and added the result as Figure 1B.

� Figure 1B. DNA-TGN/6MP intensity ratio and rate of cytopenia according to pharmacogenetic subgroups. (Line presents median with interquartile range.)

�

Do authors think that the results of DNA-TGN can be reflected to 6MP dosing at clinic immediately? It takes at least two weeks after initiating 6MP administration. If so, it seems not practical.

�- As the maintenance treatment lasts for more than 18 to 30 months, 6MP dosing based on genotypes and adjusting after administration at 2 or more weeks can help concise dosing afterwards for the long treatment period.

�- In this study, samples were collected prospectively but were analyzed after collection of sufficient samples to undergo LC-MS test for DNA-TGN. Thus, we could not change the dose according to DNA-TGN level.

�- We added the reference for the method of this study, which was published during the review period of this article. (Reference number 13).

Some minor comments are indicated below.

1. Patients with relapsed ALL are included in the study subjects. Some relapsed patients may have received maintenance therapy with 6MP previously. The episode could affect 6MP metabolism in somatic cells as well as leukemia cells.

� - Thank you for the attentive advice. In our study, there were 3 patients enrolled after relapse. The three patients had different genotypes: one patient with NUDT15 variant, another patient with TPMT variant, and a patient with wild type of both genes. Because the proportion of relapsed patients was small among the total, it was difficult to observe different metabolism in relapsed patients through our study. However, it was mentioned in the discussion that drug metabolism may change in relapsed patients. Also, we added a reference about this content. (Blood 2020;135:41-55.)

� Since drug metabolism may differ in relapsed patients, it would be better to analyze relapsed patients separately if the number of patients is sufficient. [22] (line 451-452)

�- Thank you for your comment. 6MP intensity means the explanation of the former. The description has been revised as follows.

�- 6MP intensity was defined as the ratio between the prescribed daily 6MP dose (mg/m2/day) and the standard daily dose (50 mg/m2/day). (line 164-165)

3. What is reasons for sex difference among the subjects? How is the age distribution or the ratio of T-cell type malignancy? Can authors have some discussion about sex difference in 6MP-tolerance or sensitivity?

�- We don't think there may have been any specific cause for the sex difference. The incidence of ALL is usually a bit higher in boys, and it is likely that the proportion of males who visited the hospital at the time of the study was particularly high.

�- As advisement, the detailed diagnosis name including the T-/B-cell type of the patients and the age distribution at the time of enrollment are additionally listed in Table 1. (3rd and 4th row of Table 1) (line 235)

� Additional analysis was performed to determine whether there was a difference according to sex, disease, and previous recurrence.

�- Kruskal-Wallis test was done to compare the difference of DNA-TGN according to sex, diagnosis and relapse state. (line 204-205)

�- When comparing the distribution of DNA-TGN, male (vs female, P=0.001) and Pre-B ALL (vs Pre-T ALL, lymphoma, P <0.001) showed higher level of DNA-TGN. However, when comparing DNA-TGN/6MP intensity, there was no significant difference according to sex (P=0.18), disease (P=0.061), and previous relapse (P=0.675). (line 254-257)

�- We also added comments about sex difference in 6MP-tolerance.

�- Previous studies have reported that there is a difference in 6MP tolerance according to sex [21], but this study did not show a difference in DNA-TGN values compared to 6MP intensity according to sex. (line 437-439)

� - This is a very accurate point. As you mentioned, the follow-up period of our study was short, making it difficult to observe relapse or death. Since most of the patients had already acquired MRD negative at the time of DNA-TGN measurement, it was difficult to use MRD as well for clinical impact of markers.

� - Clinical practice was mainly referring to treatment-related toxicity and 6MP dose adjustment, but the expression seemed to be unclear. To clarify the meaning, we changed the 1st paragraph of introduction as follows.

� - Mercaptopurine is a long-term administered, important drug for treatment of childhood acute lymphoblastic leukemia (ALL). However, some patients experience toxicity during mercaptopurine treatment, such as severe infections related to bone marrow suppression, need of dose reduction, or frequent treatment interruptions. Mercaptopurine is known to have narrow therapeutic indices and it is also known that frequent dose change or discontinuation of mercaptopurine is associated with increased relapse of ALL. [1] Maintaining proper concentration of thiopurine metabolites is important for preventing the occurrence of relapse or toxic events after ALL treatment. (line 95-102)

5. Is there any relationship between the average or minimum WBC count and DNA-TGN in patients with NUDT15 variants?

� - Thank you for your kind comment. The correlation between DNA-TGN and WBC was analyzed in patients with NUDT15 variants, and the result is further described.

�- When separately analyzed the patients harbouring NUDT15 variant, the DNA-TGN level also showed no association with WBC. (P=0.87) (Line 314-315)

6. In Figure 1, a few patients with NUDT15 genetic variants showed more than 1.0 of 6MP intensity. What does it mean?

- In some cases, starting with a small 6MP dose and being tolerable, the 6MP dose was administered with higher dose than the target at the later part of treatment. In the Methods section, we described the treatment in more detail.

�- There were 5 patients who were tolerable to the drug and took higher than the intended dose at the end of the treatment. (Line 349-35)

Reviewer #2: Congratulations on this interesting work titled " DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukaemia patients with NUDT15 variant genotypes" However, some issues in this manuscript need to be addressed before it is made available in the public domain.

Major

1. Difficult to follow the language

The study s main objective is to investigate the toxicity and introduction is focused on relapse?

�- Thank you for the clear comment. To clarify the meaning, we changed the 1st paragraph of introduction as follows.

�- Mercaptopurine is a long-term administered, important drug for treatment of childhood acute lymphoblastic leukemia (ALL). However, some patients experience toxicity during mercaptopurine treatment, such as severe infections related to bone marrow suppression, need of dose reduction, or frequent treatment interruptions. Mercaptopurine is known to have narrow therapeutic indices and it is also known that frequent dose change or discontinuation of mercaptopurine is associated with increased relapse of ALL. [1] Maintaining proper concentration of thiopurine metabolites is important for preventing the occurrence of relapse or toxic events after ALL treatment. (line 95-102)

� - Thank you for your comment. We added citations about NUDT15 variants in Asian population.

� - Particularly, NUDT15 variants have been shown to be more prevalent in Asian populations than in other ethnic groups. [8-10] (line 121-122)

Main research question seems association of DNA TGN levels with that of NUDT15 variants and consequent toxicities—one main problem with the methodology is that all the patients were started with low dose which is however is an advantage for the investigation as they can compare the DNA TGN levels at low doses in patients with NUDT15 variants and without the variants.

� - We agree with what you pointed out. Starting 6MP from a low dose, toxicity was rarely observed. However it was confirmed that DNA-TGN appeared differently depending on the NUDT15 status. We added some discussion about this point.

� - In this study, DNA-TGN test was performed at the beginning of the maintenance period in many cases. At this period, 6MP was administered at a low dose, and therefore, the frequency of cytopenia was very small. (line 433-435)

More details on the treatment can be provided in supplementary material.

Instead of adjusting the TGN-DNA levels with 6MP intensity which is a ratio of 6MP dose given to that of standard dose. Authors should directly adjust the levels with the dose administered (normalization). Ideally higher TGN-DNA adjusted to the dose would be

�- Thank you for your thoughtful advice. As you mentioned, for standardization, it is better to apply the actual 6MP dose per body surface area, not 6MP intensity.

�- However, it is believed that DNA-TGN itself is a direct indicator of the actual therapeutic effect. To date, several papers published on the aspects of DNA-TGN in pediatric ALL have shown the value of DNA-TGN itself. In this study, we looked at the ratio because we thought that this would be a good index to see differences between groups, rather than the high meaning of the ratio itself.

�- In this study, samples were collected prospectively but were analyzed after collection of sufficient samples to undergo LC-MS test for DNA-TGN.

�- We added the reference for the method of this study, which was published during the review period of this article. (Reference number 13)

�- Also, we described more about LC-MS method.

�- DNA-TGN test was done with isotope-labeled TG-d3 and guanine-d3 as internal standards as previously described. Chromatographic etheno-TG peaks were normalized using etheno-G by calculating TG responses as etheno-TG area/etheno-G area (DNA-TG=[etheno-TG response/etheno-G response]/[etheno-TG-d3 response/etheno-G-d3 response]).[12,13] (line 178-181)

The ratio of DNA TGN/ TGN or DNA-TGN adjusted t that of 6MP dose ca be compared between the genotype groups using parametric or non-parametric tests depending upon the distribution of the data.

Whether authors have used ROC analysis to define cutoffs for the DNA TGN levels associated with that of toxicity?

� - The ratio of DNA-TGN/6MP intensity was compared between genotype groups using Mann-Whitney test. (Not normal distribution) P values are presented in a new figure, Figure 1B.

� - The rate of cytopenia was low, and rate of cytopenia did not show correlation with the genotypes. So, we could not draw the ROC curve. Instead, we tried to show the rate of cytopenia according to groups. (Figure 1B)

� - Also, as there was also a request from other reviewer to show the distribution of DNA-TGN/6MP intensity ratio according to each subgroup, the groups were divided in this new figure.

�- Fig 1. (B) DNA-TGN/6MP intensity ratio and rate of cytopenia according to pharmacogenetic subgroups. (Line presents median with interquartile range.)

Relapse and death are phenotypes that are too early to be evaluated during the study period.

� - This is a very accurate point. As you mentioned, the follow-up period of our study was short, making it difficult to observe relapse or death. Clinical respects were mainly referring to treatment-related toxicity and 6MP dose adjustment, but the expression seemed to be unclear in the manuscript.

� - At the beginning of the article, the part that mentioned about the relapse of ALL was deleted, and drug-related toxicity and difficulty in drug dose decision were emphasized.

Whether the time point of sample collection was associated with that of the levels? as I see that authors must compare within each phase rather comparing distribution across all phases. For e.g. comparison within consolidation phase? seems there is quite difference in the time points used for sample collection, why not standardizing the time point for sample collection.

� - Thank you for your careful advice. As you mentioned, we thought that there is a concern that the value of DNA-TGN will differ depending on the treatment schedule. In addition, the chemotherapeutic agents and doses were different for each patient in the treatment stage before maintenance treatment (from consolidation to delayed intensification), as each patient had different risk group and different type of leukemia or lymphoma. As a result, it was complicated to analyze levels separately for each protocol. Therefore, when looking at the DNA-TGN distribution by 6MP dose, analysis was performed only on the samples collected during the maintenance period.

�- It is further described so that the content of this analysis can be clarified.

�- For the association of DNA-TGN by 6MP dose and hematologic toxicity, analysis was performed only on the samples collected during maintenance treatment to rule out effects of other drugs. (line 199-201)

�- In fact, data were collected with the intention of performing ANOVA, but the timing of blood collection was not completely consistent for each patient. (For example, 25% of patients collected blood at 2,4,8 weeks, 20% at 2,8 weeks, 10% at 2,4,16 weeks, etc.) Therefore, we conducted a linear mixed model analysis, which we thought was the best method of analysis. This was described in the manuscript as next paragraph. Thank you for the comment.

�- Linear mixed model analysis was performed to compare the pattern of change between NUDT15 variant and wild type groups. (line 2018-210)

�- However, the linear mixed model analysis did not reveal any significant differences in the pattern of DNA-TGN concentration with respect to time, between patients harbouring NUDT15 wild type and variants (P=0.238). (line 352-354)

It was not clear if the authors investigated only specific variants in NUDT15 or sequenced the whole? if only looked at specific variants then variability may be explained by the presence or absence of other variants ?

� - Thank you for your question. Previously, only references were provided for the test method and the contents were not written. We modified the manuscript to further describe the test method.

�- Direct Sanger sequencing of exons 1 and 3 of NUDT15, and direct sequencing of exon 2 to 9 of TPMT were performed after PCR. [14,15]. (line 186-187)

Conclusion is not supported by the findings in the study. In fact authors showed the relation of DNA TGN levels and NUDt15 that correlated with that of 6MP dosing, because similar TGN DNA levels obtained using the low doses in variant carriers of NUDT15. IN this case, as authors warrant may be close monitoring of the individuals based on NUDT15 genotype? for e.g. higher variability in levels seen in the variant carriers, so may be NUDT15 variant carriers need close monitoring of the levels and dose adjustments. However, variants were associated with that of 6MP dosing.

� - Thank you for your careful advice. As your comment, the fact that similar DNA-TGN levels were obtained with low dose of 6MP in patients with NUDT15 variant genotype is the most important point of the study. But as there were two previous articles which covered similar contents (although the 2 studies were done in small number of patients or animals), we wanted to show the new finding through this study. In our study, we thought that it was new to look at the changing pattern of DNA-TGN during the course of 6MP treatment.

�- It can be predicted that DNA-TGN variability may be higher in patients with NUDT15 variant because of the 6MP dose change.

�- However, the ratio of DNA-TGN divided by 6MP intensity was also higher in patients with NUDT15 variant, indicating that the variation in DNA-TGN was greater than from the dose changes.

- We wrote this explanation at line 352-330.

- This large variation in DNA-TGN and RBC TGN may be due to frequent changes in dose in the NUDT15 variant group. Therefore, we evaluated the variation in DNA-TGN/6MP intensity to eliminate the effect of dose variation. The CV for the DNA-TGN/6MP intensity ratios ranged from 23.8% to 90.5% (median 45.4%) in patients harbouring the NUDT15 variants, which was significantly higher (P=0.019) than that in patients harbouring wildtype NUDT15 (5.6% to 71.8%; median 32.3%). (line 330-335)

minor and when analysis is modified may be needed to modify the following.

�- We appreciate for your advice. We modified the figure as your comment, presenting the dose ratio of last dose to the initial dose of 6MP. As there were only 2 patients who were treated last dose of 6MP in TPMT variant group, we presented only NUDT15 WT and NUDT15 variant group. Dots present each patient, box present median with interquartile range, and bars mean minimum to maximum value.

�

Fig 3B 6MP intensity can be shown as mean with SD bars on each cycle of maintenance-

Fig 3C also same as Fig 3B, Fig 3C is not readable and representation must be changed

�-Thank you for your advice. We changed the graph as mean with SD bars for each group, and combined two graphs (3B, 3C) in one graph (3B).

- Fig 3. Changes in 6MP dosage according to NUDT15 status. (A) ratio of starting 6MP dose and last 6MP dose of maintenance treatment. (B) dose changes in patients harbouring wild type NUDT15 and NUDT15 variants. (C) changes in the DNA-TGN/6MP intensity ratios over the course of the maintenance treatment.

Reviewer #3: Statistical Comments:

The statistical analysis part was collectively written, results are shown in Table(1),

with P-values where you have Three groups, all t-test, U-test are only for two groups

please clear that

Please Pinpoint the statistical methods used for each P-value shown, so the results will be more informative.

- We appreciate for your advice. As there were 3 groups, Fisher’s exact test was performed to know differences in sex, diagnosis, and relapse status according to genotype group. We changed manuscript as follows.

- Descriptive analyses were conducted for age, sex, diagnosis, NUDT15/TPMT genotype, 6-mercaptopurine dose, DNA-TGN and RBC TGN. Fisher’s exact test was performed to see differences in sex, diagnosis, and relapse status according to genotypes. For the association of DNA-TGN by 6MP dose and hematologic toxicity, analysis was performed only on the samples collected during maintenance treatment to rule out effects of other drugs. Correlation analysis was performed to see the correlation of DNA-TGN by 6MP dose and WBC by DNA-TGN. Fisher’s exact test was used to determine the differences in the rate of toxicity in the NUDT15 variant and wild type groups. Kruskal-Wallis test was done to compare the difference of DNA-TGN according to sex, diagnosis and relapse state. Also, same test was used to compare the difference of DNA-TGN, 6MP intensity, DNA-TGN/6MP intensity according to genotypes. To evaluate the distribution of DNA-TGN, DNA-TGN/6MP intensity and RBC TGN values between and within patients, coefficients of variation (CV) were calculated. Linear mixed model analysis was performed to compare the pattern of change between NUDT15 variant and wild type groups. (methods, line 197-210)

- In addition, where is the results for Mixed Model

- The results are shown at line 344-346, as follows:

- However, the linear mixed model analysis did not reveal any significant differences in the pattern of DNA-TGN concentration with respect to time, between patients harbouring NUDT15 wild type and variants (P=0.238). (line 351-353)

Reviewer #4:

The authors reported a prospective study of serial collection of DNA-TGN in 72 patients including their NUDT15 genotypes and found that patients with NUDT15 variant showed higher DNA-TGN variability. The results are informative, however, there are few comments listed below to improve clarity.

Major comments

1. Table 1

�- Thank you for your comment. As you mentioned, NUDT15 genotype tests were performed in patients in this study in the same way as in Reference 14. Direct sequencing alone is not enough to distinguish *1/*2 and *3/*6, and additional tests such as parental tests were not carried out. So technically, it is difficult to distinguish between *1/*2 and *3/*6. We additionally described about this fact as follows.

� - Since NUDT15 *1/*2 and *3/*6 genotypes were not distinguished technically by our method, *1/*2 strictly represents *1/*2 or *3/*6 [14]. (line 187-189)

2. Table 1

The statistical significance test (Chi-square test) used in table 1 was not suitable due to more than 20% of expected counts less than 5.

�- We appreciate for the kind review. (Initially, the groups were divided as 2 groups; NUDT15 variant and wild type. We may have used Chi-square test at that time, and did not carefully modified when changing groups. Thank you for the comment.) Instead of Chi-square test, Fisher’s exact test was performed to know differences in sex, diagnosis, and relapse status according to genotype group. We changed manuscript and table as follows. (line 198-199)

- Fisher’s exact test was performed to see differences in sex, diagnosis, and relapse status according to genotypes.

3. page 12, line 232-239

Please revise figure 2 by orders of these results you mentioned in manuscript and label figures (2A, 2B, 2C) after each result.

�- We agree with what you pointed out. We reordered figure 2 and mentioned in manuscript as follows. (line 274-281)

Patients with NUDT15 variant genotypes were treated with significantly lower 6MP intensities when compared to patients with NUDT15 wild type genotypes (P<0.001). (Fig 2A) However, there was no significant difference in the DNA-TGN levels between patients with NUDT15 variant genotypes, TPMT variant genotypes, and both of NUDT15 and TPMT wild types (P=0.261). (Fig 2B) The ratio of DNA-TGN/6MP intensity was significantly higher in patients harbouring NUDT15 variants than that in patients harbouring wild type NUDT15 (P<0.001), but no difference was observed between patients harbouring TPMT variant and wild type (P=0.743). (Fig 2C) (line 284-285)

- Fig 2. Distribution of various parameters based on genotype. (A) 6MP intensity (B) DNA-TGN (C) DNA-TGN/6MP intensity (WT; wild type, v; variant)

4. Line 257

Please correct the range of DNA-TGN.

- We deeply appreciate for your detailed review. We corrected the range of DNA-TGN as follows.

� (line 296-297)

- The DNA-TGN concentrations during these leukopenia episodes ranged from 27.8 to 504.8 fmol TG/µg DNA.

5. line 259-260

Please correct the patient number and the range of DNA-TGN.

� - Thank you for your detailed review. We apologize for writing the contents incorrectly and corrected manuscript as follows.

� (line 297-301)

- Three of the episodes developed leukopenia before 12 weeks of treatment, and their 6MP intensity was 0.17, 0.12, and 0.08, respectively. However, in the five cases of leukopenia that occurred after 12 weeks of 6MP-based maintenance therapy, the 6MP intensity ranged from 0.22 to 0.98 (median 0.4).

6. Table 2

� - Thank you for your detailed review. We added the units which were missing as follows. (Table 2)

� (line 302)

DNA-TGN RBC TGN MMPN‡ WBC ANC

(fmol TG/µg DNA) (µmol/L) (µmol/L) (/µL) (/µL)

�- We also add measurement method for MMPN in “sample collection and analysis” section, as commented.

� (line 168-171)

DNA- and RBC- TGN concentrations and MMPN were measured using liquid chromatography-tandem mass spectrometry (LC-MS).

7. Discussion line 403-407

Please make more discussion and cite references to support your proposal since that the outcomes were not correlate with DNA-TGN level in this study.

- Thank you for the advice. It seems that the existing sentence that you pointed out did not accurately express the point of the study, so the sentence was modified as follows.

�(line 458-463)

- Through this study, it was confirmed that patients with NUDT15 variant genotype could achieve a similar level of DNA-TGN even if they administered lower dose of 6MP, but subsequent dose adjustments by DNA-TGN were impossible because the DNA-TGN test was not processed in real time. We propose a larger study that starts reduced 6MP dose in patients harbouring the NUDT15 or TPMT variants, and then titrate the dose based on the DNA-TGN levels.

- In addition, the reason why the clinical results were not consistent with DNA-TGN was mentioned more in the discussion

- (line 433-435) In this study, a DNA-TGN test was often performed at the beginning of the maintenance period. At this time, 6MP was administered at a low dose, and therefore, the frequency of cytopenia was very small.

8. Clinically, the NUDT15 genotyping will be done before the administration of 6MP. DNA-TGA will be available after the patients take the 6MP. The clinicians might adjust the dose of 6MP by white cell counts and ANC. Might you comment about this?

�- We appreciate for your advice. We added a comment about the process in the manuscript.

�- (line 181-185)

� In this study, blood samples were collected prospectively, but the DNA-TGN test was not performed in real time. Therefore, the dose of 6MP was adjusted according to white blood cell (WBC) count, aspartate aminotransferase (AST), alanine transaminase (ALT), but not DNA-TGN values.

Minor comments

Pleases add p-value in figure 2 and figure 3A

�- Thank you for your advice. The order of Figure 2 was changed, following the advice. And we added the P value also.

� (line 284-285)

Fig 2. Distribution of various parameters based on genotype. (A) 6MP intensity (B) DNA-TGN (C) DNA-TGN/6MP intensity (WT; wild type, v; variant)

Fig 3A was changed as presenting ratio of last 6MP dose vs initial dose according to groups, following the advice of another reviewer. And we added the P value also.

� Fig 3. Changes in 6MP dosage according to NUDT15 status. (A) ratio of starting 6MP dose and last 6MP dose of maintenance treatment

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PLoS One. doi: 10.1371/journal.pone.0245667.r003

Decision Letter 1

A M Abd El-Aty

10 Dec 2020

PONE-D-20-27853R1

DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukaemia patients with NUDT15 variant genotypes

PLOS ONE

Dear Dr. Koo,

==============================

ACADEMIC EDITOR: Still reviewers' are raising major concern over the revised form of the MS. Do go through the comments and amend the MS accordingly. Furthermore

1- Authors' qualifications (MD, PhD) are not needed, delete

2- Justify the text throughout the MS

3- Line 75: Avoid using "will" as well as throughout the text

4- Add 2 more keywords

5- Periods shouldn't be before Ref. No. It should be after Ref, No. For instance: of mercaptopurine is associated with increased relapse of ALL. [1] SHOULD be of mercaptopurine is associated with increased relapse of ALL [1]. Revise throughout the text

6- Don't use "We, our". Use impersonal phrasing throughout the text

7- Line 177: liquid chromatography-tandem mass spectrometry (LC-MS) is not correct. This should be LC-MS/MS. Do you use LC-MS or LC-MS/MS?

8- Line 177: Details regarding analysis, including method optimization and performance should be added as Suppl. materials

9- Study strength and limitations should be in a separate section headed as mentioned. It should be ahead of Conclusion. Start with strength followed by limitations

10- Conclusion should be in a separate section. What are the clinical relevance and future perspective? Add this to conclusion section

==============================

Please submit your revised manuscript by Jan 24 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

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A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

A. M. Abd El-Aty

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #1: (Major comments)

The authors concluded that the dose adjustment of 6MP doses based on WBC count was not effective to maintain DNA-TGN constantly.

Can they exclude the cytotoxic effect of co-administered MTX?

They showed that DNA-TGN correlates positively with 6MP dose in those with wild type NUDT-15. Therefore, they can say the additional effect of MTX on WBC count may be minimal. But the size of cohort with NUDT-15 variants is much smaller than those with wild type NUDT-15. Can they show that patients with genetic variants related with MTX toxicity were not included in the cohort with NUDT-15 variants?

In discussion section, the authors speculated a reason of non-correlation between DNA-TGN or RBC-TGN and WBC. In this part, effect of MTX should be discussed.

Let me allow me give the similar question to the review for the first submission. The measurement of DNA-TGN was carried out LC-MS with radio-isotpoic reagents. They suggested the need for close DNA-TGN monitoring to allow for the development of more finely tuned treatment plan. Is it practicable for monitoring of individual patients coming to clinic weekly or biweekly?

DNA-TGN reflects the damage of DNA during the relatively short-time after 6MP-administration while RBC-TGN means the accumulation of 6MP metabolites in cytoplasm representing the relatively long-term effect of 6MP. Can the author discussed the difference of the two parameters and difference in the impact of the parameters on WBC count during 6MP-treatment?

(Minor comments)

Conclusion of Abstract. The last sentence ; will be important for what? Clinical outcome or adverse effects?

Page 5. L111; Erythrocyte → erythrocyte

Page 8. L171; at 2 weeks; at 2nd week (?)

Reviewer #2: introduction modified statements were not referenced appropriately

statistical analysis suggestions were not appropriately implemented , normalization of DNA TGN levels to the dose of 6MP , authors claim several papers here in this reply on DNA TGN levels, and in the introduction they say there are only two reports available- conflicting statements. Normalization is required when we wanted to see effect of genetic variant as dose changes by itself could change the levels of DNA TGN. so it has to be normalized to be able tp compare between genotype groups.

they were also not sure about the timing of the sample collecting sued for analysis-- this was not disclosed clearly

now the manuscript has been improved a lot , however need more precisions to accurately represent the data

Reviewer #3: (No Response)

Reviewer #4: There were several questions from different reviewers and the editor. All questions were answered properly. I have no other comments.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

PLoS One. 2021 Jan 22;16(1):e0245667. doi: 10.1371/journal.pone.0245667.r004

Author response to Decision Letter 1

15 Dec 2020

Response to reviewers (2nd)

We sincerely appreciate the insights and constructive suggestions. We have made corrections based on the reviewer’s comment and suggestions. We sincerely hope that the manuscript is now suitable for publication in this journal and would be pleased to respond to any further queries regarding this submission. Thank you for your consideration.

==============================================================

Response to the academic editor

1- Authors' qualifications (MD, PhD) are not needed, delete

→Thank you for the comment. We have also removed qualifications from the title page.

2- Justify the text throughout the MS

→ Thank you for the comment. We justified the text throughout the manuscript.

3- Line 75: Avoid using "will" as well as throughout the text

→ Thank you for the advice which helps make the improved manuscript. We followed the advice.

4- Add 2 more keywords

→ Thank you for the comment. We added 2 more keywords.

→ Thank you for the advice which helps make the improved manuscript. We changed the format according to the advice.

6- Don't use "We, our". Use impersonal phrasing throughout the text

→Thank you for the comment. We followed the advice.

7- Line 177: liquid chromatography-tandem mass spectrometry (LC-MS) is not correct. This should be LC-MS/MS. Do you use LC-MS or LC-MS/MS?

→ Thank you for the detailed review. We changed the word according to the comment.

8- Line 177: Details regarding analysis, including method optimization and performance should be added as Suppl. Materials on

→ Thank you for your comment. The test method implemented by our center is described in detail in the paper dealing DNA-TGN test development, so the paper is cited. (reference number 17) If it is better to write the contents of the references as supplementary data, we will implement it.

9- Study strength and limitations should be in a separate section headed as mentioned. It should be ahead of Conclusion. Start with strength followed by limitations

→ Thank you for the advice. We changed the structure as recommended.

10- Conclusion should be in a separate section. What are the clinical relevance and future perspective? Add this to conclusion section

→ Thank you for the constructive advice. We changed the structure and added the content to conclusion section.

==============================================================

Response to the reviewers

Reviewer #1: (Major comments)

The authors concluded that the dose adjustment of 6MP doses based on WBC count was not effective to maintain DNA-TGN constantly. Can they exclude the cytotoxic effect of co-administered MTX? They showed that DNA-TGN correlates positively with 6MP dose in those with wild type NUDT-15. Therefore, they can say the additional effect of MTX on WBC count may be minimal. But the size of cohort with NUDT-15 variants is much smaller than those with wild type NUDT-15. Can they show that patients with genetic variants related with MTX toxicity were not included in the cohort with NUDT-15 variants?

In discussion section, the authors speculated a reason of non-correlation between DNA-TGN or RBC-TGN and WBC. In this part, effect of MTX should be discussed.

→ The authors appreciate the comments.

→ About genetic variants related with MTX toxicity, genotyping of methylenetetrahydrofolate reductase (MTHFR) was done. Fifty-five of the 71 patients (77%) enrolled in the study were tested for MTHFR, but 16 patients (23%) did not undergo MTHFR study. Due to the large missing data, it was difficult to analyze the relationship between the MTHFR genotype and the leukocyte count.

→ In addition, in this study, MTX was administered in full dose except in cases where toxicity was evident during maintenance treatment. As MTX was usually administered at a constant dose, it was thought that the degree of toxicity due to MTX would not be variable. Therefore, the degree of toxicity according to the MTX dose was not included in this study.

→ This background is further described in the discussion section.

→ (line 440-¬444) Since methotrexate (MTX) is continuously taken in addition to 6MP during maintenance treatment, treatment-related toxicity can be caused by both drugs. However, in this study, MTX was not included in the analysis because it was considered that there would be little variation in toxicity due to MTX since the dose of MTX was kept near constant in each patient.

→ Thank you for the important advice.

→ The DNA-TGN test was performed by the LC-MS/MS method using radio-isotope agents, but the substance was not injected into the patient. A technique using radio-isotope agents was used for the collected blood.

→ As the use of kits and materials for each test is burdensome for the research cost, in this study, the patient's blood was collected, prepared, and stored for testing. The test for DNA-TGN was performed when more than a certain number of samples were collected. Therefore, it took time to check the test results, making it difficult to adjust the treatment according to DNA-TGN test results. When DNA-TGN test is stably set up in the laboratory and cost for study is established, it is expected that DNA-TGN test will be possible to carry out frequently. In the future, it would be helpful to perform the DNA-TGN test at few weeks later from taking 6MP maintenance (when thought to be reached the plateau), when the 6MP dose is changed, or when toxicity occurs.

→ These comments have been briefly added to the discussion section.

→ (line 430-432) Considering the timing of the toxicity onset, it would be helpful to test DNA-TGN after 2 weeks or more, when the dose of 6MP is changed, or when toxicity occurs, rather than immediately after starting 6MP.

→ Thank you for the valuable comment.

→ DNA-TGN is a final active metabolite of all the thiopurines, which is produced by incorporation of 6-TGN into DNA. 6-TGN accumulation in DNA is an inevitable consequence of thiopurine treatment and the central to the therapeutic effects. It is believed that the S phase of the cell cycle must pass once before 6TGN is incorporated DNA, then at least one more S phase to allow replication of the 6-TGN substituted DNA, resulting in cytotoxicity.

→ According to previous studies, 6-TGN concentration reached a plateau after 2 weeks to several weeks after starting 6MP. Although there is no clear report when DNA-TGN reached its highest concentration after taking a fixed dose of 6MP, most studies performed sampling for DNA-TGN test at 2 weeks or more of 6MP administration.

→ These characteristics and the appropriate timing of DNA TGN testing are further described in the discussion section.

→ (line 428-432) Thiopurine is known to present delayed cytotoxic effect. At least two or more S phase of cell cycles are needed for 6TGN to incorporate into DNA, and this 6TGN-substituted DNA replicates and present mismatch, eventually leading to cell death [4]. Considering the timing of the toxicity onset, it would be helpful to test DNA-TGN after 2 weeks or more, when the dose of 6MP is changed, or when toxicity occurs, rather than immediately after starting 6MP.

(Minor comments)

Conclusion of Abstract. The last sentence; will be important for what? Clinical outcome or adverse effects?

→ Thank you for your comment. To clarify the expression, it has been modified as follows.

→ (line 74-76) Particularly in patients with NUDT15 variants who need to reduce the 6MP dose, DNA-TGN could be applied as a useful marker to monitor the therapeutic effect of 6MP.

Page 5. L111; Erythrocyte → erythrocyte

→ Thank you for the detailed comment. We edited the word according to the comment.

→ (line 110-112) Other studies have evaluated the use of erythrocyte TGN (RBC TGN) as an indicator of 6MP metabolism, but these studies have found that RBC TGN is not a robust prognostic marker for relapse [3].

Page 8. L171; at 2 weeks; at 2nd week (?)

→ Thank you for the comment. We edited the manuscript according to the comment.

→ after 2 weeks for the protocol taking 6MP for 2 weeks, after 2 and 4 weeks for the protocol taking 6MP for 4 weeks, after 2 and 4 and 8 weeks for the protocol taking 6MP for 8 weeks, and after 2 and 4 weeks of 1st cycle of maintenance treatment. (line 170-173)

Reviewer #2:

Introduction modified statements were not referenced appropriately

→ Thank you for your careful advice. As your comment, some introduction statements were not referenced. We added the references as follows.

→ (line 117-118) Several genes have been linked to the metabolism of 6MP used in the treatment of ALL; this include TPMT and NUDT15 [6].

→ (line 127-128) To date, three papers have been published on DNA-TGN measured focusing in patients with NUDT15 variant genotype. [13-15]

Statistical analysis suggestions were not appropriately implemented, normalization of DNA TGN levels to the dose of 6MP, Normalization is required when we wanted to see effect of genetic variant as dose changes by itself could change the levels of DNA TGN. So it has to be normalized to be able tp compare between genotype groups.

→ Thank you for valuable comments. I think I did not fully understand the meaning at first revision that the data needs normalization. At 2nd revision, normalization of DNA TGN levels to 6MP intensity was done according to the advice.

→ When the analysis was done after normalization, the P values (which were not statistically significant) changed. Changes in the analysis method were described in the method section, and the changed statistical results were reflected in the manuscript and figure.

→ (line 206-207) To see the difference in DNA-TGN/6MP intensity according to genotype, normalization of DNA-TGN/6MP intensity was done.

→ (line 276-278) The ratio of DNA-TGN/6MP intensity was significantly higher in patients harbouring NUDT15 variants than that in patients harbouring wild type NUDT15 (P<0.001), but no difference was observed between patients harbouring TPMT variant and wild type (P=0.323). (Fig 2C)� Figure 2C

Authors claim several papers here in this reply on DNA TGN levels, and in the introduction they say there are only two reports available- conflicting statements.

→ There have been reports of DNA-TGN levels in several papers, but there were only few papers reporting the DNA-TGN in patients with the NUDT15 variants. It seems that it was written inaccurately so that the meaning does not delivered well, so I modified it as follows.

→ In addition, when we have reviewed the literature based on the reviewer’s suggestion, we found one more article that partially overlaps the previously found articles, so citation was added. Thank you for the attentive advice.

→ (line 127-130) To date, three papers have been published on DNA-TGN measured focusing in patients with NUDT15 variant genotype. [13-15] These studies have reported that patients with the NUDT15 variant genotype showed higher DNA-TGN level and toxicity when 6MP was given in regular dose.

→ (line 403-406) Three previously published papers have demonstrated that higher DNA-TGN concentrations coincided with a higher incidence of cytopenia in patients harbouring the NUDT15 variants when the patients received the standard dose of 6MP [13-15].

They were also not sure about the timing of the sample collecting sued for analysis-- this was not disclosed clearly

→ Thank you for the important advice. We additionally described the conditions of the samples used in the analysis in more detail.

→ (line 210-213) When performing CV and linear mixed model analyses to determine the degree of variability, only the values collected during the maintenance treatment of patients who took DNA-TGN samples more than three times during the maintenance period were used in the analysis.

→ The sentence below was previously written, but the order of the sentences was arranged differently to better understand them.

→ (line 201-203) For the association of DNA-TGN by 6MP dose and hematologic toxicity, analysis was performed only on the samples collected during maintenance treatment to rule out effects of other drugs.

Reviewer #3:

(No Response)

→ We appreciate your evaluation.

Reviewer #4:

There were several questions from different reviewers and the editor. All questions were answered properly. I have no other comments.

→ We sincerely appreciate the positive evaluation of the manuscript.

PLoS One. doi: 10.1371/journal.pone.0245667.r005

Decision Letter 2

A M Abd El-Aty

29 Dec 2020

PONE-D-20-27853R2

DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukemia patients with NUDT15 variant genotypes

PLOS ONE

Dear Dr. Koo,

==============================

ACADEMIC EDITOR:

1- Again, don't use "Will" throughout the text

2- Don't use "We, our". Use impersonal phrasing throughout the text

3- Revise Section heading to "Study strength and limitations" instead of the current one

4- Again, comments (8) 8- Line 177: Details regarding analysis, including method optimization and performance should be added as Suppl. Materials

5- Please check grammar and syntax errors with the assistance from native speaker

==============================

Please submit your revised manuscript by Feb 12 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

A. M. Abd El-Aty

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. 2021 Jan 22;16(1):e0245667. doi: 10.1371/journal.pone.0245667.r006

Author response to Decision Letter 2

31 Dec 2020

Response to the academic editor

We sincerely appreciate the detailed review with suggestions. We have made corrections based on the comments. We sincerely hope that the manuscript is now suitable for publication in this journal and would be pleased to respond to any further queries regarding this submission. Thank you for your consideration.

1- Again, don't use "Will" throughout the text

� Thank you for the detailed comment. We removed “will” from the text.

�- In addition to the few papers on DNA-TGN so far, this paper can (<- will) help establish the standard for therapeutic dose adjustment of 6MP based on DNA-TGN. (line 451)

�- In the future, larger studies are (<- will be) needed to reduce the 6MP dose in patients with NUDT15 or TPMT variants and then titrate the dose based on DNA-TGN levels. (line 469)

2- Don't use "We, our". Use impersonal phrasing throughout the text

� Thank you for the advice. We edited the sentences which included “we, our” as follows.

� - This study was able to demonstrate the efficacy of DNA-TGN in monitoring the treatment effects of 6MP, particularly in patients harbouring NUDT15 variant genotype. (line 481)

� - This appears to be the first study to analyze prospective serial DNA-TGN samples in a large number of patients—including those, which carry NUDT15 variant genotypes—treated with reduced doses of 6MP. (line 455)

3- Revise Section heading to "Study strength and limitations" instead of the current one

� Thank you for the advice. We changed the heading to “The significance of the study”.

4- Again, comments (8) 8- Line 177: Details regarding analysis, including method optimization and performance should be added as Suppl. Materials

� Thank you for the advice which helps make the improved manuscript. We added the details regarding sample analysis, including method optimization and performance as supplementary material.

- S1 Appendix. Detailed description of study methods.

5- Please check grammar and syntax errors with the assistance from native speaker

� - Thank you for the constructive advice. The manuscript underwent additional proofreading in English. English proofreading certificate is uploaded, also.

Attachment

Submitted filename: DNATGN_ResponseToReviewers_3rd_submit.docx

Click here for additional data file.^{(19.6KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0245667.r007

Decision Letter 3

A M Abd El-Aty

4 Jan 2021

PONE-D-20-27853R3

DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukemia patients with NUDT15 variant genotypes

PLOS ONE

Dear Dr. Koo,

==============================

ACADEMIC EDITOR: Again, authors' have to amend the MS very carefully based upon the comments stated below

1- This is the 3rd time authors' are asked to revise the last paragraph before conclusion to "Study strength and limitations". Unfortunately, this is not done, although they have said done. Heading MUST be changed to what has been stated and related text should be in one paragraph

2- Line 466-472: This is a redundancy for conclusion, avoid repetition

3- Where is the results of method validation? Linearity, LOD, LOQ, Recovery, and RSD values. This should be added in the Suppl. materials

4- Suppl. Materials should be checked as well for grammar and syntax errors

5- All amendments should be highlights yellow. Don't use track-changes mode

==============================

Please submit your revised manuscript by Feb 18 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

A. M. Abd El-Aty

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

PLoS One. 2021 Jan 22;16(1):e0245667. doi: 10.1371/journal.pone.0245667.r008

Author response to Decision Letter 3

5 Jan 2021

Response to the academic editor

We sincerely appreciate the detailed review with suggestions. Also, we apologize for some misunderstandings and improper corrections. We have made corrections based on the comments again. We sincerely hope that the manuscript is now suitable for publication in PLOS ONE. It will be of great help if a quick decision is possible. Thank you for your consideration.

==============================

ACADEMIC EDITOR: Again, authors have to amend the MS very carefully based upon the comments stated below

Response: We apologize for the misunderstanding and improper correction. We changed the heading to “Study strength and limitations” as recommended, and made the text into one paragraph.

2- Line 466-472: This is a redundancy for conclusion, avoid repetition

Response: We truly agree with your comment. As mentioned by the editor, we deleted the contents that overlap with the contents of the conclusion section.

3- Where is the results of method validation? Linearity, LOD, LOQ, Recovery, and RSD values. This should be added in the Suppl. Materials

Response: We do appreciate the detailed suggestion. We added the results of method validation for LC-MS/MS of DNA-TG quantification. In this study, LOB was experimentally confirmed, but LOD was not. (As two of the guidelines for LC-MS/MS method validation recommended LOD but was not recommended by other guidelines, LOD was not confirmed in this study.) Linearity, LOB, LLOQ, recovery, and RSD valued are presented.

4- Suppl. Materials should be checked as well for grammar and syntax errors

Response: Thank you for the comment. Supplementary index was checked for grammar and syntax errors, also.

5- All amendments should be highlights yellow. Don't use track-changes mode

Response: We appreciate for the advice. We highlighted the amendments instead of using track-changes mode.

Attachment

Submitted filename: DNATGN_ResponseToReviewers_4th_submit.docx

Click here for additional data file.^{(19.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0245667.r009

Decision Letter 4

A M Abd El-Aty

6 Jan 2021

DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukemia patients with NUDT15 variant genotypes

PONE-D-20-27853R4

Dear Dr. Koo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

A. M. Abd El-Aty

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0245667.r010

Acceptance letter

A M Abd El-Aty

12 Jan 2021

PONE-D-20-27853R4

DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukemia patients with NUDT15 variant genotypes

Dear Dr. Koo:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Prof. A. M. Abd El-Aty

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Dataset

(XLSX)

Click here for additional data file.^{(48.3KB, xlsx)}

S1 Appendix. Detailed description of study methods.

(DOCX)

Click here for additional data file.^{(28.7KB, docx)}

Attachment

Submitted filename: DNATGN_response to reviewers_1112.docx

Click here for additional data file.^{(384.4KB, docx)}

Attachment

Submitted filename: DNATGN_ResponseToReviewers_3rd_submit.docx

Click here for additional data file.^{(19.6KB, docx)}

Attachment

Submitted filename: DNATGN_ResponseToReviewers_4th_submit.docx

Click here for additional data file.^{(19.9KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

DNA-thioguanine nucleotide as a treatment marker in acute lymphoblastic leukemia patients with NUDT15 variant genotypes

Hee Young Ju

Ji Won Lee

Hee Won Cho

Ju Kyung Hyun

Youngeun Ma

Eun Sang Yi

Keon Hee Yoo

Ki Woong Sung

Rihwa Choi

Hong Hoe Koo

Soo-Youn Lee

Roles

Abstract

Background

Methods

Results

Conclusions

Introduction

Methods

1. Study patients

2. Treatment

3. Sample collection and analysis

4. Data collection

5. Statistical analysis

Results

1. Patients and samples

Table 1. Participant and DNA-TGN sample characteristics.

2. DNA-TGN results stratified based on the NUDT15 status

Fig 1. DNA-TGN concentration versus 6MP intensity.

Fig 2. Distribution of various parameters based on genotype.

3. Toxicity during treatment

Table 2. Cases with toxicity related to treatment.

4. Inter-individual variability

5. Changes in 6MP dose during the maintenance treatment

Fig 3. Changes in 6MP dosage according to NUDT15 status.

Discussion

Study strength and limitations

Conclusion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

A M Abd El-Aty

Roles

Author response to Decision Letter 0

Decision Letter 1

A M Abd El-Aty

Roles

Author response to Decision Letter 1

Decision Letter 2

A M Abd El-Aty

Roles

Author response to Decision Letter 2

Decision Letter 3

A M Abd El-Aty

Roles

Author response to Decision Letter 3

Decision Letter 4

A M Abd El-Aty

Roles

Acceptance letter

A M Abd El-Aty

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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