The large amount of enthusiasm behind the potential applications of plasma cell-free DNA (cfDNA) analysis appears to have outpaced our biological understanding of this new method. Plasma cfDNA is a complex mixture derived from germline, foetal, infectious and malignant sources, and its analysis is increasingly becoming adopted as a method for non-invasive genotyping of advanced cancers.1 Unfortunately, notable discordance exists between genotyping of tissue and plasma cfDNA, which has previously been attributed to both variable shedding of tumour DNA,3 as well as tumour heterogeneity (particularly in the setting of drug resistance).4,5 However, these two factors are unable to fully account for recurring evidence in the literature of inaccurate plasma genotyping,6 and the researchers from this recent study hypothesised that somatic mutations within non-malignant cells, known as clonal haematopoiesis (CH), might also be a contributing source of the discordance between tumour and cfDNA genotyping.1
This study conducted in the USA involved the review of a cohort of patients with advanced non-small cell lung cancer (NSCLC) and focused on three cancer-associated genes – KRAS, JAK2, or TP53 – which could be genotyped using highly sensitive assays. Plasma cfDNA genotyping was performed either using a validated droplet digital PCR (ddPCR) assay or a commercially available plasma NGS assay (GuardantHealth, Redwood City).1
221 NSCLC patients were reviewed after having undergone plasma ddPCR for EGFR and KRAS mutations. 58 of these were found to harbour an EGFR mutation based on tumour genotyping. Two of these (3%) had plasma ddPCR which was positive for a KRAS codon 12 mutation. Peripheral blood cell (PBC) DNA testing confirmed the source of the KRAS mutation in both cases.1
A further 143 commercial plasma NGS results from 122 NSCLC patients were analysed. 14 mutations were detected in three genes associated with CH (JAK2, GNAS, IDH2), including 6 cases (4.9%) positive for a JAK2 V617F mutation, which is rarely seen on NSCLC tumour genotyping (0.26%). 5 of these 6 cases had PBC available for analysis, and ddPCR detected the JAK2 V617F mutation in each. However, 3 had tumour NGS which showed them to be JAK2 wildtype. TP53 mutations were detected in 108 of the 143 plasma NGS results. 33 of these had both PBC and tumour NGS available for comparison, and only 14 TP53 mutations could be confirmed to be tumour-derived.1
Many experts have advocated for plasma genotyping in patients with advanced NSCLC when tumour tissue, the gold standard, is unavailable for analysis. In line with the consensus, the cobas EGFR mutation test v2 was approved on 1 June 2016 by the US Food and Drug Administration (FDA).7 However, the caveat remains that clinicians ordering plasma genotyping need to be aware that mutations detected in cfDNA may not always reflect tumour genotype. Indeed, this study revealed that most JAK2 mutations, some TP53 mutations and rare KRAS mutations detected in cfDNA were due to CH rather than tumour. These findings also highlight that developing plasma cfDNA genotyping as a cancer detection tool in the future may require paired PBC genotyping so that CH-derived mutations are not misdiagnosed as occult malignancy.1
- The use of plasma cfDNA genotyping for cancer care remains poorly understood, and marked discordance between tumour and plasma genomic analysis still exists.
- This study revealed recurrent mutations in JAK2, TP53 and KRAS detected in cfDNA of advanced NSCLC cancer patients were in fact derived from clonal haematopoiesis, rather than from tumour.
- Current expert consensus advocates the use of ‘liquid biopsy’ in NSCLC patients when tumour tissue is unavailable. In these cases, clinicians must be made aware that mutations detected in cfDNA may not always reflect the tumour genotype.
- Future efforts in developing plasma cfDNA genotyping as a cancer detection tool may require paired PBC genotyping so that CH-derived mutations are not misdiagnosed as occult malignancy.
- Hu Y, Ulrich B, Supplee J, et al. False positive plasma genotyping due to clonal hematopoiesis. Clin Cancer Res. 2018. pii: clincanres.0143.2018. [PDF]
- Wan JCM, Massie C, Garcia-Corbacho J, et al. Liquid biopsies come of age: Towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017;17(4):223-38. [URL]
- Sacher AG, Paweletz C, Dahlberg SE, et al. Prospective validation of rapid plasma genotyping as a sensitive and specific tool for guiding lung cancer care. JAMA Oncol. 2016;2(8):1014-22. [URL]
- Oxnard GR, Thress KS, Alden RS, et al. Association between plasma genotyping and outcomes of treatment with osimertinib (AZD9291) in advanced non-small-cell lung cancer. J Clin Oncol. 2016;34(28):3375-82. [URL]
- Kuderer NM, Burton KA, Blau S, et al. Comparison of 2 commercially available next-generation sequencing platforms in oncology. JAMA Oncol. 2017;3(7):996-8. [URL]
- Torga G, Pienta KJ. Patient-paired sample congruence between 2 commercial liquid biopsy tests. JAMA Oncol. 2017. [URL]
- cobas EGFR Mutation Test v2 [online]. U.S. Food and Drug Administration; 2016 [cited 14 June 2018]. [URL]