Multiplex Digital PCR Research Articles

Abstract Circulating tumor DNA (ctDNA) is present in plasma of individuals with advanced cancers. ctDNA is a prognostic marker for patients with colorectal cancer (CRC) and it might also be used for predicting the response to targeted therapy. For example, mutations in KRAS indicate which patients will fail to respond to specific therapies (cetuximab, panitunimab). Although ctDNA is characterized by the presence of a somatic mutation, direct quantitative detection through a simple workflow of such mutant DNA is not feasible by current technologies because the ratio of ctDNA to wild-type DNA can be as low as 1/10,000. This study describes the use of droplet-based digital PCR for detection and quantitation of one of the seven most frequent KRAS mutations in ctDNA from plasma of patients with advanced colorectal cancer. Furthermore, we demonstrate that multiplex digital PCR enables testing samples for different mutations simultaneously. By segregating individual target DNA molecules into millions of aqueous droplets acting as independent microreactors, our procedure allows for extremely precise, sensitive, and fast quantification of mutated genes. The sensitivity of the procedure was confirmed by measuring 1/200,000 dilution of KRAS-mutated cell-line DNA in a background of wild-type DNA. Furthermore, plasma of 14 patients with metastatic CRC were tested. Our procedure enabled detection of the target mutation and the wild-type DNA, and thus, measurement of the total amplifiable DNA. DNA concentration in the plasma samples varied by two orders of magnitude and was not correlated with the proportion of mutated DNA, which varied from 42% to 0.5%. By using a duplex analysis to detect either of the two most frequent mutations of KRAS (G12D and G13D) and the wild-type DNA, the expected mutation (known by primary tumor characterization) was detected in 12 out of the 14 samples. Two samples had a low amount of amplifiable DNA leading to an inconclusive result. Five samples, positive for the G13D mutation, were also tested for the G12D mutations and were negative. Moreover, we tested 4 samples in a multiplex format allowing the simultaneous analysis of wild-type, G12D, G13D, and G12R mutant KRAS. The multiplex testing results (i.e., the identity of the mutation and the mutant-to-wild-type ratio) were identical to those obtained in the duplex analysis. Our results demonstrate that our digital PCR method enables non-invasive detection of KRAS in plasma of patients with metastatic CRC with high sensitivity and high specificity. We anticipate that the method will be employed in multiple applications in the clinic, including diagnosis, cancer recurrence monitoring, and treatment management. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-422. doi:1538-7445.AM2012-LB-422

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