Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients

Valerie Taly, Deniz Pekin, Leonor Benhaim, Steve K. Kotsopoulos, Delphine Le Corre, Xinyu Li, Ivan Atochin, Darren R. Link, Andrew D. Griffiths, Karine Pallier, Hélène Blons, Olivier Bouché, Bruno Landi, J. Brian Hutchison, Pierre Laurent-Puig

Research output: Contribution to journalArticle

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Abstract

BACKGROUND: Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample. METHODS: We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients forwhomthe primary tumor biopsy tissue DNA had been characterized by quantitative PCR. RESULTS: Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRASmutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRASmutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected. CONCLUSIONS: This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulatingDNAobtained by noninvasive blood collection.

Original languageEnglish
Pages (from-to)1722-1731
Number of pages10
JournalClinical Chemistry
Volume59
Issue number12
DOIs
Publication statusPublished - Dec 2013
Externally publishedYes

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Tumors
Colorectal Neoplasms
Plasmas
Polymerase Chain Reaction
Mutation
DNA
Multiplex Polymerase Chain Reaction
Oncogenes
Neoplasms
Biopsy
Sarcoma
Rats
Assays
Screening
Blood
Tissue
Codon

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

Cite this

Taly, V., Pekin, D., Benhaim, L., Kotsopoulos, S. K., Corre, D. L., Li, X., ... Laurent-Puig, P. (2013). Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. Clinical Chemistry, 59(12), 1722-1731. https://doi.org/10.1373/clinchem.2013.206359

Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. / Taly, Valerie; Pekin, Deniz; Benhaim, Leonor; Kotsopoulos, Steve K.; Corre, Delphine Le; Li, Xinyu; Atochin, Ivan; Link, Darren R.; Griffiths, Andrew D.; Pallier, Karine; Blons, Hélène; Bouché, Olivier; Landi, Bruno; Hutchison, J. Brian; Laurent-Puig, Pierre.

In: Clinical Chemistry, Vol. 59, No. 12, 12.2013, p. 1722-1731.

Research output: Contribution to journalArticle

Taly, V, Pekin, D, Benhaim, L, Kotsopoulos, SK, Corre, DL, Li, X, Atochin, I, Link, DR, Griffiths, AD, Pallier, K, Blons, H, Bouché, O, Landi, B, Hutchison, JB & Laurent-Puig, P 2013, 'Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients', Clinical Chemistry, vol. 59, no. 12, pp. 1722-1731. https://doi.org/10.1373/clinchem.2013.206359
Taly, Valerie ; Pekin, Deniz ; Benhaim, Leonor ; Kotsopoulos, Steve K. ; Corre, Delphine Le ; Li, Xinyu ; Atochin, Ivan ; Link, Darren R. ; Griffiths, Andrew D. ; Pallier, Karine ; Blons, Hélène ; Bouché, Olivier ; Landi, Bruno ; Hutchison, J. Brian ; Laurent-Puig, Pierre. / Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. In: Clinical Chemistry. 2013 ; Vol. 59, No. 12. pp. 1722-1731.
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AU - Li, Xinyu

AU - Atochin, Ivan

AU - Link, Darren R.

AU - Griffiths, Andrew D.

AU - Pallier, Karine

AU - Blons, Hélène

AU - Bouché, Olivier

AU - Landi, Bruno

AU - Hutchison, J. Brian

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N2 - BACKGROUND: Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample. METHODS: We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients forwhomthe primary tumor biopsy tissue DNA had been characterized by quantitative PCR. RESULTS: Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRASmutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRASmutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected. CONCLUSIONS: This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulatingDNAobtained by noninvasive blood collection.

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