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.
Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from ...paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA.
Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues.
One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants.
The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples.
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.
Targeted enrichment of specific loci of the human genome is a promising approach to enable sequencing-based studies of genetic variation in large populations. Here we describe an enrichment approach ...based on microdroplet PCR, which enables 1.5 million amplifications in parallel. We sequenced six samples enriched by microdroplet or traditional singleplex PCR using primers targeting 435 exons of 47 genes. Both methods generated similarly high-quality data: 84% of the uniquely mapping reads fell within the targeted sequences; coverage was uniform across approximately 90% of targeted bases; sequence variants were called with >99% accuracy; and reproducibility between samples was high (r(2) = 0.9). We scaled the microdroplet PCR to 3,976 amplicons totaling 1.49 Mb of sequence, sequenced the resulting sample with both Illumina GAII and Roche 454, and obtained data with equally high specificity and sensitivity. Our results demonstrate that microdroplet technology is well suited for processing DNA for massively parallel enrichment of specific subsets of the human genome for targeted sequencing.
Ultra-deep targeted sequencing (UDT-Seq) can identify subclonal somatic mutations in tumor samples. Early assays' limited breadth and depth restrict their clinical utility. Here, we target 71 kb of ...mutational hotspots in 42 cancer genes. We present novel methods enhancing both laboratory workflow and mutation detection. We evaluate UDT-Seq true sensitivity and specificity (> 94% and > 99%, respectively) for low prevalence mutations in a mixing experiment and demonstrate its utility using six tumor samples. With an improved performance when run on the Illumina Miseq, the UDT-Seq assay is well suited for clinical applications to guide therapy and study clonal selection in heterogeneous samples.
Cytosine methylation provides an epigenetic level of cellular plasticity that is important for development, differentiation and cancerogenesis. We adopted microdroplet PCR to bisulfite treated target ...DNA in combination with second generation sequencing to simultaneously assess DNA sequence and methylation. We show measurement of methylation status in a wide range of target sequences (total 34 kb) with an average coverage of 95% (median 100%) and good correlation to the opposite strand (rho = 0.96) and to pyrosequencing (rho = 0.87). Data from lymphoma and colorectal cancer samples for SNRPN (imprinted gene), FGF6 (demethylated in the cancer samples) and HS3ST2 (methylated in the cancer samples) serve as a proof of principle showing the integration of SNP data and phased DNA-methylation information into "hepitypes" and thus the analysis of DNA methylation phylogeny in the somatic evolution of cancer.
Abstract
A new method for enrichment of targeted genomic regions for next generation sequencing (NGS) is the use of single-molecule formatted PCR amplification, with the sample's target DNA ...partitioned into uniform picoliter volume droplets such that there are either zero or one target molecule in each droplet. All the droplets contain all of the primers and PCR reagents, ensuring that every target molecule from the sample is amplified, minimizing the amount of input required. Endpoint PCR using this single molecule format results in highly uniform yields that facilitate efficient use of the sequencing platform. In addition, the primers contain ‘Illumina tails’ that enable easy sample indexing and a workflow for loading directly onto a MiSeq without additional library preparation.
Here we detail the use of this method with the ThunderBolts Myeloid Cancer Panel, which targets ∼550 commonly mutated regions in 49 genes implicated in the causation, prognosis, and recurrence of myeloid disorders. The panel targets include an expert-curated set of 16 full genes and 33 mutational hotspot regions, including challenging targets such as CEBPA and NOTCH1. We present results showing sample inputs as low as 10 nanograms (no pre-amplification used) provide sequencing metrics with 100% coverage at >100x depth (>95% at >500x depth) with a normalized mean read depth of 2500x and high uniformity (>90% of amplicons within 0.2x of the mean), enabling detection of low minor allele frequencies (<5%). The workflow is easy and rapid, with <2 hours hands-on time and sample-to-results in <48 hours. In addition, we demonstrate similar performance metrics when adding additional primer pairs (ThunderBolts PLUS), ensuring continued flexibility for researchers to use the core panel plus additional key targets as they are identified and validated.
In summary, the ThunderBolts Myeloid Cancer Panel shows excellent sequencing coverage, high read depth and uniformity, and detection of low frequency mutations implicated in myeloid cancers. Starting from low input DNA samples, the workflow is easy and fast, enabling low cost targeted Illumina NGS sequencing with an expert curated panel that provides flexibility for expansion.
Citation Format: Michael L. Samuels, Steve K. Kotsopoulos, Frances Long, Holly Gettler, Omo Clement, Jeff Olson. Targeted sequencing of myeloid cancers using single-molecule enrichment in picodroplets. 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 4894. doi:10.1158/1538-7445.AM2015-4894
Abstract
Alongside conventional chemotherapies, targeted treatments have significantly improved the survival of patients with metastatic colorectal cancer (mCRC) from 6 months with best supportive ...care treatment, to 24 months with a combination of conventional and targeted chemotherapies. However, patients with KRAS mutated tumors have little or no benefit from these anti-EGFR antibodies based therapies (Panitumumab, Cetuximab) as single agents or combined with chemotherapy. These findings led to an amendment of the Marketing Authorization of these drugs that are now restricted to patients with KRAS wild-type tumor. However, the response of these patients ranged from 40 to 60%. Recent works have demonstrated that secondary resistance to such therapies was associated with emergence of KRAS mutated subclones in KRAS wild-type patients at the diagnosis time. To understand the importance of KRAS mutant subclones at the diagnosis time and to evaluate the consequence of their presence in terms of management of mCRC patients, a highly sensitive and quantitative procedure is required. Droplet-based digital PCR has recently emerged as a highly sensitive and quantitative approach for rare sequence detection.
A retrospective study was set up where tumor samples from 177 mCRC patients were analyzed using a multiplex highly sensitive droplet-based procedures and ultra-deep sequencing using a new droplet-based target enrichment. Results are compared with data obtained by conventional qPCR. Sixty-two patients were classified as responders according to RECIST criteria. All tumors (41) detected as positive using conventional procedures were also positive with our procedure. Among the samples detected as negative with conventional procedures, 23 presented a KRAS or a BRAF mutation only detected by droplet-based procedures and 6 presented additional subclones. For 20 patients, additional biopsy samples were available and presented comparable results. 167 samples were also submitted to deep sequencing and, for alleles detected at fractions superior to 1%, the mutational status of the sample were in agreement for 94% of the samples (157/167) and we observed a correlation with the observed fraction of mutant alleles with the two procedures (R2= 0.7).
We observed an inverse correlation between the proportion of mutated DNA and the frequency of anti-EGFR responses (P< 0.0001). The mean percentage of mutated DNA was 0.45% and 12.7% for responders and non-responders respectively. Progression Free Survival of patients was significantly different depending on the percentage of KRAS mutated allele within the tumor. The Progression Free Survival of patients with tumor presenting less than 5% of mutated KRAS was comparable to the one of non-mutated patients. HRs were of 1.07 (CI95% 0.6-2, NS), 2.9 (CI95% 1.7-4.9, P<0.001), 3.6 (CI95% 2.2-5.8, P<0.001) for patients with tumor containing less than 5%, between 5 and 25% or more than 25% of mutated DNA respectively.
Citation Format: Valerie Taly, Pierre Laurent-puig, Deniz Pekin, Corinne Normand, Steve K. Kotsopoulos, Philippe Nizard, Jeff Olson, Preethi Srinivasan, Delphine Le Corre, Xinyu Li, Qun Zhong, Darren R. Link, Olivier Bouché, Jean-François Emile, Bruno Landi, Valérie Boige, Brian J. Hutchison. Clinical significance of low frequency KRAS and BRAF subclones for advanced colon cancer management. abstract. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2820. doi:10.1158/1538-7445.AM2014-2820
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 for whom the 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 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.CONCLUSIONS: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.
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