Surveillance of cfDNA Hot Spot Mutations in NSCLC Patients during Disease Progression

Abstract

Non-small cell cancer (NSCLC) has been identified with a great variation of mutations that can be surveyed during disease progression. The aim of the study was to identify and monitor lung cancer-specific mutations incidence in cell-free DNA as well as overall plasma cell-free DNA load by means of targeted next-generation sequencing. Sequencing libraries were prepared from cell-free DNA (cfDNA) isolated from 72 plasma samples of 41 patients using the Oncomine Lung cfDNA panel covering hot spot regions of 11 genes. Sequencing was performed with the Ion Torrent™ Ion S5™ system. Four genes were detected with highest mutation incidence: KRAS (43.9% of all cases), followed by ALK (36.6%), TP53 (31.7%), and PIK3CA (29.3%). Seven patients had co-occurring KRAS + TP53 (6/41, 14.6%) or KRAS + PIK3CA (7/41, 17.1%) mutations. Moreover, the mutational status of TP53 as well an overall cell-free DNA load were confirmed to be predictors of poor progression-free survival (HR = 2.5 [0.8-7.7]; p = 0.029 and HR = 2.3 [0.9-5.5]; p = 0.029, respectively) in NSCLC patients. In addition, TP53 mutation status significantly predicts shorter overall survival (HR = 3.4 [1.2-9.7]; p < 0.001). We demonstrated that TP53 mutation incidence as well as a cell-free DNA load can be used as biomarkers for NSCLC monitoring and can help to detect the disease progression prior to radiological confirmation of the status.

Keywords: KRAS; TP53; cell-free DNA (cfDNA) mutation load; liquid biopsy; non-small cell lung cancer (NSCLC).

Figure 1

Summary of patients according to non-small cell lung cancer (NSCLC) stage and cell-free DNA (cfDNA) gene mutations in T0 group samples. Mutations are color-coded in gradient from pathogenic to benign after type of mutation: red (missense); blue (in-frame indel); orange (synonymous); brown (nonsense); green (splice site); violet (intronic region); yellow (non-frame indel). NSCLC patient stages are color-coded in blue gradient from darkest—IV stage to I stage. Grey colored squares represent non-detected (ND) mutations.

Figure 2

Changes in mutation count and variant allele frequency (VAF) according to sample collection time point in the T0, T1, and TX groups. (A) Mutation status and count distribution in T0, T1, and TX groups according to sample collection time; mutation count is color-coded in gradient from white—0 mutations to dark blue—6; grey—samples not collected. (B) Most frequently mutated actionable gene VAFs, according to sample collection time point in the T0, T1, and TX groups; each dot represents a sample, with violin plot corresponding to estimated density, and the median VAF is shown in a red vertical line; vertical dotted lines are at 2, 5, and 10% VAF.

Figure 3

Hazard ratios (HR) [95% confidence interval (CI)] of genes with mutation status for progression-free survival (PFS) and overall survival (OS). (A) Progression free survival; (B) Overall survival.

Figure 4

Kaplan–Meier estimates of progression–free and overall survival. (A,B) Patients categorized according to mutation count with 0, 1–2, and ≥3 mutations; mutation count is color-coded in blue—0, in orange—1−2 and in red—≥3 mutations. (C,D) Estimates according to mutant TP53 status; blue color indicates negative TP53 status and red—positive.

Figure 5

Kaplan–Meier estimates of progression-free survival (PFS) (A) and overall survival (OS) (B) according to median cfDNA load in high (>50 mutant cfDNA molecules) and low cfDNA load (≤50 mutant cfDNA molecules) patient groups; blue color indicates low cfDNA load (≤50 mutant cfDNA molecules) and red—high cfDNA load (>50 mutant cfDNA molecules).