Circulating tumor DNA as a practical diagnostic tool for post-treatment monitoring

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The ultimate goal of assessing tumor markers from a blood test is to reflect the tumor burden in the body. Conventional serum markers have been used as a part of daily practice for a long time despite the fact that their role may have changed as emerging new therapies were developed. Circulating tumor DNA (ctDNA) is considered to be an excellent alternative for monitoring tumor burden in a sophisticated, quantitative manner.

Although ctDNA is tumor-derived material, the variant allele frequency (VAF) in blood is very low, and therefore next generation sequencing (NGS) may not be a suitable approach for monitoring ctDNA. Nevertheless, investigators have shown that NGS can measure very low concentrations of ctDNA using various barcoding techniques with deep sequence. However, using ctDNA as a tumor marker requires frequent assessment, ideally every three months, with the possibility that levels can be lower than the detection limit of NGS. Here, the costs of the monitoring technology directly limit the feasibility of ctDNA as tumor marker.

Digital PCR (dPCR) is a technique capable of detecting mutations at a sensitivity greater than 100-fold over NGS. In addition, the cost per assay is much more affordable than NGS. With dPCR, patients can take periodical blood tests that will collectively show dynamics of ctDNA over time post-treatment. A specific dPCR probe detects only a single alteration per assay, although a parallel assay using multiple probes may be performed to increase assay credibility. In the case of double colorectal cancer, we demonstrated that mutations identified in the primary tumor (origin in the right colon) showed synchronous ctDNA dynamics, whereas mutations identified in another tumor (origin in the left colon) showed stable, low levels of ctDNA.

We were able to interpret the ctDNA dynamics from 19 time points over nearly 1,000 post-operative days, which was a comparable frequency to those for serum tumor markers used in daily practice. In esophageal cancers, we have also found that using ctDNA as a tumor marker can provide information important for >90% of post-treatment patients in terms of: (i) early relapse prediction; (ii) treatment efficacy evaluation; and (iii) non-relapse corroboration1. These elements for clinical validity can only be achieved in a daily practice setting with affordable and sensitive techniques, such as dPCR.

There is a concern that dPCR assesses only a few mutations per tumor (although this can be increased when funding is unlimited) due to mutational heterogeneity. Therefore, ctDNA monitoring should ideally be used for mutations involved in relatively early phases of tumor development. We investigated the degree of mutational heterogeneity of colorectal tumors by multiregional sequencing. We found that a single biopsy is sufficient to select high VAF mutations that are ideal for ctDNA monitoring (except for hypermutated tumors). Overall, we recommend frequent ctDNA monitoring by dPCR when high VAF mutations are identified from the tumor for most post-treatment colorectal cancer patients.

1          Iwaya, T., Endo, F., Takahashi, F., Tokino, T., Sasaki, Y., Nishizuka, S. S. Frequent Tumor Burden Monitoring of Esophageal Squamous Cell Carcinoma With Circulating Tumor DNA Using Individually Designed Digital Polymerase Chain Reaction. Gastroenterology. 2021 Jan;160(1):463-465.e4.

Satoshi Nishizuka

Professor, Iwate Medical University