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Mutational testing on liquid biopsies for treatment decisions in metastatic colorectal cancer – comparison of ddPCR and MassARRAY methods
Background
The aim was to investigate if mutational analysis of circulating DNA (cfDNA) in plasma is feasible for selection of patients with metastatic colorectal cancer (mCRC) for anti-EGFR treatment. Secondary, to investigate emergence of mutations in cfDNA during therapy, and to compare the fractions of mutations detected by the ddPCR and MassARRAY methods.
Methods
This was a prospective clinical feasibility study using circulating tumor DNA (ctDNA) for treatment selection in mCRC. Inclusion criteria: mCRC; indication for systemic palliative treatment; measureable disease (RECIST v1.1); age ≥ 18. Blood samples were drawn at inclusion, during treatment and follow up. Treatment regimens according to standard. Samples for prospective analyzes were collected in Streck tubes, shipped to central laboratory and DNA analyzed by a multiplex ddPCR (Bio-Rad) testing for 30 somatic mutations in KRAS/NRAS/BRAF with a limit of detection of app. 0.01% as previously published. Feasibility endpoints were; quality and turn-around time of ctDNA analysis. Failure parameters were; low quality of samples (lymphocyte contamination > 5%, major loss 3 working days (WD), results delivered > 7 WD. For retrospective analyzes plasma samples were collected in EDTA tubes and analyzed by a mass spectrometric based multiplexed platform (MassARRAY® Agena Bioscience). The UltraSEEK MassARRAY Colon Panel v2 was used to test for 104 somatic mutations in KRAS/NRAS/BRAF/EGFR/PIK3CA with a limit of detection of app. 0.01%. All mutations targeted by the ddPCR panel were included. Mutational testing on tumor tissue was done according to standard.
Results
Forty-nine patients were included. Average time from sampling to results by ddPCR were 2.7 WD. One sample failed quality parameters due to transportation to lab > 3 WD. Concordance between mutational status in tissue and plasma by ddPCR was 70%. Disconcordant (30%) were wild type (wt) in plasma, but mutated in tumor tissue. Comparing the concordant and disconcordant cases there was no difference in treatment response (p=0.46) but longer progression free survival (HR=2.1 95% CI 1.05-4.27, p=0.04) and a tendency to longer overall survival (HR=2.3 95% CI 0.97-5.32, p=0.06) among disconcordant cases. Concordance between mutational status in tissue and plasma evaluated by MassARRAY was 80%. When comparing mutational testing by ddPCR and MassARRAY at inclusion, all but one mutation detected by ddPCR were detected by MassARRAY. In four patients, who were wt by ddPCR, MassARRAY detected a mutation, which were in the ddPCR panel and also present in the tumor tissue. MassARRAY detected mutations in seven patients, which were not covered by the ddPCR panel. In one patient we observed the emergence of a single mutation (EGFR S492R, by MassARRAY) at the end-of-study.
Conclusions
Using cfDNA for mutational detection by ddPCR in daily clinic proved feasible. Treatment response and survival among disconcordant cases were not inferior to concordant cases. Mutational testing by MassARRAY seemed to have a better concordance with tissue status than ddPCR and future studies should investigate MassARRAY for selection of patients with mCRC who will benefit from anti-EGFR treatment. Emergence of a new mutation during systemic palliative treatment was a rare event.
Clinical trial identification
NCT03750175.
Legal entity responsible for the study
The author.
Funding
This project was funded by grants from The Danish Cancer Society (Kræftens Bekæmpelse; grant nos. R99-A6323-14 -S25 and R269-A15652), Novo Nordisk Foundation (grant no. 9297), and Health Research Foundation of Central Denmark Region (grant no. A1602).
Disclosures
All authors have declared no conflicts of interest.
