Abstract 6704: A digital-PCR primer/probe library for pan-cancer ctDNA monitoring established from public databases for somatic mutations
Authors: Hiraki Hayato , Yashima-Abo Akiko , Iwaya Takeshi , Nishizuka Satoshi S. 
Published: April 2023

Background: Circulating tumor DNA (ctDNA), a new class of tumor marker, has been demonstrated the clinical validity of: 1) early relapse prediction, 2) therapeutic efficacy evaluation, and 3) relapse-free corroboration in the context of cancer treatment. Variant allele frequencies (VAFs) of ctDNA were under 0.1% even in most advanced cancer patients. Since the detection limit of next-generation sequencing (NGS) is above 1%, higher sensitivity is needed for sensitive ctDNA monitoring. Digital PCR (dPCR) offers excellent sensitivity at an affordable cost compared with NGS. A mutation-specific primer/probe (P/P) is required for the dPCR assay, but a limited number of P/Ps are commercially available.

Materials and Methods: To provide essential probes for the rapid monitoring of sensitive ctDNA, we developed an off-the-shelf (OTS) dPCR P/P library (OTS-Probes) that includes probes that can detect &gt;1,000 mutations found in human cancer. The majority of the mutations to be detected by these P/P sets have been selected using an originally developed statistical scoring from hot spot mutations reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. To evaluate the coverage of OTS-Probes in human cancer specimens, the Cancer Genome Atlas (TCGA) and the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) datasets were used. TCGA is an international landmark cancer genomic program in the U.S., and C-CAT is a Japanese cancer genome profiling database.

Results: At least one mutation was matched to the OTS-Probes for ctDNA monitoring in lung, colorectum, stomach, liver, pancreas, breast, uterus, and prostate from TCGA and C-CAT cancer specimens, reflecting 63% and 77% of corresponding tumor types, respectively. The mutation coverage of the OTS-Probes tends to be lower in TCGA than in C-CAT, partially due to the fact that information about the TERT promoter mutation is absent in TCGA. We found that some ARID1A deletion mutations that have a high count in COSMIC were not observed in either the TCGA or the C-CAT database. We also analyzed the coverage of the OTS-Probes in different organs and driver genes. Currently, more than 250 P/P sets have been designed, synthesized, and validated with human tumor DNA or synthesized DNA fragments. Importantly, nearly 98% of P/P sets work with the same PCR condition. With our proprietary oligo synthesis method with modified bases, only a small percentage of P/P sets required optimization of the annealing temperature.

Conclusion: We developed a dPCR P/P library called OTS-Probes. Our analysis suggests that 60-80% of cancer patients could immediately apply at least one P/P set for sensitive ctDNA monitoring by dPCR. A quarter of the P/P library has already been validated by tumor DNA or synthesized DNA fragments. OTS-Probes is a great tool for ctDNA- related studies and for clinical use when a higher sensitivity than NGS can offer is required.

Citation Format: Hayato Hiraki, Akiko Yashima-Abo, Takeshi Iwaya, Satoshi S. Nishizuka. A digital-PCR primer/probe library for pan-cancer ctDNA monitoring established from public databases for somatic mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6704.