Picodroplet Digital PCR Research Articles

Early Evaluation of Circulating Tumor DNA as Marker of Therapeutic Efficacy in Metastatic Colorectal Cancer Patients (PLACOL Study).

Purpose: Markers of chemotherapy efficacy in metastatic colorectal cancer (mCRC) are essential for optimization of treatment strategies. We evaluated the applicability of early changes in circulating tumor DNA (ctDNA) as a marker of therapeutic efficacy.Experimental Design: This prospective study enrolled consecutive patients with mCRC receiving a first- or second-line chemotherapy. CtDNA was assessed in plasma collected before the first (C0), second (C1) and/or third (C2) chemotherapy cycle, using picodroplet-digital PCR assays based either on detection of gene mutation (KRAS, BRAF, TP53) or hypermethylation (WIF1, NPY). CT scans were centrally assessed using RECIST v1.1 criteria. Multivariate analyses were adjusted on age, gender, ECOG performance status (PS), metastatic synchronicity, and treatment line.Results: Eighty-two patients with mCRC treated in first- (82.9%) or second- (17.1%) line chemotherapy were included. Patients with a high (>10 ng/mL) versus low (≤0.1 ng/mL) ctDNA concentration at C0 had a shorter overall survival (OS; 6.8 vs. 33.4 months: adjusted HR, 5.64; 95% CI, 2.5-12.6; P < 0.0001). By analyzing the evolution of the ctDNA concentration between C0 and C2 or C1 (C2or1), we classified the patients in two groups (named "good" or "bad ctDNA responders"). In multivariate analysis, patients belonging to the group called "good ctDNA responder" (n = 58) versus "bad ctDNA responder" (n = 15) had a better objective response rate (P < 0.001), and a longer median progression-free survival (8.5 vs. 2.4 months: HR, 0.19; 95% CI, 0.09-0.40; P < 0.0001) and OS (27.1 vs. 11.2 months: HR, 0.25; 95% CI, 0.11-0.57; P < 0.001).Conclusions: This study suggests that early change in ctDNA concentration is a marker of therapeutic efficacy in patients with mCRC. Clin Cancer Res; 23(18); 5416-25. ©2017 AACR.

Multiplex Ultrasensitive Genotyping of Patients with Non-Small Cell Lung Cancer for Epidermal Growth Factor Receptor (EGFR) Mutations by Means of Picodroplet Digital PCR

Epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of effectiveness of treatment with EGFR tyrosine kinase inhibitors (TKIs). Three most common EGFR mutations (L858R, exon 19 deletion, and T790M) are known to be major selection markers for EGFR-TKIs therapy. Here, we developed a multiplex picodroplet digital PCR (ddPCR) assay to detect 3 common EGFR mutations in 1 reaction. Serial-dilution experiments with genomic DNA harboring EGFR mutations revealed linear performance, with analytical sensitivity ~0.01% for each mutation. All 33 EGFR-activating mutations detected in formalin-fixed paraffin-embedded (FFPE) tissue samples by the conventional method were also detected by 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 EGFR 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).

Predictive impact of complete molecular response in plasma: A phase II, liquid biopsy study in EGFR mutated NSCLC patients treated with afatinib (WJOG 8114LTR).

9027 Background: Liquid biopsy has been approved as an alternative method to detect clinically relevant EGFR mutations in NSCLC. However, it is unknown whether the early assessment of molecular response in plasma could be a surrogate of clinical response and/or longer efficacy. Here, we conducted a prospective, multi-institutional study of liquid biopsy in EGFRmutated NSCLC patients (WJOG 8114LTR). Methods: Chemotherapy naïve, advanced NSCLC patients with EGFR-sensitizing mutation received afatinib monotherapy (40 mg/body) until progressive disease (PD) or unacceptable toxicity. Plasma DNA was obtained from patients at baseline, weeks 2, 4, 8, 12, 24, 48, and at PD. Three types of clinically relevant EGFRmutations (exon 19 deletion, exon 20 T790M and exon 21 L858R) will be analyzed using plasma DNA with multiplexed, pico-droplet digital PCR assay (RainDrop system, RainDance Technologies, Billerica, MA). Complete molecular response (CMR) was defined as mutant allele event/frequency of exon 19 deletion or exon 21 L858R below the cutoff for the positivity by digital PCR in plasma. This study was registered at UMIN (ID: 000015847). Results: Fifty-seven patients were registered in the study. Efficacy of afatinib was comparable to previous reports (overall response rate: 78.6%, and median progression-free survival (mPFS): 14.2 months). At baseline, 62.5% of patients were positive for EGFR mutation in plasma. Plasma positive patients had slightly shorter PFS than plasma negative patients, but not significant (p = 0.24, log-rank). Among those who were positive at baseline, CMR was achieved in 60.6% of patients at two weeks, and 87.5% at four weeks, respectively. Patients with CMR at two weeks had significantly longer PFS than those without one (13.6 versus 7.5 months, p = 0.0001). Patients with CMR at four weeks also demonstrated longer PFS than those without one (13.6 versus 5.1 months, p &lt; 0.0001). Among patients who achieved CMR by week four, time to achieve CMR did not affect PFS ( p= 0.59). Conclusions: This is the first report on the predictive impact of early CMR in plasma for longer therapeutic efficacy of afatinib in EGFR mutated NSCLC patients. Clinical trial information: 000015847.

Clinical Application of Picodroplet Digital PCR Technology for Rapid Detection of EGFR T790M in Next-Generation Sequencing Libraries and DNA from Limited Tumor Samples

Although next-generation sequencing (NGS) is a robust technology for comprehensive assessment of EGFR-mutant lung adenocarcinomas with acquired resistance to tyrosine kinase inhibitors, it may not provide sufficiently rapid and sensitive detection of the EGFR T790M mutation, the most clinically relevant resistance biomarker. Here, we describe a digital PCR (dPCR) assay for rapid T790M detection on aliquots of NGS libraries prepared for comprehensive profiling, fully maximizing broad genomic analysis on limited samples. Tumor DNAs from patients with EGFR-mutant lung adenocarcinomas and acquired resistance to epidermal growth factor receptor inhibitors were prepared for Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets sequencing, a hybrid capture-based assay interrogating 410 cancer-related genes. Precapture library aliquots were used for rapid EGFR T790M testing by dPCR, and results were compared with NGS and locked nucleic acid-PCR Sanger sequencing (reference high sensitivity method). Seventy resistance samples showed 99% concordance with the reference high sensitivity method in accuracy studies. Input as low as 2.5 ng provided a sensitivity of 1% and improved further with increasing DNA input. dPCR on libraries required less DNA and showed better performance than direct genomic DNA. dPCR on NGS libraries is a robust and rapid approach to EGFR T790M testing, allowing most economical utilization of limited material for comprehensive assessment. The same assay can also be performed directly on any limited DNA source and cell-free DNA.

Abstract 1376: Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients

Abstract BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as a reliable predictor of response to EGFR tyrosine kinase inhibitor (TKI) treatment. Two common EGFR mutations (L858R, and exon19 deletion) and EGFR T790M mutation are clinically important for decision-making in clinical practice. Picodroplet digital PCR (ddPCR) has recently emerged as an accurate and ultra-sensitive method for tumor genotyping and have a great advantage over conventional mutation detection methods. We have previously reported far more frequent incidence of pretreatment EGFR T790M mutation than previous reports utilizing ddPCR method. Here, we developed a multiplex ddPCR assay to detect three clinically relevant EGFR mutations in one reaction. METHODS: Positive and negative control plasmids for the EGFR mutation assay were prepared by cloning DNA fragments containing wild-type or mutant EGFR. Genomic DNAs from lung cancer cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then were used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. We then evaluated the utility of multiplex ddPCR to detect the three clinically relevant mutations in EGFR from FFPE samples of patients with non-small cell lung cancer. 45 FFPE samples were also genotyped for EGFR mutations by conventional qPCR-based methods for validation. The concordance between duplex and multiplex ddPCR assay was also evaluated. RESULTS: Serial dilutions experiments using genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. All of the 33 EGFR-activating mutations detected in FFPE samples by conventional qPCR-based method were also detected by multiplex ddPCR assay. Owing to its ultra-high sensitivity, additional T790M mutation at ultra-low allele frequency (&amp;lt;0.1%) was also detected in the same reaction. The regression analysis between duplex and multiplex assay demonstrated a correlation coefficient (R2) of 0.9986 for L858R, 0.9844 for exon19 deletion, and 0.9959 for T790M, respectively. CONCLUSIONS: Using ddPCR technology, we established multiplex and ultra-sensitive genotyping platform for three clinically relevant EGFR mutations. Results of a proof-of-principle study using clinical samples indicated that the clinical utility of multiplex ddPCR assay to screen for multiple EGFR-activating mutations concurrently with low-frequency T790M mutation. Citation Format: Yasuhiro Koh, Tomoya Kawaguchi, Masaru Watanabe, Shun-ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Seiichi Kakegawa, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Kazuhiro Sakamoto, Akihide Matsumura. Ultra-sensitive picodroplet digital PCR assay for multiplex genotyping of mutations in epidermal growth factor receptor (EGFR) in non-small cell lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1376.

Abstract CT078: A prospective biomarker study using digital PCR method in EGFR-mutated, advanced lung adenocarcinoma patients treated with afatinib (West Japan Oncology Group 8114LTR)

Abstract Background: In epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer (NSCLC), EGFR-tyrosine kinase inhibitor (TKI) demonstrates durable response, but its efficacy duration varies among patients. Early remission of mutated cells was reported as a surrogate of longer efficacy in chronic myeloid leukemia. Similar approach can be done among advanced solid malignancies using highly sensitive method, like digital PCR. In addition, early detection of resistant mutation (EGFR exon20 T790M) is important in EGFR mutated NSCLC, because third generation EGFR-TKIs which specifically bind to EGFR with this mutation have been developed. Thus, monitoring genetic changes during treatment becomes important. Because it is difficult to obtain tumor tissue repeatedly from advanced NSCLC patients, liquid biopsy can be an alternative method to detect cancer less invasively. Although some reports have mentioned the utility of liquid biopsy in EGFR mutated NSCLC, most were single-institutional, retrospective studies. Methods: West Japan Oncology Group (WJOG) 8114LTR is a multi-institutional, prospective study using highly-sensitive liquid biopsy in advanced NSCLC patients. Chemotherapy naïve, advanced NSCLC patients with EGFR sensitive mutation will receive afatinib monotherapy (40 mg/body) until progressive disease (PD) or unacceptable toxicity. Serum DNA will be obtained from patients at 0, 2, 4, 8, 12, 24, 48 weeks, and at PD. Three types of EGFR mutation (exon 19 deletion, exon 20 T790M and exon 21 L858R) will be analyzed using serum DNA with pico-droplet digital PCR (RainDrop® system, RainDance Technologies, Billerica, MA). Primary endpoint of this study is the concordance of EGFR mutation status between tissue and plasma before treatment. Secondary endpoints are overall response rate, progression-free survival and safety. Exploratory analyses include: 1. concordance of EGFR mutation status at PD between tissue and plasma, 2. longitudinal quantitative monitoring of EGFR mutation status in plasma, and its correlation with clinical efficacy, 3. comprehensive molecular analyses before and after afatinib treatment using next generation sequencing. Total number of patients will be fifty-five. This study is the first study to explore the utility of liquid biopsy in EGFR mutated NSCLC treated with afatinib. Using our highly sensitive method, we can explore whether early remission of EGFR mutational load in plasma is significant, or not. In addition, this study will clarify the molecular mechanism of disease progression on first-line afatinib in EGFR mutated NSCLC. This study was registered at UMIN (ID: 000015847). Patients’ accrual: This study was started in Feb, 2015, and has already completed final patient's accrual in Nov 2015. Now, we are collecting samples from patients on treatment, and will launch measurement and analyses in early 2016. Citation Format: Hiroaki Akamatsu, Yasuhiro Koh, Satoshi Morita, Nobuyuki Yamamoto, Kazuhiko Nakagawa. A prospective biomarker study using digital PCR method in EGFR-mutated, advanced lung adenocarcinoma patients treated with afatinib (West Japan Oncology Group 8114LTR). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT078.

Evaluation of picodroplet digital PCR for quantitative detection of HCV RNA

Evaluation of picodroplet digital PCR for quantitative detection of HCV RNA

Abstract 4908: Establishment of multiplexed ultra-sensitive detection of epidermal growth factor receptor mutations using picodroplet digital PCR

Abstract BACKGROUND: The epidermal growth factor receptor (EGFR) mutations have been used as the strongest predictor of EGFR tyrosine kinase inhibitor (TKI) treatment. Three most common EGFR mutations (L858R, exon19 deletion, and T790M) are known to be major selection marker for EGFR-TKI therapy. Noninvasive techniques for tumor genotyping may be needed to fully realize the potential of genotype-directed cancer care as a “Liquid biopsy”. The low abundance of mutants and limited amount and quality of available cell-free DNA (cfDNA) in plasma samples render it difficult to reliably detect multiple mutations with current platforms and methods. Here, we develop a multiplex dPCR assay to detect three common EGFR mutations in one reaction using picodroplet digital PCR (ddPCR) technology, which enable us to provide 3-fold throughput and save the cost as well as DNA samples. METHODS: Positive and negative control plasmids for the EGFR assay were prepared by cloning DNA fragments containing wild-type or the EGFR mutations. The appropriate concentration of plasmid DNA was determined empirically to yield a mixture in which the number of copies of mutant DNA was ca. 0.01-1.00% of the number of wild-type EGFR fragments. Genomic DNA from the lung tumor cell lines H1975, PC-9/ZD, A549 and wild-type human genomic DNA were digested with CviQ1, and then used to quantitatively assess each EGFR mutant sequence in the multiplex assay panels. RESULTS: Serial dilutions experiments using plasmid as well as genomic DNA harboring EGFR mutations revealed a linear performance with an analytical sensitivity of approximately 0.01% for each mutation. The regression analysis between duplex and multiplex assay demonstrated a correlation coefficient (R2) of 0.9983for L858R, 0.9911 for exon19 deletion, and 0.9781 for T790M. The limit of blank (LOB) was defined by the frequency of positive droplets measured in 400 ng of wild-type DNA control samples. The number of false events of mutant droplets detected per analysis is: 8 for L858R, 6 for Exon 19 deletion, 6 for T790M from human normal gDNA, and 9 for L858R, 4 for Exon 19 deletion, 2 for T790M from A549 gDNA. Thus, the LOB was determined as a 10 events/assay. CONCLUSIONS: Using ddPCR, we established multiplex and ultra-sensitive genotyping platform for three common EGFR mutations. Results of a proof-of-principle study using preclinical model indicated that the clinical utility of multiplex ddPCR to detect for multiple EGFR mutations. Further evaluation using clinical samples is warranted. Citation Format: Hiroaki Akamatsu, Yasuhiro Koh, Masaru Watanabe, Takashi Kikuchi, Masanori Nakanishi, Kazuto Matsunaga, Nobuyuki Yamamoto. Establishment of multiplexed ultra-sensitive detection of epidermal growth factor receptor mutations using picodroplet digital PCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4908. doi:10.1158/1538-7445.AM2015-4908

Abstract 617: Ultrasensitive detection of the pretreatment EGFR T790M mutation in non-small cell lung cancer patients with an EGFR-activating mutation using picodroplet digital PCR

Abstract Purpose: The resistance of the epidermal growth factor receptor (EGFR) to tyrosine kinase inhibitors (TKIs) is a major concern in non-small-cell lung cancer (NSCLC) treatment. T790M mutation in EGFR accounts for nearly 50% of the acquired resistance to EGFR-TKIs. Earlier studies suggested that EGFR T790M mutation was also detected in TKI-naive NSCLCs in a small cohort and the prevalence of a de novo EGFR T790M mutation in patients with EGFR-mutant NSCLC remains unclear. Here, we use an ultra-sensitive droplet digital PCR (ddPCR) technique to address the incidence and clinical significance of pretreatment EGFR T790M mutation in a larger cohort. Experimental design: ddPCR was established as follows: EGFR wild-type or T790M mutation-containing DNA fragments were cloned into plasmids. Candidate threshold was identified using wild-type plasmid, normal human genomic DNA, and human A549 cell line DNA, which expresses wild-type EGFR. Surgically resected tumor tissues from 373 early-stage NSCLC patients with EGFR-activating mutations detected by Cycleave PCR method were then examined for the presence of pretreatment EGFR T790M mutation using ddPCR. Results: Preclinical model data revealed a linear performance for this ddPCR method (R2 = 0.998) with an analytical sensitivity of ∼0.001%. The overall incidence of the pretreatment EGFR T790M mutation was 79.9% (298/373) and the frequency ranged from 0.009% to 26.9%, while only five patients were diagnosed positive for EGFR T790M mutation by Cycleave PCR method. The EGFR T790M mutation was detected more frequently in patients with a larger tumor size (p = 0.019) and those with common EGFR-activating mutations (p = 0.022), as compared to the others. Among the 69 patients who had co-incident mutations in EGFR and p53, 61 patients (88.4%) had the EGFR T790M mutation (p = 0.096). Moreover, all 11 patients with mutations in PIK3CA and EGFR had the EGFR T790M mutation (p = 0.097). These results, while not statistically significant, suggest that tumors with the pretreatment EGFR T790M mutation may manifest greater genomic complexity. Conclusions: To our knowledge, this is the largest study thus far using an ultra-sensitive method for detecting the pretreatment EGFR T790M mutation that has demonstrated associations between the incidence of the pretreatment T790M mutation and clinicopathological as well as genetic features in early-stage NSCLC. The ultra-sensitive ddPCR assay revealed that pretreatment EGFR T790M mutation was found in the majority of NSCLC patients with EGFR-activating mutations. ddPCR should be utilized for detailed assessment of the impact of the low frequency pretreatment EGFR T790M mutation on treatment with EGFR-TKIs, allowing the possibility of developing strategies for personalized cancer therapies in NSCLC patients. Citation Format: Yasuhiro Koh, Tomoya Kawaguchi, Masaru Watanabe, Shun-ichi Isa, Masahiko Ando, Akihiro Tamiya, Akihito Kubo, Hideo Saka, Sadanori Takeo, Hirofumi Adachi, Tsutomu Tagawa, Seiichi Kakegawa, Motohiro Yamashita, Kazuhiko Kataoka, Yukito Ichinose, Yukiyasu Takeuchi, Kazuhiro Sakamoto, Akihide Matsumura. Ultrasensitive detection of the pretreatment EGFR T790M mutation in non-small cell lung cancer patients with an EGFR-activating mutation using picodroplet digital PCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 617. doi:10.1158/1538-7445.AM2015-617

TG1050, a novel immunotherapeutic for the treatment of chronic hepatitis B, can control HBsAg and provoke HBsAg seroconversion in HBV-persistent mouse models

s / Journal of Clinical Virology 69 (2015) 223–246 227 evaluated with serial dilutions (106–100 IU/mL; Roche). The quantitative correlation and lower limit of detection were evaluated by use of clinical samples. Findings: Picodroplet digital PCR showed a high degree of linearity (R2 =0.9891) and accuracy (SD≤0.31 log copies/mL; CV≤15%) across their detectable ranges. The quantitative correlation between the picodroplet digital PCR and the Roche TaqMan HCV test was high (R2 =0.9439) across their detectable ranges (106–15 IU/mL). We also found discordance in detection of low HCVRNA samples between the twomethods. In 18 clinical samples (HCV RNA 15 copies/mL, 44.4% with an HCV RNA levels < 15 copies/mL, and in 33.3% of samples the target was not detected. The discordance in low RNA samples needs to be verified using a large sample size. Interpretation: Picodroplet digital PCR could provide a useful method for HCV quantification, especially for low HCV RNA levels, but the approach needs to be further optimised. http://dx.doi.org/10.1016/j.jcv.2015.06.018

Performance of picodroplet digital PCR for quantitative detection of HCV RNA

Performance of picodroplet digital PCR for quantitative detection of HCV RNA

Clinical relevance of KRAS-mutated subclones detected with picodroplet digital PCR in advanced colorectal cancer treated with anti-EGFR therapy.

KRAS mutations are predictive of nonresponse to anti-EGFR therapies in metastatic colorectal cancer (mCRC). However, only 50% of nonmutated patients benefit from them. KRAS-mutated subclonal populations nondetectable by conventional methods have been suggested as the cause of early progression. Molecular analysis technology with high sensitivity and precision is required to test this hypothesis. From two cohorts of patients with mCRC, 136 KRAS, NRAS, and BRAF wild-type tumors with sufficient tumor material to perform highly sensitive picodroplet digital PCR (dPCR) and 41 KRAS-mutated tumors were selected. All these patients were treated by anti-EGFR therapy. dPCR was used for KRAS or BRAF mutation screening and compared with qPCR. Progression-free survival (PFS) and overall survival (OS) were analyzed according to the KRAS-mutated allele fraction. In addition to the confirmation of the 41 patients with KRAS-mutated tumors, dPCR also identified KRAS mutations in 22 samples considered as KRAS wild-type by qPCR. The fraction of KRAS-mutated allele quantified by dPCR was inversely correlated with anti-EGFR therapy response rate (P < 0.001). In a Cox model, the fraction of KRAS-mutated allele was associated with worse PFS and OS. Patients with less than 1% of mutant KRAS allele have similar PFS and OS than those with wild-type KRAS tumors. This study suggests that patients with mCRC with KRAS-mutated subclones (at least those with a KRAS-mutated subclones fraction lower or equal to 1%) had a benefit from anti-EGFR therapies.

Multiplex Picodroplet Digital PCR to Detect KRAS Mutations in Circulating DNA from the Plasma of Colorectal Cancer Patients

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. 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 for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR. 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 KRAS-mutated 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 KRAS-mutated 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. This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.