Quantitative Detection of Cancer Biomarkers in Picoliter Droplets.

Valerie Taly1, Deniz Pekin1,3, Corinne Normand1, Zakaria El Harrak1, Thevy Hor1, Li Xinyu2, Ivan Atochin2, Steve Kotsopoulos2, Delphine Le Corre1, Leonor Benhaim1, J. Brian Hutchison2, Darren R. Link2, Helene Blons1, Philippe Nizard1, Pierre Laurent-Puig1
1Université Paris Descartes; INSERM UMR-S775; Centre Universitaire des Saints-Pères, 45 rue des Saints-Pères, 75270 Paris Cedex 06, France;2RainDance Technologies, Lexington, MA 02421, Massachusetts, USA; 3Université de Strasbourg; CNRS UMR 7006

Gene alterations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells. These DNA biomarkers are especially important for the diagnosis, prognosis, treatment and follow-up of patients. Target-oriented treatments have dramatically improved prognosis of patients with cancer and new biomarkers of resistance or sensitivity to such treatment have been highlighted (1). These drugs are now limited to subgroups of patients including, for example, patients bearing KRAS wild-type colorectal adenocarcinomas for anti-EGFR antibodies based treatments. However it has also been recently demonstrated that resistance occurs through the selection of resistant subclones. In case of resistance of colorectal cancer to cetuximab and panitunimab subclones, such subclones bear KRAS mutations.
Plain use of gene alterations as biomarkers in clinical oncology requires a highly sensitive, and ideally quantitative, strategy that allow the detection of the tumour specific modifications in a background of non-mutated DNA from normal cells (2). Procedures classically used in clinics cannot detect less than ~1% mutant genes in a background of non-mutated DNA from normal cells. Among the challenge to be assessed in clinical oncology, the ability to detect with a high sensitivity and quantify mutations in complex samples (including tumors or plasma) will allow, within other, to detect subclones in order to predict resistance and build therapeutic strategies to overcome potential resistance as well as to perform follow up of the efficiency of treatment by measuring disappearance of the targeted clones. Moreover, such procedure will permit to use non-invasive strategies that could greatly facilitate patient follow up (3).
We first developed a procedure based on using droplet-based microfluidics to perform digital PCR in millions of picoliter droplets (4). By segregating individual target DNA-molecules in billions of aqueous droplets acting as independent microreactors, this procedure allows extremely precise, sensitive and fast quantification of mutated genes to be carried out. The procedure has then been extended the detection of multiple mutations in parallel (5). Finally, to demonstrate the pertinence of our procedures to overcome the clinical oncology challenges, two studies have been built. When the first one addresses our ability to detect rare subclones in colorectal tumors and to understand the impact of these subclones on responses and survival of the patients treated by anti-EGFR therapies, the second one aims at demonstrated the possibility to detect KRASmutations directly in plasma of patients with advanced colorectal cancers.
1. Sawyers CL, Nature 2008, 452(7187), 548-552; 2. Diehl F, et al., Curr Opin Oncol 2007, 19(1), 36-42; 3. Taly V, et al., Trends Mol Med 2012, 18(7), 405-416. 4. Pekin D, et al., Lab Chip 2011, 11(13), 2156-2166. 5. Zhong Q. et al., Lab Chip 2011, 11(13), 2167-2174.

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