Droplet digital PCR of circulating tumor cells from colorectal cancer patients can predict KRAS mutations before surgery.
In colorectal cancer (CRC), KRAS mutations are a strong negative predictor for treatment with the EGFR-targeted antibodies cetuximab and panitumumab. Since it can be difficult to obtain appropriate tumor tissues for KRAS genotyping, alternative methods are required. Circulating tumor cells (CTCs) are believed to be representative of the tumor in real time. In this study we explored the capacity of a size-based device for capturing CTCs coupled with a multiplex KRAS screening assay using droplet digital PCR (ddPCR). We showed that it is possible to detect a mutant ratio of 0.05% and less than one KRAS mutant cell per mL total blood with ddPCR compared to about 0.5% and 50-75 cells for TaqMeltPCR and HRM. Next, CTCs were isolated from the blood of 35 patients with CRC at various stage of the disease. KRAS genotyping was successful for 86% (30/35) of samples with a KRAS codon 12/13 mutant ratio of 57% (17/30). In contrast, only one patient was identified as KRAS mutant when size-based isolation was combined with HRM or TaqMeltPCR. KRAS status was then determined for the 26 available formalin-fixed paraffin-embedded tumors using standard procedures. The concordance between the CTCs and the corresponding tumor tissues was 77% with a sensitivity of 83%. Taken together, the data presented here suggest that is feasible to detect KRAS mutations in CTCs from blood samples of CRC patients which are predictive for those found in the tumor. The minimal invasive nature of this procedure in combination with the high sensitivity of ddPCR might provide in the future an opportunity to monitor patients throughout the course of disease on multiple levels including early detection, prognosis, treatment and relapse as well as to obtain mechanistic insight with respect to tumor invasion and metastasis.
Sorbonne Universités, UPMC Univ. Paris 06, F-75005, Paris, France; Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Department of Oncology and Endocrine Biochemistry, Paris, France; Cancer Biology and Therapeutics, Centre de Recherche Saint-Antoine, Institut National de la Santé et de la Recherche Médicale (INSERM) U938, Institut Universitaire de Cancérologie (IUC), Université Pierre et Marie Curie (UPMC), Sorbonne Universities, Paris, France. Electronic address: jerome.denis@aphp.fr.
Jérôme Alexandre
JA
Denis
Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Department of Digestive and Hepato-Pancreato-Biliary Surgery, Paris, France.
Alexia
A
Patroni
Sorbonne Universités, UPMC Univ. Paris 06, F-75005, Paris, France; Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Department of Oncogenetics and Molecular Angiogenetics, Paris, France.
Erell
E
Guillerm
Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Department of Oncology and Endocrine Biochemistry, Paris, France.
Dominique
D
Pépin
ScreenCell SA, Sarcelles, France.
Naoual
N
Benali-Furet
ScreenCell SA, Sarcelles, France.
Janine
J
Wechsler
Sorbonne Universités, UPMC Univ. Paris 06, F-75005, Paris, France; Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Department of Digestive and Hepato-Pancreato-Biliary Surgery, Paris, France.
Gilles
G
Manceau
Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Department of Oncology and Endocrine Biochemistry, Paris, France.
Maguy
M
Bernard
Sorbonne Universités, UPMC Univ. Paris 06, F-75005, Paris, France; Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Department of Oncogenetics and Molecular Angiogenetics, Paris, France.
Florence
F
Coulet
Cancer Biology and Therapeutics, Centre de Recherche Saint-Antoine, Institut National de la Santé et de la Recherche Médicale (INSERM) U938, Institut Universitaire de Cancérologie (IUC), Université Pierre et Marie Curie (UPMC), Sorbonne Universities, Paris, France.
Annette K
AK
Larsen
Sorbonne Universités, UPMC Univ. Paris 06, F-75005, Paris, France; Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Department of Digestive and Hepato-Pancreato-Biliary Surgery, Paris, France.
Mehdi
M
Karoui
Sorbonne Universités, UPMC Univ. Paris 06, F-75005, Paris, France; Assistance Publique-Hôpitaux de Paris, Pitié-Salpêtrière Hospital, Department of Oncology and Endocrine Biochemistry, Paris, France; INSERM, UMR_S 1166, Institute of Cardiometabolism and Nutrition, ICAN, Paris, France.
Jean-Marc
JM
Lacorte
D000328:Adult; D001483:Base Sequence; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000601:surgery*; D005260:Female; D018628:Gene Dosage; D005838:Genotype; D006801:Humans; D008297:Male; D009154:Mutation / Q000235:genetics*; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D015203:Reproducibility of Results
C117307:KRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)
Circulating tumor cells; Colorectal cancer; Digital PCR; KRAS; Liquid biopsy