The Detection of Minor Clones with Somatic KIT D816V Mutations Using Droplet Digital PCR in Pediatric De Novo AML: AML-05 Trial from the Japanese Pediatric Leukemia/Lymphoma Study Group
Authors: Sasaki Koji , Uchiyama Yuri , Ikeda Junji , Yoshitomi Masahiro , Shimosato-Wada Yuko , Tokumasu Mayu , Matsuo Hidemasa , Yoshida Kenichi , Oki Kentaro , Yamato Genki , Hara Yusuke , Kinoshita Akitoshi , Tomizawa Daisuke , Taga Takashi , Adachi Souichi , Tawa Akio , Horibe Keizo , Matsumoto Naomichi , Ito Shuichi , Hayashi Yasuhide , Shiba Norio 
Published: November 2019

Introduction
Recent medical advances and development of comprehensive genetic understanding dramatically improve the clinical outcome of whole pediatric cancers, particularly in pediatric acute lymphoblastic lymphoma. However, approximately 50% of patients have disease relapse, and overall survival (OS) of pediatric acute myeloid leukemia (AML) is less than 70% as of the major therapeutic challenges. AML is caused by various chromosomal aberrations, gene mutations/epigenetic modifications, and deregulated/overregulated gene expressions, leading to increased proliferation and decreased hematopoietic progenitor cell differentiation. AML with RUNX1-RUNX1T1 gene fusions are generally classified as a low-risk group and resulted in favorable prognosis. However approximately 30% of the patients relapsed within 3 years. Conversely, KIT mutations were found in approximately 30% of AML cases with RUNX1-RUNX1T1 and thought to be a risk factor for relapse, particularly when occurring in D816V within KIT exon 17. Recently, droplet digital PCR (ddPCR), a method for measuring target nucleic acid sequence quantity, has been used to determine low-prevalence somatic mutations that were not detectable using Sanger sequencing. It shows the possibility that there are some of Pediatric AML cases which were not detected minor clones with somatic KIT mutation by using ordinary PCR. In this study, we explored KIT D816V mutations including the cases which are not detected by Sanger sequencing and found the prognosis of them by using Japanese pediatric AML cases.
Methods
We reanalyzed somatic KIT mutations (p.D816V) in the DNA extracted from 335 pediatric AML patients with RUNX1-RUNX1T1 who participated in the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) AML-05 trial using ddPCR . In this trial, we conducted the tests as follows,: PCR mixture containing 10 μL 2x ddPCR Supermix for probes, 900 nM target-specific PCR primers, and 250 nM mutant-specific (FAM) and wild-type-specific (HEX) probes.20 µL of PCR mixture and 70 μL Droplet generation oil were mixed, and droplet generation was performed using a Bio-Rad QX100 Droplet Generator. The droplet emulsion was thermally cycled in the following conditions: denaturing at 95 °C for 10 min, 40 cycles of PCR at 94 °C for 30 s and at 57 °C for 2 min, and a final extension at 98 °C for 10 min. PCR amplification in the droplets was confirmed using Bio-Rad QX200 Droplet Reader. ThresholdThe threshold was determined by comparing the non-template ddPCR results. All the data were evaluated above the threshold. We also performed targeted gene mutation analysis of KIT in all patients using Sanger sequencing.
Results
We identified 24 KIT D816V mutations (7.2%) in the 335 pediatric AML. Variant allele frequency (VAF) was 0.1%-46.9%.It is noteworthy that 12 out of 24 KIT D816V mutations were undetected in our previous study using Sanger sequencing. Fourteen out of these 24 patients were AML with RUNX1-RUNX1T1, 5 cases with inv(16), and 5 cases with other alterations. Ten of the 14 RUNX1-RUNX1T1 (71%) patients were newly identified using ddPCR. Six of these 14 RUNX1-RUNX1T1 patients had relapsed, and D816V mutations were only detected using ddPCR in 4 of these 6 relapsed cases. The mean VAF of KIT D816V was 3.8% (0.1%-13.4%) in the 10 undetected patients with RUNX1-RUNX1T1. Two of the 5 patients with inv(16) were newly identified, and 1 had relapsed. All 5 cases with other alterations were already identified using Sanger sequencing. The mean VAF of KIT D816V in the 3 patients with inv(16) was 44.8% (42.7%-46.9%), detected using Sanger sequencing, whereas the mean VAF of KIT D816V was 8.6% (6.5% and 10.7%) in the undetected patients with inv(16). The mean VAF of KIT D816V with other alterations was 28.1% (16.2%-42.9%).
Conclusion
We identified 12 KIT D816V mutations using ddPCR that were undetected using Sanger sequencing. ddPCR may be useful for detecting accurate frequencies of mutations that were undetected using Sanger sequencing. Potential co-existing gene mutations may contribute different significance of leukemogenesis and relapse.

Disclosures
Ito: Asahi Kasei Pharma: Consultancy, Other: Grants; Astellas Pharma: Consultancy, Other: Grants; Teijin: Other: Grants; Novartis Pharma: Consultancy; Zenyaku Kogyo Co. Ltd: Consultancy; Chugai Pharma: Consultancy, Other: Grants; Japan Blood Products Organization: Other: Grants; Pfizer inc.: Other: Grants.