Liquid Biopsy for Disease Monitoring in Non-Small Cell Lung Cancer: The Link between Biology and the Clinic

Abstract

Introduction: Cell-free DNA (cfDNA) analysis offers a non-invasive method to identify sensitising and resistance mutations in advanced Non-Small Cell Lung Cancer (NSCLC) patients. Next-generation sequencing (NGS) of circulating free DNA (cfDNA) is a valuable tool for mutations detection and disease's clonal monitoring.

Material and methods: An amplicon-based targeted gene NGS panel was used to analyse 101 plasma samples of advanced non-small cell lung cancer (NSCLC) patients with known oncogenic mutations, mostly EGFR mutations, serially collected at different clinically relevant time points of the disease.

Results: The variant allelic frequency (VAF) monitoring in consecutive plasma samples demonstrated different molecular response and progression patterns. The decrease in or the clearance of the mutant alleles was associated with response and the increase in or the emergence of novel alterations with progression. At the best response, the median VAF was 0% (0.0% to 3.62%), lower than that at baseline, with a median of 0.53% (0.0% to 9.9%) (p = 0.004). At progression, the VAF was significantly higher (median 4.67; range: 0.0-36.9%) than that observed at the best response (p = 0.001) and baseline (p = 0.006). These variations anticipated radiographic changes in most cases, with a median time of 0.86 months. Overall, the VAF evolution of different oncogenic mutations predicts clinical outcomes.

Conclusion: The targeted NGS of circulating tumour DNA (ctDNA) has clinical utility to monitor treatment response in patients with advanced lung adenocarcinoma.

Keywords: adenocarcinoma; cell-free DNA; clinical outcomes; liquid biopsy; lung cancer; next-generation sequencing; tumour-free DNA.

Figure 1

(A) Variant allelic fraction between tissue and ctDNA at baseline. Box represents the interquartile range of values and whiskers the median and the 25th and 75th percentile values (* p < 0.001, Mann–Whitney test). (B) Correlation between ctDNA concentration and variant allelic fraction (r = −0.319; p = 0.289).

Figure 2

Box plots depicting the detectable variants of the mutated genes (A) and cfDNA concentration (B) at baseline, best response, and progression. Box represents the interquartile range of values and whiskers the median and the 25th and 75th percentile values (n = 13, * p = 0.001, Kruskal–Wallis test). The allelic fraction decreased to its lowest level at best response to treatment and increased to its highest level when the disease progressed.

Figure 3

Progression with T790M in EGFR patients. (A–E), serial monitoring of cfDNA in five patients that developed T790M mutation as resistance mechanism to 1st/2nd G TKIS. The emergence and rise of the T790M (grey line) were accompanied by an increase in the original sensitising mutation (blue line), except for (B). In (B), dual Del19 and T790M were detected at diagnosis, and when progression occurred, there was a rise of the T790M clone, with a clearance of both clones when 3rd generation TKI was initiated. In (E), cfDNA analysis also revealed residual amounts of pathogenic TP53 mutations, ALK C1156Y and PIK3CA E545K, not detected initially. (F) Comparison of the VAF ctDNA of the EGFR sensitising mutation and the T790M at progression. The black arrow on the top of the graph indicates the treatment administrated during that time. ChT, chemotherapy; TKI, Tyrosine Kinase Inhibitor; PD, Progressive Disease; PR, Partial Response.

Figure 4

Progression with an increase in the sensitising mutation. (A,B) Serial ctDNA measurements revealed a VAF decrease with treatment and before radiological progression. There was an increase in the original mutation without other detectable alterations. In (A), tissue re-biopsy at 24 months was negative for T790M.

Figure 5

Complete ctDNA monitoring of a patient with Del19 EGFR mutation and representative CT scans at indicated time points. With both 1st and 3rd G TKIs, clinical response was accompanied by molecular decrease in the driver mutation. Clinical progression was preceded by an increase in del19 and the appearance of the T790M. After the initiation of the 3rd G TKI, partial response was observed with ctDNA clearance of both clones. At the recurrence time, the patient lost the T790M, del19 reappeared, and the C767S in cis was detected. Plasmatic alterations at progression were both confirmed with a tissue biopsy represented by *. Red circles refer to main tumour lesions.

Figure 6

Example of “negative” patients. Despite fluctuations in cfDNA amount in (A), no mutations were detected. In (B), there was a complete clearance of the driver mutation with response to 1st G TKI, but no change nor a new alteration was stated when progression occurred.

Figure 7

CtDNA monitoring in a KRAS/STK11/P53 mutant patient. At the 3rd cycle of chemotherapy (ChT), there was a proportional decrease in all detected variants. One month before RECIST progressive disease (PD), the patient displayed an increase in all variants, becoming exponential before death occurs.

Figure 8

CtDNA monitoring in BRAF 600E mutant patients. In (A), 1st line chemotherapy (Cht) with maintenance was stopped due to optic neuropathy. In the treatment-free interval, there was an increase in ctDNA, followed by ctDNA clearance after 2nd line treatment, which was performed until local progression and brain metastasis (24th month). Increase in the BRAF allelic frequency preceded PD in one month. In (B), the patient progressed on 1st line chemotherapy with leptomeningeal metastasis at month 4, not accompanied by VAF increase. With vemurafenib, there was a neurological and thoracic improvement with BRAF V600E ctDNA clearance. The patient developed a large pleural effusion and clinical deterioration escorted by ctDNA increase and death.