This makes great difference in terms of accurate calculation of variant frequency of the mutant allele of interest. a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for an exon 19 deletion, and 0.9959 for T790M. Using ddPCR, we designed a multiplex ultrasensitive genotyping platform for 3 common mutations. Results of this proof-of-principle study on clinical samples indicate clinical utility of multiplex ddPCR for screening for multiple EGFR mutations concurrently with an ultra-rare pretreatment mutation (T790M). mutation, Droplet digital PCR, NonCsmall cell lung cancer 1.?Introduction Targeted molecular therapy has improved the treatment of nonCsmall cell lung cancer (NSCLC). Superiority of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) to platinum-based chemotherapy in terms of progression-free survival (PFS) in EGFR-mutated lung cancers has been reported in several phase III trials as a first-line treatment (Zhou et al., 2011, Rosell et al., 2012, Mok et al., 2009, Mitsudomi et al., 2010, Maemondo et al., 2010). EGFR-TKIs (gefitinib, erlotinib, or afatinib) have been demonstrated to be effective for NSCLC patients with EGFR-activating mutations such as exon19 deletion or exon 21 L858R mutations (Lynch et al., 2004, Paez et al., 2004). Evidence shows, however, that most responders eventually develop acquired resistance to EGFR-TKIs (Kobayashi et al., 2005, Yu et al., 2013, Ohashi et al., 2013). Among these patients, a secondary missense T790M mutation is observed in nearly half of all cases resistant to EGFR-TKIs (Ohashi et al., 2013). This T790M mutation was also detected in tumors as a minor cellular clone before exposure to EGFR-TKIs and was found concurrently with other EGFR-activating mutations (Inukai et al., 2006). This pretreatment T790M mutation is present Rabbit Polyclonal to POLE1 in 1C8% of cases according to conventional DNA sequencing like Sanger sequencing (Wu et al., 2011, Sequist et al., 2008, Li et al., 2014, Fujita et al., 2012) and in 2C79% of cases according to more sensitive detection methods like Scorpion Amplification Refractory Mutation System (SARMS) technology with an EGFR-activating mutation (Su et al., SR-13668 2012, Rosell et al., 2011, Maheswaran et al., 2008, Costa et al., 2014, Yu et al., 2014). Patients with pretreatment T790M mutation detected by less sensitive methods show a lower response rate and shorter PFS (Inukai et al., 2006, Wu et al., 2011, Sequist et al., 2008). Recent studies revealed that patients with a pretreatment T790M mutation detected by a highly sensitive method also have shorter PFS (Su et al., 2012, Rosell et al., 2011, Maheswaran et al., 2008, Costa et al., 2014, Ding et al., 2014), suggesting that a low-level pretreatment T790M mutation can be used for optimizing treatment with EGFR-TKIs. Therefore, the ability of molecular analytical technologies to detect EGFR mutants at the subclone level before EGFR-TKI treatment is critically important for enabling more personalized therapies in NSCLC. Picodroplet digital PCR (ddPCR) recently emerged as a highly sensitive method for detection of gene mutations and is based on compartmentalization of DNA into picoliter-size droplets (Taly et al., 2012). Our previous report showed detection of 0.001% prevalence of the T790M mutation among tumor cells (Watanabe et al., 2015). Several examples of ddPCR application to highly sensitive detection of mutations were published recently (Pekin et al., 2011, Oxnard et al., 2014, Ono et al., 2014, Iwama et al., 2015, Sacher et al., 2016). Multiplexing of mutation detection in a single assay is desirable for genotype testing in the clinic; promising results have also been demonstrated using ddPCR (Zhong et al., 2011, Didelot et al., 2013, Taly et al., 2013, Laurent-Puig et al., 2015, Zonta SR-13668 et al., 2016). The multiplex procedure has been adapted to quantitative detection of 7 common mutations of (in codons 12 and 13) in plasma samples and primary.Assessment of the Multiplex ddPCR Assay To assess performance of our multiplex ddPCR assay, a plasmid containing a mutant sequence was added to the solution of the plasmid containing a wild-type sequence, and then the multiplex ddPCR assay was performed. this multiplex assay. Owing to the higher sensitivity, an additional mutation (T790M; including an ultra-low-level mutation, ?0.1%) was detected in the same reaction. Regression analysis of the duplex assay and multiplex assay showed a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for an exon 19 deletion, and 0.9959 for T790M. Using ddPCR, we designed a multiplex ultrasensitive genotyping platform for 3 common mutations. Results of this proof-of-principle study on clinical samples indicate clinical utility of multiplex ddPCR for screening for multiple EGFR mutations concurrently with an ultra-rare pretreatment mutation (T790M). mutation, Droplet digital PCR, SR-13668 NonCsmall cell lung cancer 1.?Introduction Targeted molecular therapy has improved the treatment of nonCsmall cell lung cancer (NSCLC). Superiority of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) to platinum-based chemotherapy in terms of progression-free survival (PFS) in EGFR-mutated lung cancers has been reported in several phase III trials as a first-line treatment (Zhou et al., 2011, Rosell et al., 2012, Mok et al., 2009, Mitsudomi et al., 2010, Maemondo et al., 2010). EGFR-TKIs (gefitinib, erlotinib, or afatinib) have been demonstrated to be effective for NSCLC patients with EGFR-activating mutations such as exon19 deletion or exon 21 L858R mutations (Lynch et al., 2004, Paez et al., 2004). Evidence shows, however, that most responders eventually develop acquired resistance to EGFR-TKIs (Kobayashi et al., 2005, Yu et al., 2013, Ohashi et al., 2013). Among these patients, a secondary missense T790M mutation is observed in nearly half of all cases resistant to EGFR-TKIs (Ohashi et al., 2013). This T790M mutation was also detected in tumors as a minor cellular clone before exposure to EGFR-TKIs and was found concurrently with other EGFR-activating mutations (Inukai et al., 2006). This pretreatment T790M mutation is present in 1C8% of cases according to conventional DNA sequencing like Sanger sequencing (Wu et al., 2011, Sequist et al., 2008, Li et al., 2014, Fujita et al., 2012) and in 2C79% of cases according to more sensitive detection methods like Scorpion Amplification Refractory Mutation System (SARMS) technology with an EGFR-activating mutation (Su et al., 2012, Rosell et al., 2011, Maheswaran et al., 2008, Costa et al., 2014, Yu et al., 2014). Patients with pretreatment T790M mutation detected by less sensitive methods show a lower response rate and shorter PFS (Inukai et al., 2006, Wu et al., 2011, Sequist et al., 2008). Recent studies revealed that patients with a pretreatment T790M mutation detected by a highly sensitive method also have shorter PFS (Su et al., 2012, Rosell et al., 2011, Maheswaran et al., 2008, Costa et al., 2014, Ding et al., 2014), suggesting that a low-level pretreatment T790M mutation can be used for optimizing treatment with EGFR-TKIs. Therefore, the ability of molecular analytical technologies to detect EGFR mutants at the subclone level before EGFR-TKI treatment is critically important for enabling more personalized therapies in NSCLC. Picodroplet digital PCR (ddPCR) recently emerged as a highly sensitive method for detection of gene mutations and is based on compartmentalization of DNA into picoliter-size droplets (Taly et al., 2012). Our previous report showed detection of 0.001% prevalence of the T790M mutation among tumor cells (Watanabe et al., 2015). Several examples of ddPCR application to highly sensitive detection of mutations were published recently (Pekin et al., 2011, Oxnard et al., 2014, Ono et al., 2014, Iwama et SR-13668 al., 2015, Sacher et al., 2016). Multiplexing of mutation detection in a single assay is desirable for genotype testing in the clinic; promising results have also been demonstrated using ddPCR (Zhong et al., 2011, Didelot et al., 2013, Taly et al., 2013, Laurent-Puig et al., 2015, Zonta et al., 2016). The multiplex procedure has been adapted to quantitative detection of 7 common mutations of (in codons 12 and 13) in plasma samples and primary tumor samples from patients with metastatic colorectal cancer (mCRC) (Taly et al., 2013, Laurent-Puig et al., 2015). Zonta et al., developed several multiplex panels for EGFR (several three- and four-plex) in reference standard DNA samples. Here, we report the advantage of our 6-plex ddPCR assay that detects 3 clinically.