Detection of KRAS mutations in liquid biopsies from metastatic colorectal cancer patients using droplet digital PCR, Idylla, and next generation sequencing.
Author(s): Holm M, Andersson E, Osterlund E, Ovissi A, Soveri LM, Anttonen AK, Kytölä S, Aittomäki K, Osterlund P, Ristimäki A
Publication: PLoS One, 2020, Vol. 15, Page e0239819
PubMed ID: 33237900 PubMed Review Paper? No
Purpose of Paper
This paper compared the detection of KRAS mutation in plasma of metastatic colorectal cancer patients by Idylla and next generation sequencing (NGS) with droplet digital PCR (ddPCR). The changes in KRAS mutant allele frequency during treatment and disease progression as measured by Response evaluation criteria in solid tumors (RECIST), carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) levels was also assessed.
Conclusion of Paper
MAF values by ddPCR at baseline varied from very low to high among the six patients for which they were available. There was a very strong negative concordance between ddPCR MAF and Idylla CT values with KRAS mutations identified in four specimens by Idylla and not ddPCR. The same mutation was identified by the hotspot NGS panel in all seven patients with a mutation detected in the final specimen by ddPCR and Idylla and no additional mutations were identified by NGS in the three patients that were negative by ddPCR and Idylla. After treatment, ddPCR MAF decreased with the patient’s initial response to treatment (RECIST, CEA, and CA19-9 levels) and remained low when stable or no evidence of disease progression but increased in all patients when only a partial response or disease progression were found. Throughout the study period, ddPCR MAF was associated with CEA values but not CA19-9.
Studies
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Study Purpose
This study compared the detection of KRAS mutation in plasma of metastatic colorectal cancer patients by Idylla and NGS with ddPCR. The changes in KRAS MAF during treatment and disease progression as measured by RECIST, CEA, and CA19-9 levels was also assessed. EDTA blood was collected from 10 patients diagnosed with KRAS mutation positive (G12 or G13) metastatic colorectal adenocarcinoma before treatment (six patients), on week 3 of treatment with bevacizumab in combination with alternating capecitabine and oxaliplatin or irinotecan, and six times (every 9 weeks) during the follow-up period. Patient response was evaluated by RECIST and assay of CEA and CA19-9 values (methods unspecified). Plasma was obtained by centrifugation (details not provided) within 30 min of collection and stored temporarily at -20°C for up to two weeks before storage at -80°C. DNA was extracted from plasma using the QiaSymphony DSP Circulating DNA Kit for ddPCR and NGS analysis. Wildtype and mutant KRAS mutations in extracted DNA were quantified using Bio-Rad ddPCR assays (mutation G12 and G13) and by NGS using a house cancer panel of 7 genes and the Ion AmpliSeq Hotspot Panel v2 (only last collection time-point) on an Ion Proton System. Additionally, 21 KRAS mutations were detected directly in plasma of nine patients using the real-time PCR based Idylla ctKRAS Mutation Test.
Summary of Findings:
MAF values by ddPCR at baseline varied from very low (0%) to high (63%) among the six patients for which they were available. The kappa agreement between mutation detection with Idylla and ddPCR was high (κ= 0.860) and there was a very strong negative concordance between ddPCR MAF and Idylla CT values (ρ=0.9461, P<0.0001). However, Idylla detected KRAS mutations at high CTs in four specimens where the MAF was 0 by ddPCR and no mutations were identified by ddPCR and not by Idylla. KRAS mutations were detected by the hotspot NGS panel in all seven patients whose final specimen was positive by NGS and Idylla and not in the three that were negative by NGS and Idylla. After treatment, MAF decreased (where possible) with patient’s initial response to treatment (RECIST, CEA, and CA19-9 levels) and remained low when stable or no evidence of disease but increased in all patients when only a partial response or disease progression were found. Throughout the study period, MAF was associated with CEA values (P<0.001) but not CA19-9.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform DNA Real-time qPCR Protein Clinical chemistry/auto analyzer DNA Digital PCR DNA Next generation sequencing Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Digital PCR Specific Technology platform Idylla
NGS