An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage

Abstract

Circulating tumor DNA (ctDNA) represents a promising biomarker for noninvasive assessment of cancer burden, but existing methods have insufficient sensitivity or patient coverage for broad clinical applicability. Here we introduce CAncer Personalized Profiling by deep Sequencing (CAPP-Seq), an economical and ultrasensitive method for quantifying ctDNA. We implemented CAPP-Seq for non-small cell lung cancer (NSCLC) with a design covering multiple classes of somatic alterations that identified mutations in >95% of tumors. We detected ctDNA in 100% of stage II–IV and 50% of stage I NSCLC patients, with 96% specificity for mutant allele fractions down to ~0.02%. Levels of ctDNA significantly correlated with tumor volume, distinguished between residual disease and treatment-related imaging changes, and provided earlier response assessment than radiographic approaches. Finally, we explored biopsy-free tumor screening and genotyping with CAPP-Seq. We envision that CAPP-Seq could be routinely applied clinically to detect and monitor diverse malignancies, thus facilitating personalized cancer therapy.

Exosome-Based Detection of EGFR T790M in Plasma from Non–Small Cell Lung Cancer Patients

“…However, a primary challenge for the liquid biopsy field is the low allelic frequencies of the mutation (often below 0.5%) and low copy numbers of the mutation target. Technical advancements in recent years have enabled detection of mutations at very low allelic frequencies (34), but cannot solve the problem of low/no copy numbers in the sample.…”

Liquid biopsy in early stage lung cancer

“…Nowadays, there are various methods to detect genetic alterations in cfDNA, including real-time polymerase chain reaction (PCR), digital PCR, amplification protocols with magnetic beads in oil emulsions (beads emulsion), amplification and magnetics (BEAMing), next-generation sequencing (NGS) and mass spectrometry (MS) genotyping (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). The limit of mutation detection of these techniques fluctuates from 15% to 0.01%, but one of the main problems is the absence of standardized methods for biospecimen collection, processing, cfDNA isolation and analysis.…”

Detection and characterization of circulating cell free tumor DNA in cancer patients with malignant solid tumors. Liquid biopsy: a new tool in molecular pathology?

“…The most promising technique at the moment was recently published by Newman et al [28], a capture-based NGS ctDNA detection method called CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) designed especially for detection of all known major classes of mutations in non-small cell lung cancer (NSCLC). Capture-based NGS methods enrich for genomic regions before sequencing and the capture library (a custom-derived collection of targetspecific oligonucleotides) can easily be adapted for any given cancer type.…”

“…Capture-based NGS methods enrich for genomic regions before sequencing and the capture library (a custom-derived collection of targetspecific oligonucleotides) can easily be adapted for any given cancer type. According to the authors, their CAPP Seq approach identified mutations in >95% of tumors with high specificity and sensitivity down to approximately 0.02% [28,29]. However, although ctDNA was detected in the majority of patients with stage II-IV disease, biomarkers could be identified in only 50% of patients with early-stage NSCLC (stage I).…”