Early assessment of circulating tumor DNA after curative-intent resection predicts tumor recurrence in early-stage and locally advanced non-small-cell lung cancer

Abstract

Circulating tumor DNA (ctDNA) has demonstrated great potential as a noninvasive biomarker to assess minimal residual disease (MRD) and profile tumor genotypes in patients with non-small-cell lung cancer (NSCLC). However, little is known about its dynamics during and after tumor resection, or its potential for predicting clinical outcomes. Here, we applied a targeted-capture high-throughput sequencing approach to profile ctDNA at various disease milestones and assessed its predictive value in patients with early-stage and locally advanced NSCLC. We prospectively enrolled 33 consecutive patients with stage IA to IIIB NSCLC undergoing curative-intent tumor resection (median follow-up: 26.2 months). From 21 patients, we serially collected 96 plasma samples before surgery, during surgery, 1-2 weeks postsurgery, and during follow-up. Deep next-generation sequencing using unique molecular identifiers was performed to identify and quantify tumor-specific mutations in ctDNA. Twelve patients (57%) had detectable mutations in ctDNA before tumor resection. Both ctDNA detection rates and ctDNA concentrations were significantly higher in plasma obtained during surgery compared with presurgical specimens (57% versus 19% ctDNA detection rate, and 12.47 versus 6.64 ng·mL^-1 , respectively). Four patients (19%) remained ctDNA-positive at 1-2 weeks after surgery, with all of them (100%) experiencing disease progression at later time points. In contrast, only 4 out of 12 ctDNA-negative patients (33%) after surgery experienced relapse during follow-up. Positive ctDNA in early postoperative plasma samples was associated with shorter progression-free survival (P = 0.013) and overall survival (P = 0.004). Our findings suggest that, in early-stage and locally advanced NSCLC, intraoperative plasma sampling results in high ctDNA detection rates and that ctDNA positivity early after resection identifies patients at risk for relapse.

Keywords: circulating tumor DNA; early-stage and locally advanced non-small-cell lung cancer; minimal residual disease; noninvasive biomarker; relapse prediction.

Fig. 1

Overview of the study design, detected tumor mutations of patients, and sensitivity of ctDNA analysis by our custom NGS panel. (A) Overview of the study design with standardized time points of blood plasma collection. (B) Overview of clinicopathological features and tumor mutations. Each column represents the data from one patient (left). Bar chart showing the frequency of detected mutations in the respective gene (right). (C) Bars showing sensitivity of ctDNA detection in pretreatment plasma samples by our custom molecular barcode‐containing capture strategy. cfDNA, circulating tumor DNA; NGS, next‐generation sequencing; ddPCR, digital‐droplet polymerase chain reaction; Indel, insertion/deletion.

Fig. 2

ctDNA in presurgical and intrasurgical plasma samples (A) Comparison of cfDNA concentrations isolated from patient’s plasma presurgery and during surgery. Lines represent the median and interquartile range. (B) Percentage of patients with positive or negative ctDNA status presurgery and during surgery. Lines represent the mean. (A, B) Mann–Whitney U‐test was used to compare the two groups. (C) Comparison of ctDNA detection rates between plasma samples obtained presurgery or during surgery in all patients, stage I–II, and stage III patients. Fisher’s exact test was performed to assess statistical differences between the two groups. (D) ctDNA concentrations of paired plasma samples from the same patients, connected through a black line. n.d., not detected; cfDNA, cell‐free DNA; ctDNA, circulating tumor DNA.

Fig. 3

The value of ctDNA for outcome prediction early after surgery. (A) Comparison of mean ctDNA VAFs during surgery and after tumor resection (1–2 weeks postsurgery). Paired values from the same patients are connected through a line. Mann–Whitney U‐test was used to assess differences between the two groups. (B) PFS in patients with positive and negative ctDNA status 1‐2 weeks after surgery. (C) Overall survival in patients with positive and negative ctDNA status 1–2 weeks after surgery. In (B and C) log‐rank tests were used to assess significance. n.d., not detected; HR, hazard ratio; ctDNA, circulating tumor DNA; VAF, variant allele frequency.

Fig. 4

ctDNA as biomarker for disease surveillance. (A) Event chart showing the course of the disease with results of radiographic assessment and ctDNA analyses for each patient. Red circle, ctDNA detected; empty circle; ctDNA not detected; black line, imaging studies showing complete response; red lines, imaging studies show detection of disease. (B–D) Change of disease burden in response to treatment and during clinical progression in three different patients. Shown is the mean AF of all monitored SNVs in plasma over serial time points. ctDNA, circulating tumor DNA; Sx, Surgery; RTx, radiotherapy; CTx, chemotherapy; PD, progressive disease; VAF, variant allele frequency.