Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR

Abstract BACKGROUND Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. METHODS We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. RESULTS The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. CONCLUSIONS This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy.

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Oncogenic alterations detected by droplet digital PCR in patients with metastatic colorectal cancer resistant to cetuximab.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.4_suppl.575 ◽

2019 ◽

Vol 37 (4_suppl) ◽

pp. 575-575

Author(s):

Rujiao Liu ◽

Zhi-Yu Chen ◽

Weijian Guo ◽

Xiaodong Zhu ◽

Mingzhu Huang ◽

...

Keyword(s):

Colorectal Cancer ◽

Metastatic Colorectal Cancer ◽

Acquired Resistance ◽

Standard Of Care ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Egfr Mutations ◽

Exon 2 ◽

First Line Therapy ◽

Line Setting

575 Background: Anti-EGFR therapy is the standard of care for metastatic colorectal cancer (mCRC) patients with RAS and BRAF genes wild type (wt). Secondary alterations of several genes have been identified as possibly resistant mechanisms to EGFR blockade, these including mutations of KRAS, NRAS, BRAF, MEK and EGFR ectodomain, as well as amplifications of HER2 and c-MET. In this study, we investigated alterations of these targeted genes for mCRC patients with acquired resistance to cetuximab treatment by using non-invasive droplet digital PCR (ddPCR) method within circulating DNA. Methods: We enrolled 38 RAS and BRAF wt patients, who progressed after failure of cetuximab contained regimens between Jul 2015 and Jan 2018. Plasma samples from all 38 patients were collected and detected for seven candidates (mutation of KRAS, NRAS, EGFR, BRAF, MEK, amplification of HER2, c-MET) by using ddPCR at the time of baseline, each evaluation by interval of 2 months and documented progression. All clinical parameters were collected simultaneously. Results: A total of 23 secondary alterations were found in 17 (17/38,44.7%) cetuximab resistant patients. The targeted gene alterations were detected as follows: 9 (9/23,39%) RAS mutations, 5 (22%) HER2 amplifications, 5 (22%) EGFR mutations, 2 (9%) c-MET amplifications, 1 (4%) BRAF and 1 MEK mutation. Among 17 patients, 6 patients had multiple alterations, including 2 patients with KRAS+EGFR mutations, 2 patients with HER2+c-MET co-amplifications and 2 patients with KRAS gene exon 2 and 3 multiple mutations. Primary sites were 15 left sides and 2 right sides, descending colon (12 cases) was the most common origins. Eleven patients were synchronous disease. Twelve patients received cetuximab as first line therapy, whereas 5 patients in the ≥ 2nd line setting. All of these 17 patients, plasma levels of oncogenic alterations detected by ddPCR were showed dynamic changing and good agreement with tumor responding status. Conclusions: Oncogenic alterations detected by ddPCR were found in almost half mCRC patients resistant to cetuximab. Dynamic changes of specific alteration may facilitate making decisions for selection of anti-EGFR mAb during the treatment course.

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Quantification of circulating free and circulating tumor DNA in pretreated EGFR mutant NSCLC to inform patient outcomes.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.9080 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. 9080-9080

Author(s):

Alexandra Pender ◽

Curtis Hughesman ◽

Elaine Law ◽

Amadea Kristanti ◽

Kelly McNeil ◽

...

Keyword(s):

Patient Outcome ◽

Limit Of Detection ◽

Resistance Mutation ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Egfr Mutations ◽

Mutational Status ◽

Egfr Mutant ◽

Egfr T790m

9080 Background: EGFR T790M testing is standard of care for EGFR mutant (EGFRm) NSCLC progressing on 1st/2nd generation TKIs to select patients for osimertinib. Circulating free DNA (cfDNA) levels are measured prior to circulating tumour DNA (ctDNA) testing using droplet digital PCR (ddPCR) to measure activating/resistant EGFR mutations. We reviewed cfDNA levels and ctDNA mutational status to determine the influence on patient outcome. Methods: Following extraction of cfDNA from plasma using the QIAamp Circulating Nucleic Acid Kit, cfDNA levels are measured with a Qubit 2.0 Fluorometer. Custom ddPCR assays were used to test for the appropriate EGFR activating mutation and the EGFR T790M resistance mutation using the Bio-Rad QX200 system. The custom designed ddPCR assays have a limit of detection of < 0.1% variant allele fraction. All patients undergoing ctDNA testing from February-December 2018 were identified. Baseline characteristics and follow up data were collected retrospectively. OS was calculated from date of metastatic diagnosis to death/last follow-up. Results: 142 patients with EGFR mutant adenocarcinoma had EGFR ctDNA testing: results 52% indeterminant, 32% T790M, 16% activating EGFRm only. At the time of testing: median age 66, 64% female, 57% never smokers 53% Asian; systemic treatment (tx) 62% first line only, 25% two lines and 13% ≥ three lines. First TKI therapy: 32% afatanib, 66% gefitinib, 2% erlotinib. Median cfDNA concentration was 5.65 ng/ml (range 0.50-217.72). The 5 yr OS was 72% below cfDNA median and 25% above the median. Tx after ctDNA testing for below and above cfDNA median: 52 vs 33% original TKI, 34 vs 55% osimertinib, 14 vs 12% other systemic tx. Multivariate analysis shows that even accounting for age, sex and ctDNA mutation result, cfDNA concentration remains an independent predictor of outcome (HR 2.36, 95% CI 1.08-5.18, p = 0.032). Conclusions: cfDNA concentration can predict patient outcome in patients with EGFR mutant NSCLC progressing on TKI regardless of ctDNA testing results. Clinicians may consider switching to chemotherapy for patients with high cfDNA and without detectable EGFR T790M ctDNA to avoid missing the window for therapy instead of awaiting repeat EGFR T790M testing

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A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency

Genome Biology ◽

10.1186/s13059-021-02316-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Wendell Jones ◽

Binsheng Gong ◽

Natalia Novoradovskaya ◽

Dan Li ◽

Rebecca Kusko ◽

...

Keyword(s):

Quality Control ◽

Allele Frequency ◽

Liquid Biopsy ◽

Limit Of Detection ◽

Reference Sample ◽

Digital Pcr ◽

Detection Sensitivity ◽

Analytical Accuracy ◽

And Performance ◽

Reference Samples

Abstract Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

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Liquid biopsy in peritoneal fluid and plasma as a prognostic factor in advanced colorectal and appendiceal tumors after complete cytoreduction and hyperthermic intraperitoneal chemotherapy

Therapeutic Advances in Medical Oncology ◽

10.1177/1758835920981351 ◽

2020 ◽

Vol 12 ◽

pp. 175883592098135

Author(s):

Irene López-Rojo ◽

Susana Olmedillas-López ◽

Pedro Villarejo Campos ◽

Víctor Domínguez Prieto ◽

Javier Barambio Buendía ◽

...

Keyword(s):

Prognostic Factor ◽

Intraperitoneal Chemotherapy ◽

Liquid Biopsy ◽

Peritoneal Fluid ◽

Hyperthermic Intraperitoneal Chemotherapy ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Before And After ◽

Colorectal Origin ◽

Systemic Relapse

Background: Positive cytology has been identified as an independent negative prognostic factor in patients with peritoneal metastases (PM) of colorectal origin. Liquid biopsy in plasma may detect increasing levels of circulating tumor DNA (ctDNA) and could help predict systemic relapse in patients with colorectal cancer, but little is known about the role of liquid biopsy in peritoneal fluid. The aim of this study was to evaluate the prognostic value of peritoneal fluid and plasma liquid biopsy in patients undergoing complete cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CC-HIPEC). Methods: A longitudinal prospective study was designed in patients with KRAS-mutated colorectal or appendiceal primary tumor, including PM of colorectal origin, pseudomyxoma peritonei and patients at high risk of developing PM (selected for second-look surgery). Eleven patients were recruited according to inclusion and exclusion criteria. ctDNA from plasma and peritoneal fluid before and after HIPEC was studied by droplet digital PCR looking for KRAS mutation. A close follow-up was scheduled (mean of 28.5 months) to monitor for systemic and peritoneal recurrences. Results: All patients with positive plasma postHIPEC had systemic relapse and four patients died as a result, while those with negative plasma postHIPEC did not relapse. Patients with negative peritoneal ctDNA after CC-HIPEC did not present peritoneal relapse. Of six patients with positive peritoneal ctDNA postHIPEC, two presented peritoneal recurrence and four systemic relapses. Conclusions: Treatment with CC-HIPEC does not always neutralize ctDNA in peritoneal fluid, and its persistence after treatment may predict adverse outcome. Despite being a proof of concept, an adequate correlation between liquid biopsy in plasma and peritoneal fluid with both systemic and peritoneal relapse has been observed.

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Diagnostic accuracy of droplet digital PCR for detection of EGFR T790M mutation in circulating tumor DNA

Cancer Management and Research ◽

10.2147/cmar.s161382 ◽

2018 ◽

Vol Volume 10 ◽

pp. 1209-1218 ◽

Cited By ~ 16

Author(s):

Rui Zhang ◽

Bojiang Chen ◽

Xiang Tong ◽

Ye Wang ◽

Chengdi Wang ◽

...

Keyword(s):

Diagnostic Accuracy ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Droplet Digital Pcr ◽

T790m Mutation ◽

Tumor Dna ◽

Egfr T790m

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Development and Clinical Validation of a Droplet Digital PCR Method for Detection of Acinetobacter baumannii and Klebsiella pneumonia in Patients with Suspected Bloodstream Infections

10.22541/au.162337143.35366523/v1 ◽

2021 ◽

Author(s):

Yang Zheng ◽

Jun Jin ◽

Ziqiang Shao ◽

Jingquan Liu ◽

Run Zhang ◽

...

Keyword(s):

Blood Culture ◽

Acinetobacter Baumannii ◽

Limit Of Detection ◽

Early Stage ◽

Bloodstream Infections ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Klebsiella Pneumonia ◽

Clinical Validation ◽

Blood Samples

The relatively long turnaround time and low sensitivity of traditional blood culture may delay the effective antibiotic therapy in patients with bloodstream infection (BSI). To reduce the morbidity and mortality of BSI, a rapid and sensitive pathogen detection method is urgently required. Acinetobacter baumannii and Klebsiella pneumonia are two major microorganisms responsible for BSI. Here we reported a novel droplet digital PCR (ddPCR) method that can detect A. baumannii and K. pneumonia in whole blood samples within 4 h, with a specificity of 100% for each strain and limit of detection at 0.93 copies/microliter for A. baumannii and 0.27 copies/microliter for K. pneumonia. Clinical validation in 170 patients with suspected BSIs showed that, compared with blood culture that reported 4 (2.4%) A. baumannii cases and 7 (4.1%) K. pneumonia cases, ddPCR detected 23 (13.5%) A. baumannii cases, 26 (15.3%) K. pneumonia cases, and 4 (2.4%) dual infection cases, including the 11 positive patients reported by blood culture. In addition, the positive patients reported by ddPCR alone (n = 42) had significantly lower serum concentrations of procalcitonin and lactate, SOFA and APACHE II scores, and 28-day mortality than those reported by both blood culture and ddPCR (n = 11), suggesting that patients with less severe manifestations can potentially benefit from the guidance of ddPCR results. In conclusion, our study suggests that ddPCR represents a sensitive and rapid method to identify causal pathogens in blood samples and to guide the treatment decisions in the early stage of BSI.

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Abstract A66: Analytical validation of 7 droplet digital PCR assays detecting TERT, BRAF, and NRAS hotspot mutations in plasma-derived circulating tumor DNA (ctDNA)

10.1158/1557-3265.liqbiop20-a66 ◽

2020 ◽

Author(s):

Mahrukh M. Syeda ◽

Broderick Corless ◽

Iman Osman ◽

Cindy Spittle ◽

George Karlin-Neumann ◽

...

Keyword(s):

Digital Pcr ◽

Circulating Tumor Dna ◽

Droplet Digital Pcr ◽

Analytical Validation ◽

Hotspot Mutations ◽

Pcr Assays ◽

Tumor Dna

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Plasma next generation sequencing and droplet digital PCR‐based detection of epidermal growth factor receptor (EGFR) mutations in patients with advanced lung cancer treated with subsequent‐line osimertinib

Thoracic Cancer ◽

10.1111/1759-7714.13154 ◽

2019 ◽

Vol 10 (10) ◽

pp. 1879-1884 ◽

Cited By ~ 7

Author(s):

Pei N. Ding ◽

Therese Becker ◽

Victoria Bray ◽

Wei Chua ◽

Yafeng Ma ◽

...

Keyword(s):

Lung Cancer ◽

Epidermal Growth Factor Receptor ◽

Next Generation Sequencing ◽

Growth Factor Receptor ◽

Digital Pcr ◽

Droplet Digital Pcr ◽

Egfr Mutations ◽

Advanced Lung Cancer ◽

Epidermal Growth ◽

Generation Sequencing

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Limited Sensitivity of Circulating Tumor DNA Detection by Droplet Digital PCR in Non-Metastatic Operable Gastric Cancer Patients

Cancers ◽

10.3390/cancers11030396 ◽

2019 ◽

Vol 11 (3) ◽

pp. 396 ◽

Cited By ~ 4

Author(s):

Luc Cabel ◽

Charles Decraene ◽

Ivan Bieche ◽

Jean-Yves Pierga ◽

Mostefa Bennamoun ◽

...

Keyword(s):

Gastric Cancer ◽

Gastric Adenocarcinoma ◽

Metastatic Gastric Cancer ◽

Digital Pcr ◽

Circulating Tumor Dna ◽

Allelic Frequency ◽

Droplet Digital Pcr ◽

Perioperative Chemotherapy ◽

Plasma Samples ◽

Tumor Dna

This study was designed to monitor circulating tumor DNA (ctDNA) levels during perioperative chemotherapy in patients with non-metastatic gastric adenocarcinoma. Plasma samples were prospectively collected in patients undergoing perioperative chemotherapy for non-metastatic gastric adenocarcinoma (excluding T1N0) prior to the initiation of perioperative chemotherapy, before and after surgery (NCT02220556). In each patient, mutations retrieved by targeted next-generation sequencing (NGS) on tumor samples were then tracked in circulating cell-free DNA from 4 mL of plasma by droplet digital PCR. Thirty-two patients with a diagnosis of non-metastatic gastric adenocarcinoma were included. A trackable mutation was identified in the tumor in 20 patients, seven of whom experienced relapse during follow-up. ctDNA was detectable in four patients (N = 4/19, sensitivity: 21%; 95% confidence interval CI = 8.5–43%, no baseline plasma sample was available for one patient), with a median allelic frequency (MAF) of 1.6% (range: 0.8–2.3%). No patient with available plasma samples (N = 0/18) had detectable ctDNA levels before surgery. After surgery, one of the 13 patients with available plasma samples had a detectable ctDNA level with a low allelic frequency (0.7%); this patient experienced a very short-term distant relapse only 3 months after surgery. No ctDNA was detected after surgery in the other four patients with available plasma samples who experienced a later relapse (median = 14.4, range: 9.3–26 months). ctDNA monitoring during preoperative chemotherapy and after surgery does not appear to be a useful tool in clinical practice for non-metastatic gastric cancer to predict the efficacy of chemotherapy and subsequent relapse, essentially due to the poor sensitivity of ctDNA detection.

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