Detection of Ubiquitous and Heterogeneous Mutations in Cell-Free DNA from Patients with Early-Stage Non-Small-Cell Lung Cancer
A partir d'échantillons sanguins prélevés sur 50 patients atteints d'un cancer du poumon non à petites cellules de stade précoce, cette étude évalue la faisabilité d'une technique combinant PCR et séquençage à haut débit pour identifier des mutations d'intérêt clinique dans l'ADN libre circulant
Background The aim of this pilot study was to assess whether both ubiquitous and heterogeneous somatic mutations could be detected in cell-free DNA (cfDNA) from patients with early-stage non-small-cell lung cancer (NSCLC).
Patients and methods Three stage I and one stage II primary NSCLC tumors were subjected to multi-region whole-exome sequencing and validated with AmpliSeq. A subset of ubiquitous and heterogeneous single-nucleotide variants (SNVs) were chosen. Multiplexed PCR using custom-designed primers, coupled with next-generation sequencing (mPCR-NGS), was used to detect these SNVs in both tumor DNA and cfDNA isolated from plasma obtained prior to surgical resection of the tumors. The limit of detection for each assay was determined using cfDNA from 48 presumed-normal healthy volunteers.
Results Tumor DNA and plasma-derived cfDNA was successfully amplified and sequenced for 37/50 (74%) SNVs using the mPCR-NGS method. Twenty-five (68%) were ubiquitous and 12 (32%) were heterogeneous variants. Variant detection by mPCR-NGS and WES-AmpliSeq in tumor tissue were well correlated (R2=0.8722, p<0.0001). Sixteen (43%) of 37 SNVs were detected in cfDNA. Eleven ubiquitous and four heterogeneous variants with variant allele frequencies (VAFs) of 0.15%–23.3%. There was a statistically significant linear relationship between VAFs for tumor and cfDNA (R2=0.5144; p=0.0018). For all four patients, at least two variants were detected in plasma. The estimated number of copies of variant DNA present in each sample ranged from 5 to 524. The average number of variant copies required for detection (VCRD) was 3.16 (range: 0.2–7.6 copies).
Conclusions mPCR-NGS detected intratumor heterogeneity in early-stage NSCLC tumors, and detected 43% of the variants in cfDNA, including 25% of the heterogeneous variants. Further validation of mPCR-NGS for detection of variants in plasma will be needed to define the use of circulating biomarkers, such as cfDNA, to monitor tumor clonal dynamics in clinical practice.
Annals of Oncology , résumé, 2016