Quantitative Identification of Mutant Alleles Derived from Lung Cancer in Plasma Cell-Free DNA via Anomaly Detection Using Deep Sequencing Data

Abstract

The detection of rare mutants using next generation sequencing has considerable potential for diagnostic applications. Detecting circulating tumor DNA is the foremost application of this approach. The major obstacle to its use is the high read error rate of next-generation sequencers. Rather than increasing the accuracy of final sequences, we detected rare mutations using a semiconductor sequencer and a set of anomaly detection criteria based on a statistical model of the read error rate at each error position. Statistical models were deduced from sequence data from normal samples. We detected epidermal growth factor receptor (EGFR) mutations in the plasma DNA of lung cancer patients. Single-pass deep sequencing (>100,000 reads) was able to detect one activating mutant allele in 10,000 normal alleles. We confirmed the method using 22 prospective and 155 retrospective samples, mostly consisting of DNA purified from plasma. A temporal analysis suggested potential applications for disease management and for therapeutic decision making to select epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI).

Analytical Validation of BRAF Mutation Testing from Circulating Free DNA Using the Amplification Refractory Mutation Testing System

“…This recommendation is evidence based and supported by 6 studies, 232,234,235,242,243,247 comprising 2 MAs, 243,247 2 PCSs, 235,242 and 2 PRCSs. 232,234 The identified studies used various EGFR detection methods, but all verified the results from cfDNA with results from tumor tissue. Using reported true-positive, false-positive, truenegative, and false-negative data from 4 studies, a metaanalysis was conducted to determine a pooled estimate of sensitivity and specificity for cfDNA detection of EGFR mutation ( Figure 3).…”

“…231 Several studies 232,233 have shown comparable, if not superior, analytical sensitivity with droplet digital PCR, and NGS methods 234,235 can also provide this level of sensitivity, given the appropriate assay design. It is important, however, to recognize that unmodified Sanger sequencing, which was an acceptable method in the original 2013 guideline, 1 does not provide adequate sensitivity for this application.…”

“…Although not formally proven, this potential advantage of cfDNA is particularly important in the setting of secondary clinical resistance, 236 to enable broad sampling of different tumor subclones. 232,240,241 Analytical methods for cfDNA have high analytical specificity, with very low (<5%e20%) false-positive rates, 232,234,235,242,243 such that demonstration of a mutation, in the proper clinical context, can be used to guide treatment with a targeted inhibitor. However, sensitivity of cfDNA analysis is lower (60%e70%), 232,234,235,242,243 such that the absence of mutation finding does not exclude the possibility of a mutation.…”

“…Pooled estimate of sensitivity and specificity based on bivariate analysis of included studies. Four included studies compared tumor tissue samples with plasma samples using the same detection system, 234,235,242,243 and a fifth study 232 obtained plasma samples from patients with known EGFR and KRAS tumor mutation status. ARMs, amplification refractory mutation system; ddPCR, droplet digital PCR; FN, false-negative; FP, false-positive; PCR, polymerase chain reaction; PNA/LNA, peptide nucleic acidelocked nucleic acid; TN, true-negative; TP, true-positive.…”

Neueste technologische Entwicklungen für die Analyse von zirkulierender Tumor-DNA

“…Ein Algorithmus, entwickelt von Narayan et al, in dem eine Konsensussequenz nach einer Paired-End-Sequenzierung über den überlappenden Bereich gebildet wird, konnte die Fehlerrate von 0,31 % pro Base auf 0,07 % verringern [56]. Mit demselben Ansatz konnte eine japanische Gruppe LODs von 0,01, 0,01 und 0,05 % für die Detektion der EGFR Hotspot Mutationen L858R, L861Q, und T790M aus Plasma erzielen [57,58].…”