Development of Multiplex Drop-Off Digital PCR Assays for Hotspot Mutation Detection of KRAS, NRAS, BRAF, and PIK3CA in the Plasma of Colorectal Cancer Patients

Abstract

The detection of mutations in KRAS, NRAS, BRAF, and PIK3CA has become essential in managing the treatment of metastatic colorectal cancer (CRC) with the approval of new targeted therapies. We developed novel multiplex drop-off digital PCR (MDO-dPCR) assays by combining amplitude-/ratio-based multiplexing with drop-off/double drop-off strategies that allow for the detection of at least the 69 most frequent hotspot mutations in all four genes with only three reactions. The analytical performance of the assays was assessed using synthetic oligonucleotides, which were further validated on plasma cell-free DNA samples from a large cohort of CRC patients and compared with next-generation sequencing data. The MDO-dPCR assays showed a high sensitivity with a limit of detection ranging from 0.084% to 0.182% in mutant allelic frequency. The screening of plasma cell-free DNAs from 106 CRC patients identified mutations in 42.45% of them, with a sensitivity of 95.24%, a specificity of 98.53%, and an accuracy of 96.98% for mutation detection, and a strong correlation of measured mutant allelic frequencies compared with next-generation sequencing results. The high sensitivity and comprehensive mutation coverage of the MDO-dPCR assays make them suitable for rapid and cost-effective detection of KRAS, NRAS, BRAF, and PIK3CA mutations in the plasma of CRC patients, and could be useful in early response assessment and longitudinal disease monitoring. The detection of mutations in KRAS, NRAS, BRAF, and PIK3CA has become essential in managing the treatment of metastatic colorectal cancer (CRC) with the approval of new targeted therapies. We developed novel multiplex drop-off digital PCR (MDO-dPCR) assays by combining amplitude-/ratio-based multiplexing with drop-off/double drop-off strategies that allow for the detection of at least the 69 most frequent hotspot mutations in all four genes with only three reactions. The analytical performance of the assays was assessed using synthetic oligonucleotides, which were further validated on plasma cell-free DNA samples from a large cohort of CRC patients and compared with next-generation sequencing data. The MDO-dPCR assays showed a high sensitivity with a limit of detection ranging from 0.084% to 0.182% in mutant allelic frequency. The screening of plasma cell-free DNAs from 106 CRC patients identified mutations in 42.45% of them, with a sensitivity of 95.24%, a specificity of 98.53%, and an accuracy of 96.98% for mutation detection, and a strong correlation of measured mutant allelic frequencies compared with next-generation sequencing results. The high sensitivity and comprehensive mutation coverage of the MDO-dPCR assays make them suitable for rapid and cost-effective detection of KRAS, NRAS, BRAF, and PIK3CA mutations in the plasma of CRC patients, and could be useful in early response assessment and longitudinal disease monitoring. Colorectal cancer (CRC) follows lung and breast cancers as the third most common neoplasm worldwide, and is the second leading cause of cancer death worldwide.1Sung H. Ferlay J. Siegel R.L. Laversanne M. Soerjomataram I. Jemal A. Bray F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J Clin. 2021; 71: 209-249Crossref PubMed Scopus (37538) Google Scholar CRC is known to be initiated by an accumulation of several mutations in a subset of driver genes involved in regulatory pathways, such as KRAS, NRAS, BRAF, and PIK3CA. Approximately 40% to 70% of CRC patients harbor mutations in these four driver genes, for which Food and Drug Administration–approved targeted therapies are available.2Raskov H. Soby J.H. Troelsen J. Bojesen R.D. Gogenur I. Driver gene mutations and epigenetics in colorectal cancer.Ann Surg. 2020; 271: 75-85Crossref PubMed Scopus (45) Google Scholar,3Guo F. Gong H. Zhao H. Chen J. Zhang Y. Zhang L. Shi X. Zhang A. Jin H. Zhang J. He Y. Mutation status and prognostic values of KRAS, NRAS, BRAF and PIK3CA in 353 Chinese colorectal cancer patients.Sci Rep. 2018; 8: 6076Crossref PubMed Scopus (65) Google Scholar Activating mutations in exons 2, 3, and 4 of KRAS and NRAS are a negative predictor of benefit from anti–epidermal growth factor receptor antibodies,4Lievre A. Bachet J.B. Boige V. Cayre A. Le Corre D. Buc E. Ychou M. Bouche O. Landi B. Louvet C. Andre T. Bibeau F. Diebold M.D. Rougier P. Ducreux M. Tomasic G. Emile J.F. Penault-Llorca F. Laurent-Puig P. 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P. and in colorectal cancer: an and Clin Oncol. 2020; PubMed Scopus Google Scholar that allow for the detection of are on PCR or next-generation sequencing assays coverage of genes or of to several genes with sensitivity allelic for detection of the high and the for analysis a for clinical sequencing in detection of mutations in and 2020; Full Full PubMed Scopus Google Scholar such as mutation and digital PCR are rapid and cost-effective of the detection and of a of known mutations with high sensitivity for detection, for mutation and for and for M. A. Laurent-Puig P. V. digital PCR and sequencing for a cancer 2018; PubMed Scopus Google K.S. R. S. S. H. T. M. mutation detection in from a of leading to the clinical of Cancer. Full Full PubMed Scopus Google Scholar make for The approval of the for in clinical of an mutation in lung cancer Mutation by the Food and Drug in that the of in analysis is in clinical an of and clinical mutations are a in the of analysis is to multiplex screening assays for the analysis of are drop-off of the was developed to detection of mutations a using a an in the of the hotspot and a drop-off with a the wild-type of the hotspot the of a is as a a mutant in the hotspot is with a of the drop-off as a of or leading to of mutant and drop-off has to hotspot mutations in BRAF R. D. A. R. J. C. C. detection and of BRAF status in colorectal cancer using digital PCR and a novel wild-type negative Full Full PubMed Scopus Google Scholar and KRAS C. A. M. S. A. A. A. M. O. Ychou M. J.Y. C. hotspot mutations by digital 2018; PubMed Scopus Google Scholar in C. A. M. S. A. A. A. M. O. Ychou M. J.Y. C. hotspot mutations by digital 2018; PubMed Scopus Google Scholar in lung and E. L. M. J.Y. A. S. C. I. A. R. A. L. C. digital PCR for mutations detection in 2020; PubMed Scopus Google Scholar and PIK3CA J. Le F. V. F. L. H. A. L. C. C. V. De T. of multiplex digital PCR assays for the detection of PIK3CA mutations in the plasma of metastatic breast cancer patients.Sci Rep. 2021; PubMed Scopus Google Scholar in breast an of the drop-off was developed to mutations at by a with of the of the as a for and as a for the mutation of a to a a a mutation at the by the drop-off was for mutation screening of in NRAS, and disease in C. M. S. A. M. S. drop-off digital a for mutation screening and disease in using or cell-free 2021; Full Full PubMed Scopus Google Scholar and A. M. R. F. F. S. H. J. J. J. E. digital PCR to mutations in and plasma from patients with 2018; PubMed Scopus Google Scholar to the multiplex of or by the of with the or the of with leading to in or such as the and digital PCR assays for detection of the or most common KRAS mutations in V. D. L. Le Corre D. X. I. H. Bouche O. Landi B. J.B. Laurent-Puig P. digital PCR to KRAS mutations in DNA from the plasma of colorectal cancer 2013; PubMed Scopus Google Scholar or lung A. I. S. G. I. D. J. M. S. J. of KRAS mutant lung cancer using a digital PCR PubMed Scopus Google Scholar mutations in lung F. Zhang M. H. S. Zhao Y. Zhang C. J. B. B. Zhang X. digital PCR for assessment of mutations in cell-free DNA from advanced lung cancer Rep. PubMed Scopus Google Scholar a for detection of PIK3CA mutations and in breast J. Le F. V. F. L. H. A. L. C. C. V. De T. of multiplex digital PCR assays for the detection of PIK3CA mutations in the plasma of metastatic breast cancer patients.Sci Rep. 2021; PubMed Scopus Google Scholar and digital PCR assays for mutation screening of and in M. S. D. S. R.D. digital PCR of mutations in large and PubMed Scopus Google Scholar further the of and the of cell-free DNA required for screening clinical mutations in developed a novel multiplex drop-off (MDO-dPCR) that detection of all in KRAS exons 2, 3, and NRAS exons and 3, PIK3CA and and BRAF mutation with only three reactions. the and analytical performance of as as the from a large cohort of plasma samples from CRC patients in with an of patients with CRC treatment in the of at were for was from at the of the for plasma samples from patients the of the and were from further The for using the MDO-dPCR assays were compared with by for all of the 106 CRC The were by and in a of clinical or from the was by the of in with the of and was from all of was from using and was at for and for at and were at samples were from to 4 plasma using the and were in The were using the on a and were at and the of the was assessed using the DNA on a The DNA from for and the the of the DNA a of to and the of the DNA be for or for were for of the MDO-dPCR to and of the MDO-dPCR assays were using and for gene specificity using and were assessed by and by DNA were by DNA and all were for with for the MDO-dPCR MDO-dPCR KRAS KRAS KRAS MDO-dPCR NRAS NRAS MDO-dPCR BRAF PIK3CA PIK3CA are in and are in the drop-off multiplex drop-off digital wild-type in a new are in and are in the drop-off multiplex drop-off digital wild-type were as positive with most frequent mutations on and of KRAS and of NRAS and of PIK3CA and BRAF of all of mutations identified on these of KRAS, NRAS, and BRAF, for large samples in the of in Cancer were by with the and were by of the DNA from were to by with to the and as negative were on the PCR to the PCR was from PCR for 4 MDO-dPCR with and in the multiplex PCR DNA and DNA samples were in to a of on to limit with were by on a to at least to of PCR was with for to a which was a to to of were to a PCR and on a the for of for and for and for and a positive a of from at and were in were on a The of and mutant DNA of MDO-dPCR was with the in with for to on the The were as is the of the mutation is the of DNA of the and is the of the of all mutations the with or to the of detection with were the from the and for of of detection and of the of were with for mutation of the MDO-dPCR assays by of from at with on of using the and were the for and of for mutation in to and for at the The analytical sensitivity was assessed using of in DNA at of with of and for mutation and The detection of a was positive the measured of the mutation was were by analysis with a using from CRC was with the CRC as B. S. F. M. H. Y. Y. Zhao Y. Y. B. T. and clinical of a novel for the of DNA in metastatic colorectal cancer Med. PubMed Scopus Google X. F. M. Chen X. H. H. Y. M. Y. B. T. of next-generation sequencing and for mutations in from metastatic colorectal cancer Clin 2021; PubMed Scopus Google Scholar the a of in exons 2, 3, and 4 of KRAS, exons and of NRAS, exons and of and of DNA were on an and sequencing were using The of is at an of for B. S. F. M. H. Y. Y. Zhao Y. Y. B. T. and clinical of a novel for the of DNA in metastatic colorectal cancer Med. PubMed Scopus Google Scholar were with were using the or the for and the for values were a of was using and the correlation and of were to as the positive predictive negative predictive and accuracy of MDO-dPCR assays were by multiplex assays with the of and drop-off strategies were C. A. M. S. A. A. A. M. O. Ychou M. J.Y. C. hotspot mutations by digital 2018; PubMed Scopus Google C. M. S. A. M. S. drop-off digital a for mutation screening and disease in using or cell-free 2021; Full Full PubMed Scopus Google Scholar The KRAS MDO-dPCR drop-off and a drop-off for the detection of hotspot mutations using a drop-off hotspot and a the for the detection of hotspot mutations using a drop-off and a on the a third for detection of and hotspot mutations the using a drop-off hotspot and a drop-off The NRAS MDO-dPCR drop-off for the detection of hotspot mutations using a drop-off hotspot and a the the for the detection of hotspot mutations using a drop-off hotspot and a the The MDO-dPCR drop-off for the detection of PIK3CA mutations and a for BRAF mutation of the drop-off is for detection of and hotspot mutations the using a drop-off hotspot and a drop-off the for detection of and hotspot mutations the using a drop-off hotspot and a drop-off these three MDO-dPCR assays and of all mutations of KRAS, NRAS, and BRAF identified for samples in the of in Cancer of MDO-dPCR were using of and mutant to the and for a and mutant with and with were to to on the of mutant the showed an of independent mutation detection for the drop-off of MDO-dPCR were using of and mutant to to and mutant on the with to showed that the MDO-dPCR assays could mutant and that mutation detection was The for mutation of the MDO-dPCR assays was the analysis of of with of as in the and The were and for detection of KRAS KRAS KRAS and KRAS in the KRAS MDO-dPCR and for NRAS and NRAS detection of the NRAS MDO-dPCR and and for detection of BRAF PIK3CA PIK3CA PIK3CA and PIK3CA in the MDO-dPCR sensitivity DNA were using of mutant in a of The DNA were in with from to for mutation as in the and detection of a was the measured of the mutation was the from in from 0.084% to 0.182% for the KRAS MDO-dPCR 0.084% to for the NRAS MDO-dPCR and to for the MDO-dPCR showed that the MDO-dPCR assays the of sensitivity required to The of the MDO-dPCR assays were a of from to for mutation with of mutant in a of The DNA were in for the at of and which were in The correlation from and of detection from to The for from to for at of from to for at of from to for at of from to for at of and from to for at of showed that the MDO-dPCR assays could and mutation at a of The specificity of the MDO-dPCR assays were by DNA in with mutant of mutation at a of and of from to mutation in the of high of DNA high specificity of the MDO-dPCR The MDO-dPCR assays were on from plasma samples of 106 CRC all 106 patients, the was were and cancer for of with with and with and the metastatic status of the 4 patients was The for all 106 plasma samples was plasma to patients of at least with metastatic patients a detection of to patients with disease of was correlation was plasma status and and with patients positive negative was as the from the and to the was as the from the to the and the or was patients with or was patients with or was patients with or was patients with plasma cell-free was as the from the and to the was as the from the to the and the The or was patients with in a new cell-free of mutations were four patients mutations and three patients mutations and patients mutation to the mutation by MDO-dPCR were in of plasma KRAS of KRAS of KRAS of NRAS of NRAS of BRAF of PIK3CA of PIK3CA of PIK3CA of and PIK3CA of The of the to was for all mutations to with of the mutations at and for mutation KRAS to KRAS to KRAS to NRAS to NRAS to BRAF PIK3CA to PIK3CA to PIK3CA and PIK3CA to of patients at advanced and showed for at and was patients with the of with advanced with to and in The performance of the MDO-dPCR assays was further validated by the with MDO-dPCR with with the targeted for exons 2, 3, and 4 of KRAS, exons and of NRAS, exons and of and of BRAF on the samples of 106 CRC patients The for mutation detection the was of all of the mutation 106 and results. The were the with positive only by MDO-dPCR and positive only by mutations were at with a of and a of to The was in patients at and in at advanced and the was was the status and these are to or of be by analytical sensitivity of the the mutation frequencies for patients by were with the frequencies in the of in Cancer with MDO-dPCR a sensitivity of 95.24%, a specificity of 98.53%, a positive predictive of a negative predictive of and an accuracy of 96.98% for mutation detection on plasma the of all mutations as measured by the were compared for all patients and a high correlation was with a of and of from to the and in estimates compared with and for these DNA by MDO-dPCR and the Mutation in of the of in Cancer for mutation only the mutations and by the multiplex drop-off digital PCR as in of the of in Cancer for mutation only the mutations and by the multiplex drop-off digital PCR as in of the of in Cancer for mutation only the mutations and by the multiplex drop-off digital PCR as in of the of in Cancer for mutation only the mutations and by the multiplex drop-off digital PCR as in of in multiplex drop-off digital next-generation The of the of in Cancer for mutation only the mutations and by the multiplex drop-off digital PCR as in in a new of MDO-dPCR with for DNA Mutation in 106 Colorectal Cancer values were on the mutation of for all of the mutations by the multiplex drop-off digital PCR assays for samples in the of in Cancer KRAS NRAS BRAF and PIK3CA values were on the mutation of for all of the mutations by the multiplex drop-off digital PCR assays for samples in the of in Cancer KRAS NRAS BRAF and PIK3CA values were on the mutation of for all of the mutations by the multiplex drop-off digital PCR assays for samples in the of in Cancer KRAS NRAS BRAF and PIK3CA of in multiplex drop-off digital next-generation negative predictive positive predictive values were on the mutation of for all of the mutations by the multiplex drop-off digital PCR assays for samples in the of in Cancer KRAS NRAS BRAF and PIK3CA in a new of in multiplex drop-off digital next-generation of in multiplex drop-off digital next-generation negative predictive positive predictive The growth of novel targeted therapies and treatment strategies for the of CRC to an for assays that are and for the detection and of targeted gene has to be a with sensitivity and for the assessment of using of from in these clinical at the of cancer is for disease are for early detection of disease and of to targeted therapies that could clinical in to an multiplex a of with to the clinical and three and combining amplitude-/ratio-based multiplexing and drop-off/double drop-off which allow for rapid and cost-effective screening of all KRAS exons 2, 3, and NRAS exons and PIK3CA and and BRAF with mutation coverage of and for of the four genes in developed drop-off assays in of C. A. M. S. A. A. A. M. O. Ychou M. J.Y. C. hotspot mutations by digital 2018; PubMed Scopus Google Scholar and C. M. S. A. M. S. drop-off digital a for mutation screening and disease in using or cell-free 2021; Full Full PubMed Scopus Google Scholar of J. Le F. V. F. L. H. A. L. C. C. V. De T. of multiplex digital PCR assays for the detection of PIK3CA mutations in the plasma of metastatic breast cancer patients.Sci Rep. 2021; PubMed Scopus Google Scholar and BRAF R. D. A. R. J. C. C. detection and of BRAF status in colorectal cancer using digital PCR and a novel wild-type negative Full Full PubMed Scopus Google Scholar in or amplitude-/ratio-based multiplex assays that a of three to four for V. D. L. Le Corre D. X. I. H. Bouche O. Landi B. J.B. Laurent-Puig P. digital PCR to KRAS mutations in DNA from the plasma of colorectal cancer 2013; PubMed Scopus Google A. I. S. G. I. D. J. M. S. J. of KRAS mutant lung cancer using a digital PCR PubMed Scopus Google Scholar and J. Le F. V. F. L. H. A. L. C. C. V. De T. of multiplex digital PCR assays for the detection of PIK3CA mutations in the plasma of metastatic breast cancer patients.Sci Rep. 2021; PubMed Scopus Google Scholar are the to developed a multiplex drop-off that with only three at least 69 of the most frequent mutations in all four driver genes and in The MDO-dPCR assays and a of 0.084% to 0.182% for for DNA at PCR the MDO-dPCR assays mutation hotspot at a of with a and high of the accuracy of mutation the MDO-dPCR assays an analytical sensitivity and specificity with drop-off and multiplex assays for common driver R. D. A. R. J. C. C. detection and of BRAF status in colorectal cancer using digital PCR and a novel wild-type negative Full Full PubMed Scopus Google Scholar, C. A. M. S. A. A. A. M. O. Ychou M. J.Y. C. hotspot mutations by digital 2018; PubMed Scopus Google Scholar, E. L. M. J.Y. A. S. C. I. A. R. A. L. C. digital PCR for mutations detection in 2020; PubMed Scopus Google Scholar, J. Le F. V. F. L. H. A. L. C. C. V. De T. of multiplex digital PCR assays for the detection of PIK3CA mutations in the plasma of metastatic breast cancer patients.Sci Rep. 2021; PubMed Scopus Google Scholar, C. M. S. A. M. S. drop-off digital a for mutation screening and disease in using or cell-free 2021; Full Full PubMed Scopus Google Scholar, A. M. R. F. F. S. H. J. J. J. E. digital PCR to mutations in and plasma from patients with 2018; PubMed Scopus Google Scholar, V. D. L. Le Corre D. X. I. H. Bouche O. Landi B. J.B. Laurent-Puig P. digital PCR to KRAS mutations in DNA from the plasma of colorectal cancer 2013; PubMed Scopus Google Scholar, A. I. S. G. I. D. J. M. S. J. of KRAS mutant lung cancer using a digital PCR PubMed Scopus Google Scholar The of high analytical and comprehensive detection coverage of a large of clinical mutations in only three for of the make a suitable to for mutations in of these mutations were identified in the plasma of 42.45% of CRC patients, with and of patients KRAS, NRAS, BRAF, and PIK3CA were with the in the of in Cancer for and in the B. S. F. M. H. Y. Y. Zhao Y. Y. B. T. and clinical of a novel for the of DNA in metastatic colorectal cancer Med. PubMed Scopus Google Scholar, X. F. M. Chen X. H. H. Y. M. Y. B. T. of next-generation sequencing and for mutations in from metastatic colorectal cancer Clin 2021; PubMed Scopus Google Scholar, F. Cunningham D. De D. E. R. A. J. B. S. J. D. G. J. I. of KRAS, NRAS, BRAF, PIK3CA and mutations in a large of advanced cancer J Cancer. 2020; PubMed Scopus Google Scholar the detection with with and of patients positive at and sensitivity of MDO-dPCR assays for early CRC of by with using targeted in CRC patients, showed high and a strong correlation of measured The of as with PCR is the for to the mutation the hotspot all mutations in exons 2, 3, and 4 of KRAS and A. Bachet J.B. Boige V. Cayre A. Le Corre D. Buc E. Ychou M. Bouche O. Landi B. Louvet C. Andre T. Bibeau F. Diebold M.D. Rougier P. Ducreux M. Tomasic G. Emile J.F. Penault-Llorca F. Laurent-Puig P. KRAS mutations as an independent prognostic factor in patients with advanced colorectal cancer treated with cetuximab.J Clin Oncol. 2008; 26: 374-379Crossref

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