Ultrasensitive Measurement of Hotspot Mutations in Tumor DNA in Blood Using Error-suppressed Multiplexed Deep Sequencing

Overview

Journal Cancer Res

Specialty Oncology

Date 2012 May 15

PMID 22581825

Citations 94

Authors

Azeet Narayan

Nicholas J Carriero

Scott N Gettinger

Jeannie Kluytenaar

Kevin R Kozak

Torunn I Yock

Nicole E Muscato

Pedro Ugarelli

Roy H Decker

Abhijit A Patel

Affiliations

Soon will be listed here.

Abstract

Detection of cell-free tumor DNA in the blood has offered promise as a cancer biomarker, but practical clinical implementations have been impeded by the lack of a sensitive and accurate method for quantitation that is also simple, inexpensive, and readily scalable. Here we present an approach that uses next-generation sequencing to quantify the small fraction of DNA molecules that contain tumor-specific mutations within a background of normal DNA in plasma. Using layers of sequence redundancy designed to distinguish true mutations from sequencer misreads and PCR misincorporations, we achieved a detection sensitivity of approximately 1 variant in 5,000 molecules. In addition, the attachment of modular barcode tags to the DNA fragments to be sequenced facilitated the simultaneous analysis of more than 100 patient samples. As proof-of-principle, we showed the successful use of this method to follow treatment-associated changes in circulating tumor DNA levels in patients with non-small cell lung cancer. Our findings suggest that the deep sequencing approach described here may be applied to the development of a practical diagnostic test that measures tumor-derived DNA levels in blood.

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