Next-Generation Sequencing and PCR Analysis of ctDNA Correlate with Clinical Outcomes and Emerging Mutations Linked to Drug Resistance

August 2020, Vol 1, No 1

One of the challenges that clinicians face when trying to diagnose cholangiocarcinoma (CCA) is obtaining enough tumor tissue to conduct molecular-profiling studies. A repeated biopsy could potentially be performed, but some tumors may be difficult or dangerous to reach to obtain the necessary tissue. As an alternative, blood testing for circulating tumor-cell DNA (ctDNA) can identify important molecular markers that could be missed if a repeated biopsy is difficult to perform.

An important drawback of ctDNA testing is the potential difficulty in detecting DNA variations in the blood when the amount of ctDNA is too low. Despite this hindrance, oncologists and pathologists have embraced the ctDNA testing, because of its value in screening for and early detection of primary cancers or cancer recurrence, the assessment of a cancer treatment’s effectiveness, and the identification of treatment-resistant genetic variations.

At the 2020 American Association for Cancer Research annual meeting, Morten Lapin, PhD, MS, Department of Investigational Cancer Therapeutics, M.D. Anderson Cancer Center, Houston, TX, and colleagues presented the results of their recent study. In their study, they sought to determine if the ctDNA from patients with advanced CCA and isocitrate dehydrogenase (IDH) mutation was concordant with tissue samples of the same patients, and to establish whether ctDNA can be associated with clinical outcomes and can detect emerging mutations associated with therapeutic resistance.

The study included 32 patients with an IDH1 or IDH2 mutation. The detection of ctDNA in the patients in this study was done at baseline, while the patients were receiving therapy with an IDH inhibitor, and at disease progression during active therapy. Molecular profiling of ctDNA was accomplished using droplet digital polymerase chain reaction (ddPCR), which produces more precise, reproducible, and statistically significant results than quantitative PCR. The baseline and disease progression ctDNA samples were tested using targeted digital next-generation sequencing for oncogenic aberrations in 74 genes.

The baseline results from the ctDNA analysis were compared with the molecular profiles of archival biopsy tissue to establish concordance and sensitivity. The variant allele frequency—a surrogate measure of the proportion of DNA molecules carrying a specific DNA variant—was compared with patients’ clinical outcomes.

Samples from patients with progressive disease were analyzed for the emergence of mutations that are plausibly associated with treatment resistance. The rates of known simultaneous mutations other than IDH were also analyzed.

The detection of IDH1 and IDH2 mutations was well aligned between the patients who had ddPCR and next-generation sequencing testing (84% and 83%, respectively). When the researchers examined the samples taken at baseline for IDH mutation–positive ctDNA, patients with variant allele frequency values of less than the median had longer times to treatment nonresponse than patients with higher than the median variant allele frequency.

Similarly, patients with a low amount of ctDNA for all detected molecular alterations detected by variant allele frequency at baseline had longer times to treatment nonresponse than patients with high combined variant allele frequency.

In addition, changes in the quantity of IDH mutation–positive ctDNA in serially collected samples evaluated by ddPCR demonstrated that patients with a decrease in variant allele frequency of IDH-positive ctDNA compared with patients who had no change or an increase in IDH-positive ctDNA had a trend toward longer survival (P = .06) but not longer time to treatment nonresponse (P = .4).

In patients whose disease progressed during therapy, no IDH isoform switching was detected. However, next-generation sequencing of ctDNA did expose mutations—most often the TP53 and ARID1 mutations—that were not detected at baseline in these patients.

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