140 - Efficacy of digital PCR for the determination of NUDT15 diplotype in patients with pediatric acute lymphoblastic leukemia

Publication Number: 2134.140

Reiji Fukano, Department of Pediatrics, University of Occupational and Environmental Health, Japan, KitaKyushu, Fukuoka, Japan; Yasunori Iida, Yamaguchi University, Ube, Yamaguchi, Japan; Takuya Ichimura, Department of Pediatrics, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan; Sachiyo Kamimura, University of Miyazaki Faculty of Medicine, Miyazaki, Miyazaki, Japan; Takuro Nishikawa, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Kagoshima, Japan; Shuichi Ozono, Kurume University School of Medicine, Department of Pediatrics, Kurume, Fukuoka, Japan; Yutaka Suehiro, Yamaguchi University, Ube, Yamaguchi, Japan; Hiroshi Moritake, University of Miyazaki, Miyazaki, Miyazaki, Japan; Yasuhiro Okamoto, Kagoshima University, Kagoshima, Kagoshima, Japan; Takahiro Yamasaki, Department of Oncology and Laboratory Medicine, Ube, Yamaguchi, Japan; Hideki Nakayama, NHO Kyushu Cancer Center, Japan, Fukuoka, Fukuoka, Japan; Masahiro Migita, National Hospital Organization Kumamoto Medical Center, Kumamoto, Kumamoto, Japan; Shunji Hasegawa, Yamaguchi University, Ube, Yamaguchi, Japan

Background: Patients with NUDT15 variants are sensitive to 6-mercaptopurine (6-MP); hence, dosage of 6-MP should be lowered for these patients owing to observed myelosuppressive effects. The bi-allelic NUDT15 variants caused extreme intolerance of 6-MP, however, diplotyping of NUDT15 variants has not yet been established.

Objective: We aimed to develop a practical diplotyping method using digital PCR (dPCR) to enhance clinical outcomes and reduce complications.

Design/Methods: We analyzed NUDT15 variants of exon 1 and 3 for 38 pediatric patients who were diagnosed with acute lymphoblastic leukemia between January 2010 and December 2021. We genotyped NUDT15 variants of exon 1 and 3 by Sanger sequencing. The tolerated 6-MP dose for each patient was evaluated according to the NUDT15 variants. We extracted germline RNA and synthesized cDNA from patients with multiple variants and performed dPCR with four variant-specific probes using the QuantStudio Absolute Q Digital PCR System to determine the NUDT15 diplotype. Specific probes included: Wild-type exon 1 probe labeled with VIC; c.52G>A variant probe for exon 1 labeled with FAM; Wild-type probe for exon 3 labeled with JUN; and c.415C>T variant probe for exon 3 labeled with ABY.

Results: Nine patients had single NUDT15 variants, and two had multiple variants. Patients with multiple variants required significantly lower 6-MP doses compared with those carrying wild-type or single-variant genotypes (Figure 1). Of the two patients with multiple variants, one had both c.52G>A and c.415C>T variants. Prior to the diplotyping, we confirmed the specificity of the probes for their intended sequences using artificially synthesized plasmids (Figure 2). Subsequently, we performed dPCR for the patient sample. Signals for VIC and ABY were double positive, suggesting that wild-type exon 1 and the c.415C>T variant in exon 3 were present on the same allele (Figure 3a). Wild-type exons 1 and 3 appeared to reside on opposite alleles, as indicated by distinct fluorescence populations (Figure 3b). The c.52G>A variant in exon 1 and the c.415C>T variant in exon 3 were located on opposite alleles (Figure 3c), whereas the c.52G>A variant in exon 1 and wild-type exon 3 were found on the same allele (Figure 3d). These findings indicated a compound heterozygous variant.

Conclusion(s): Our method enables detailed diplotype analysis and establishes dPCR as a valuable tool for assessing NUDT15 variants. This approach has potential for tailoring treatment strategies for pediatric ALL and could substantially reduce treatment-related toxicity and enhance patient management outcomes.

Average dose of 6-MP during maintenance therapy evaluated by the number of variants.
Patients with multiple variants had a significantly lower average dose than those with wild-type or single variants. Single-variant cases were classified as monoallelic variants and multiple-variant cases were classified as biallelic variants based on diplotyping, aligning with previously reported findings. Abbreviation: 6-MP, 6-mercaptopurine.

Validation of sequence-specific probes created for digital PCR.
The FAM probe for the exon 1 c.52G>A variant showed positive results only with the plasmid containing the sequence and negative results with the plasmid lacking the sequence. Similarly, other fluorescent probes demonstrated binding to their respective target sequences.

Results of digital PCR using the specific probe for the patient with multiple variants.
a) Signals for VIC and ABY were double positive, suggesting that wild-type exon 1 and the c.415C>T variant in exon 3 were present on the same allele. b) Wild-type exons 1 and 3 appeared to reside on opposite alleles, as indicated by distinct fluorescence populations. c) The c.52G>A variant in exon 1 and the c.415C>T variant in exon 3 were located on opposite alleles. d) The c.52G>A variant in exon 1 and wild-type exon 3 were found on the same allele.