Liquid biopsy in non-small cell lung cancer: Is it ready for prime time yet?

Abstract

The past decade has seen major changes in the treatment paradigm of non-small cell lung cancer (NSCLC) with the development of tyrosine kinase inhibitors (TKIs) targeting driver mutations. The technological advancement and access to molecular genomic studies have propelled uptake of rapidly expanding molecular classification and made therapeutic intervention available for patients in the clinic. Despite a high response rate with first-generation EGFR TKIs, the majority of patients progress within 12 months, with the most common acquired resistance mutation found in p.Thr790Met point mutation (T790M) in the EGFR gene.1, 2 Osimertinib, a third-generation EGFR-TKI inhibiting both EGFR-TKI-sensitizing and EGFR T790M resistance mutations, showed superior efficacy over chemotherapy in patients with secondary T790M mutation following progression with a first-generation TKI.3 This has prompted the clinical need to repeat biopsy in those patients. However, the need for repeat biopsies to identify resistance mechanisms is often constrained by limited tumor access or intra-patient and intra-tumor heterogeneity. Liquid biopsies such as analysis of plasma circulating tumor DNA (ctDNA) are minimally invasive and allow repeat sampling, which becomes particularly attractive when treatment decisions need to be based on these biopsy findings. Multiple mutation testing platforms for ctDNA have been used, such as Cobas EGFR mutation kit, digital droplet PCR (ddPCR), and Next Generation Sequencing (NGS). Hence, studies to identify the optimal platform for ctDNA testing is vital for further progress. Plasma ctDNA from the AURA3 study was retrospectively analyzed for EGFR mutations to compare the sensitivity of the Cobas EGFR mutation Test v2 with ddPCR and NGS assays. The study reported the positive percent agreement (PPA) of plasma T790M was comparable between ddPCR (58%) and NGS (66%), demonstrating their higher sensitivity compared to cobas assays (51%).4 Patients with positive T790M mutation determined by plasma ctDNA benefited from Osimertinib in this study, with subsequent studies examining the different platforms of assessing plasma T790M ctDNA mutation status and the efficacy of Osimertinib.5 In this issue, Lee et al. 6 report the analytical performances of real-time PCR assays for plasma EGFR test using various platforms including Cobas EGFR mutation Test v2 (Roche), PANA Mutyper-R-EGFR kit (PANAGENE) and ddPCR in 75 NSCLC patients. An EGFR mutation was identified in 54 patients; 52 of 54 detected in tissue and 43 of 54 in plasma. The analytical performances of the three commonly found EGFR mutations were assessed using matched tissues and plasma samples in 68 patients who underwent simultaneous tissue and plasma analysis. The sensitivity of the Cobas was statistically higher (P = 0.031) than that of the PANAMutyper. The sensitivity of specific mutations exon 19 deletion/L585R/T790M was 95.7%/87.5%/75.0% for Cobas, 69.6%/75%/62.5% for PANAMutyper and (not evaluated)/75%/75% for ddPCR. The analytical performance of ddPCR for exon 19 deletion was not evaluated as it has different coverage for exon 19 deletions compared to other techniques. Importantly, the specificity was 100% across all technology and specific EGFR mutations except for one suspected false-positive case of an L858R mutation using Cobas. Furthermore, the authors demonstrated a good correlation in VAF between real-time PCR and ddPCR (Pearson's r = 0.830). From this report, it is clear that there is variability in sensitivity between different assays, but real-time PCRs offer broader coverage compared to ddPCR as in evaluating Exon 19 deletion. However, we would argue that single-gene testing such as studied in the current study is becoming less relevant, particularly given that detection of secondary T790M mutation is not necessary with Osimertinib moving to the first line setting with the report of the FLAURA study. 7 Therefore, what is the current role of plasma ctDNA detection in the clinical setting? In the diagnostic setting, patients who have limited biopsy samples to allow molecular characterization may benefit from examination of the ctDNA. However, a platform that can detect a broader range of potentially targetable mutations would be ideal, including EGFR, ALK (Anaplastic Lymphoma kinase), ROS1 (ROS proto-oncogene1), RET (rearranged during transfection), MET (Mesenchymal-epithelial transition), HER2 (human epidermal growth factor receptor 2), BRAF (B-raf proto-oncogene), KRAS (KRAS proto-oncogene), and NTRK (Neurotrophic tyrosine receptor kinase). Liquid biopsy can also detect possible mechanism of resistance following acquired resistance after first-line Osimertinib in patients who do not have an accessible biopsy site at the time of progression. These can include appearance of MET, HER2 and PI3Kinase. Due to the need for broader mutation testing for NSCLC, we are advancing towards NGS as the primary testing modality. It is worth noting that while NGS was not tested in the current study, the analysis from the AURA3 study showed NGS has broader coverage than other methodologies. Other emerging use of liquid biopsy is to monitor treatment response and resistance, as residual ctDNA and dynamic changes of detectable ctDNA may be detected earlier than radiological disease progression.8 For instance, the detection of T790M mutation in plasma in patients on EGFR-TKI predates radiological progression by up to 344 days, although its impact on treatment decision is yet to be determined.9 The current NCCN (National Comprehensive Cancer Network) guideline recommends using a broad, panel-based approach like NGS and considering RNA-based NGS if any driver oncogenes are not identified, especially in nonsmokers, to improve detection of fusion events. It does not support the use of ctDNA in lieu of tissue diagnosis due to higher false-negative rates of up to 30%. However, NCCN guideline does support the use of ctDNA if the patient is not medically fit for an invasive procedure, in case of insufficient material following pathological confirmation on the tissue sample, or incomplete diagnostic biomarker assessment due to limited tissue availability.10, 11 The 2021 IASLC (International Association for the Study of Lung Cancer) consensus statement also recognizes plasma ctDNA as a valid tool for genotyping of advanced NSCLC. Low multiplex-based approaches are considered appropriate only when plasma NGS is unavailable. IASLC consensus statement also regarded single-gene testing as incomplete if oncogene driver mutation is not detected and recommends serial testing for additional actionable biomarkers in line with other guidelines.12 However, the adoption of NGS in liquid biopsy has been relatively slow, partly due to the lack of standardization of methodologies and commercially available assays. Affordability of using NGS in liquid biopsy also remains a key issue in slow adoption, even in developed countries such as Singapore.13 Overall, we believe liquid biopsy has a potential role in lung cancer management. However, single-gene platform such as the ones reported in the current issue is unlikely to be helpful, given the broad range of mutations of clinical interest in lung cancer, both at the time of diagnosis and progression following targeted therapy. As such, when liquid biopsy is being considered in instances where there is inadequate tissue biopsy, NGS appears to be the most advantageous in this setting. The real-world uptake will be determined by access and high-quality evidence from methodological studies and clinical outcome data before it can evolve into a practice-changing modality for better patient care.