TP53 digital PCR probe library for circulating tumor DNA detection in the post-therapeutic monitoring of gastrointestinal cancers

Circulating tumor DNA (ctDNA) is considered a new class of biomarkers in post-therapeutic monitoring. As the variant allele frequency (VAF) of ctNDA is generally less than 1%, digital PCR (dPCR) is one of the most practical monitoring methods. However, specific primers/probes for dPCR must be designed individually as tumor mutations vary between individual patients. To apply ctDNA monitoring for a wide range of human cancers, we designed a TP53 dPCR probe library. According to the COSMIC database, there are 1,284 unique mutations among a total of 25,376 recorded mutations in the p53 DNA binding domain, where approximately 90% of the mutations have been found. The majority (77%) of the 1,284 unique mutations are not recurrent, and thus 42, 132, and 426 unique mutations account for 50%, 70%, and 90% of the total mutations, respectively. Our initial TP53 dPCR probe library designed by Hypercool Primer & Probe^TM technology, contains 132 unique mutations to cover 70% of the mutations. The dPCR library allows "off-the-shelf", highly sensitive ctDNA tracking once the TP53 mutation has been identified in the target tumor (e.g., surgical specimens). In our practice, TP53 mutations were found in 97% (34/35) of esophageal, 70% (7/10) of stomach, and 93% (13/14) of colorectal tumors. In the course of the post-therapeutic follow-up with >500 patient DNA samples, 98% of the probes were confirmed to have detected the target mutation. When monitored monthly, ctDNA was elevated approximately six months prior to a relapse diagnosis by CT scan, whereas no ctDNA elevation was observed in the non-relapsed cases over 1–3 years. Overall, the sensitivity of the VAF using dPCR with the probes was as low as 0.001%, and the monthly monitoring was practically feasible. These results suggest that the TP53 dPCR probe library can be immediately used in the post-therapeutic monitoring of gastrointestinal cancers.