single-droplet-digital-polymerase-chain-reaction-for-comprehensive

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Institut Curie, PSL Research University

Circulating tumor cells (CTCs) isolated from blood can be probed for the expression of treatment targets. Immunofluorescence is often used for both the enumeration of CTC and the determination of protein expression levels related to treatment targets. Accurate and reproducible assessment of such treatment target expression levels is essential for their use in the clinic. To enable this, an open source image analysis program named ACCEPT was developed in the EU-FP7 CTCTrap and CANCER-ID programs. Here its application is shown on a retrospective cohort of 132 metastatic breast cancer patients from which blood samples were processed by CellSearch® and stained for HER-2 expression as additional marker. Images were digitally stored and reviewers identified a total of 4084 CTCs. CTC's HER-2 expression was determined in the thumbnail images by ACCEPT. 150 of these images were selected and sent to six independent investigators to score the HER-2 expression with and without ACCEPT. Concordance rate of the operators' scoring results for HER-2 on CTCs was 30% and could be increased using the ACCEPT tool to 51%. Automated assessment of HER-2 expression by ACCEPT on 4084 CTCs of 132 patients showed 8 (6.1%) patients with all CTCs expressing HER-2, 14 (10.6%) patients with no CTC expressing HER-2 and 110 (83.3%) patients with CTCs showing a varying HER-2 expression level. In total 1576 CTCs were determined HER-2 positive. We conclude that the use of image analysis enables a more reproducible quantification of treatment targets on CTCs and leads the way to fully automated and reproducible approaches.

Quantifying HER-2 expression on circulating tumor cells by ACCEPT.

Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown  remains limited.

Charles Decraene

Circulating Tumor Biomarkers Laboratory,
SiRIC,
Translational Research Department,
CNRS UMR144,
Circulating Tumor Biomarkers Laboratory, SiRIC, Translational Research Department

Institut Curie, PSL Research University

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Charles Decraene

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Circulating Tumor Biomarkers Laboratory,SiRIC,Translational Research Department,CNRS UMR144,Circulating Tumor Biomarkers Laboratory, SiRIC, Translational Research Department

Institut Curie, PSL Research University

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Circulating Tumor Biomarkers Laboratory,
SiRIC,
Translational Research Department,
CNRS UMR144,
Circulating Tumor Biomarkers Laboratory, SiRIC, Translational Research Department