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Molyneux S.D.,Ontario Cancer Institute | Waterhouse P.D.,Ontario Cancer Institute | Shelton D.,Digital biology Center | Shao Y.W.,Ontario Cancer Institute | And 14 more authors.
Nature Genetics | Year: 2014

Creating spontaneous yet genetically tractable human tumors from normal cells presents a fundamental challenge. Here we combined retroviral and transposon insertional mutagenesis to enable cancer gene discovery starting with human primary cells. We used lentiviruses to seed gain- and loss-of-function gene disruption elements, which were further deployed by Sleeping Beauty transposons throughout the genome of human bone explant mesenchymal cells. De novo tumors generated rapidly in this context were high-grade myxofibrosarcomas. Tumor insertion sites were enriched in recurrent somatic copy-number aberration regions from multiple cancer types and could be used to pinpoint new driver genes that sustain somatic alterations in patients. We identified HDLBP, which encodes the RNA-binding protein vigilin, as a candidate tumor suppressor deleted at 2q37.3 in greater than one out of ten tumors across multiple tissues of origin. Hybrid viral-transposon systems may accelerate the functional annotation of cancer genomes by enabling insertional mutagenesis screens in higher eukaryotes that are not amenable to germline transgenesis. © 2014 Nature America, Inc. All rights reserved.


Pretto D.,University of California at Davis | Maar D.,Digital Biology Center | Yrigollen C.M.,University of California at Davis | Regan J.,Digital Biology Center | Tassone F.,University of California at Davis
Clinical Chemistry | Year: 2015

Background: The diagnosis of 22q11 deletion syndrome (22q11DS) is often delayed or missed due to the wide spectrum of clinical involvement ranging from mild to severe, often life-threatening conditions. A delayed diagnosis can lead to life-long health issues that could be ameliorated with early intervention and treatment. Owing to the high impact of 22q11DS on public health, propositions have been made to include 22q11DS in newborn screening panels; however, the method of choice for detecting 22q11DS, fluorescent in situ hybridization, requires specialized equipment and is cumbersome for most laboratories to implement as part of their routine screening. We sought to develop a new genetic screen for 22q11DS that is rapid, cost-effective, and easily used by laboratories currently performing newborn screening.Methods: We evaluated the accuracy of multiplex droplet digital PCR (ddPCR) in the detection of copy number of 22q11DS by screening samples from 26 patients with 22q11DS blindly intermixed with 1096 blood spot cards from the general population (total n = 1122).Results: Multiplex ddPCR correctly identified all 22q11DS samples and distinguished between 1.5-and 3-Mb deletions, suggesting the approach is sensitive and specific for the detection of 22q11DS.Conclusions: These data demonstrate the utility of multiplex ddPCR for large-scale population-based studies that screen for 22q11DS. The use of samples from blood spot cards suggests that this approach has promise for newborn screening of 22q11DS, and potentially for other microdeletion syndromes, for which early detection can positively impact clinical outcome for those affected. © 2014 American Association for Clinical Chemistry.


Beaver J.A.,Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins | Jelovac D.,Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins | Balukrishna S.,Christian Medical College Vellore | Cochran R.L.,Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins | And 25 more authors.
Clinical Cancer Research | Year: 2014

Purpose: Detecting circulating plasma tumor DNA (ptDNA) in patients with early-stage cancer has the potential to change how oncologists recommend systemic therapies for solid tumors after surgery. Droplet digital polymerase chain reaction (ddPCR) is a novel sensitive and specific platform for mutation detection. Experimental Design: In this prospective study, primary breast tumors and matched pre-and postsurgery blood samples were collected from patients with early-stage breast cancer (n=29). Tumors (n=30) were analyzed by Sanger sequencing for common PIK3CA mutations, and DNA from these tumors and matched plasma were then analyzed for PIK3CA mutations using ddPCR. Results: Sequencing of tumors identified seven PIK3CA exon 20 mutations (H1047R) and three exon 9 mutations (E545K). Analysis of tumors by ddPCR confirmed these mutations and identified five additional mutations. Presurgery plasma samples (n=29) were then analyzed for PIK3CA mutations using ddPCR. Of the 15 PIK3CA mutations detected in tumors by ddPCR, 14 of the corresponding mutations were detected in presurgical ptDNA, whereas no mutations were found in plasma from patients with PIK3CA wild-type tumors (sensitivity 93.3%, specificity 100%). Ten patients with mutation-positive ptDNA presurgery had ddPCR analysis of postsurgery plasma, with five patients having detectable ptDNA postsurgery. Conclusions: This prospective study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. Future studies can now address whether ptDNA detected after surgery identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients. © 2014 American Association for Cancer Research.


Ludlow A.T.,Southwestern Medical Center | Robin J.D.,Southwestern Medical Center | Sayed M.,Southwestern Medical Center | Litterst C.M.,Digital Biology Center | And 3 more authors.
Nucleic Acids Research | Year: 2014

The telomere repeat amplification protocol (TRAP) for the human reverse transcriptase, telomerase, is a PCR-based assay developed two decades ago and is still used for routine determination of telomerase activity. The TRAP assay can only reproducibly detect ∼2-fold differences and is only quantitative when compared to internal standards and reference cell lines. The method generally involves laborious radioactive gel electrophoresis and is not conducive to high-throughput analyzes. Recently droplet digital PCR (ddPCR) technologies have become available that allow for absolute quantification of input deoxyribonucleic acid molecules following PCR. We describe the reproducibility and provide several examples of a droplet digital TRAP (ddTRAP) assay for telomerase activity, including quantitation of telomerase activity in single cells, telomerase activity across several common telomerase positive cancer cells lines and in human primary peripheral blood mononuclear cells following mitogen stimulation. Adaptation of the TRAP assay to digital format allows accurate and reproducible quantification of the number of telomerase-extended products (i.e. telomerase activity; 57.8 ± 7.5) in a single HeLa cell. The tools developed in this study allow changes in telomerase enzyme activity to be monitored on a single cell basis and may have utility in designing novel therapeutic approaches that target telomerase. © 2014 The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.


Taylor S.C.,Bio Rad Laboratories Inc. | Carbonneau J.,Laval University | Shelton D.N.,Digital Biology Center | Boivin G.,Laval University
Journal of Virological Methods | Year: 2015

The recent introduction of Droplet Digital PCR (ddPCR) has provided researchers with a tool that permits direct quantification of nucleic acids from a wide range of samples with increased precision and sensitivity versus RT-qPCR. The sample interdependence of RT-qPCR stemming from the measurement of Cq and δCq values is eliminated with ddPCR which provides an independent measure of the absolute nucleic acid concentration for each sample without standard curves thereby reducing inter-well and inter-plate variability. Well-characterized RNA purified from H275-wild type (WT) and H275Y-point mutated (MUT) neuraminidase of influenza A (H1N1) pandemic 2009 virus was used to demonstrate a ddPCR optimization workflow to assure robust data for downstream analysis. The ddPCR reaction mix was also tested with RT-qPCR and gave excellent reaction efficiency (between 90% and 100%) with the optimized MUT/WT duplexed assay thus enabling the direct comparison of the two platforms from the same reaction mix and thermal cycling protocol. ddPCR gave a marked improvement in sensitivity (>30-fold) for mutation abundance using a mixture of purified MUT and WT RNA and increased precision (>10 fold, p<0.05 for both inter- and intra-assay variability) versus RT-qPCR from patient samples to accurately identify residual mutant viral population during recovery. © 2015 The Authors.


Belgrader P.,Digital Biology Center | Tanner S.C.,University of Mississippi Medical Center | Regan J.F.,Digital Biology Center | Koehler R.,Digital Biology Center | And 2 more authors.
Clinical Chemistry | Year: 2013

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) testing is routinely performed by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH) analyses for all new cases of invasive breast carcinoma. IHC is easier to perform, but analysis can be subjective and variable. FISH offers better diagnostic accuracy and added confidence, particularly when it is used to supplement weak IHC signals, but it is more labor intensive and costly than IHC. We examined the performance of droplet digital PCR (ddPCR) as a more precise and less subjective alternative for quantifying HER2 DNA amplification. METHODS: Thirty-nine cases of invasive breast carcinoma containing ≥30% tumor were classified as positive or negative for HER2 by IHC, FISH, or both. DNA templates for these cases were prepared from formalin-fixed paraffin-embedded (FFPE) tissues to determine the HER2 copy number by ddPCR. ddPCR involved emulsifying hydrolysis probe-based PCR reaction mixtures containing the ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian); also known as HER2] gene and chromosome 17 centromere assays into nanoliter-sized droplets for thermal cycling and analysis. RESULTS: ddPCR distinguished, through differences in the level of HER2 amplification, the 10 HER2-positive samples from the 29 HER2-negative samples with 100% concordance to HER2 status obtained by FISH and IHC analysis. ddPCR results agreed with the FISH results for the 6 cases that were equivocal by IHC analyses, confirming 2 of these samples as positive for HER2 and the other 4 as negative. CONCLUSIONS: ddPCR can be used as a molecular-analysis tool to precisely measure copy number alterations in FFPE samples of heterogeneous breast tumor tissue.


PubMed | NYU Langone Medical Center, Digital Biology Center and Molecular MD Corporation
Type: Journal Article | Journal: Molecular oncology | Year: 2015

Melanoma lacks a clinically useful blood-based biomarker of disease activity to help guide patient management. To determine whether measurements of circulating, cell-free, tumor-associated BRAF(mutant) and NRAS(mutant) DNA (ctDNA) have a higher sensitivity than LDH to detect metastatic disease prior to treatment initiation and upon disease progression we studied patients with unresectable stage IIIC/IV metastatic melanoma receiving treatment with BRAF inhibitor therapy or immune checkpoint blockade and at least 3 plasma samples obtained during their treatment course. Levels of BRAF(mutant) and NRAS(mutant) ctDNA were determined using droplet digital PCR (ddPCR) assays. Among patients with samples available prior to treatment initiation ctDNA and LDH levels were elevated in 12/15 (80%) and 6/20 (30%) (p = 0.006) patients respectively. In patients with RECIST scores <5 cm prior to treatment initiation, ctDNA levels were elevated in 5/7 (71%) patients compared to LDH which was elevated in 1/13 (8%) patients (p = 0.007). Among all disease progression events the modified bootstrapped sensitivities for ctDNA and LDH were 82% and 40% respectively, with a median difference in sensitivity of 42% (95% confidence interval, 27%-58%; P < 0.001). In addition, ctDNA levels were elevated in 13/16 (81%) instances of non-RECIST disease progression, including 10/12 (83%) instances of new brain metastases. In comparison LDH was elevated 8/16 (50%) instances of non-RECIST disease progression, including 6/12 (50%) instances of new brain metastases. Overall, ctDNA had a higher sensitivity than LDH to detect disease progression, including non-RECIST progression events. ctDNA has the potential to be a useful biomarker for monitoring melanoma disease activity.


PubMed | Digital Biology Center, Bio Rad Laboratories Inc. and Laval University
Type: | Journal: Journal of virological methods | Year: 2015

The recent introduction of Droplet Digital PCR (ddPCR) has provided researchers with a tool that permits direct quantification of nucleic acids from a wide range of samples with increased precision and sensitivity versus RT-qPCR. The sample interdependence of RT-qPCR stemming from the measurement of Cq and Cq values is eliminated with ddPCR which provides an independent measure of the absolute nucleic acid concentration for each sample without standard curves thereby reducing inter-well and inter-plate variability. Well-characterized RNA purified from H275-wild type (WT) and H275Y-point mutated (MUT) neuraminidase of influenza A (H1N1) pandemic 2009 virus was used to demonstrate a ddPCR optimization workflow to assure robust data for downstream analysis. The ddPCR reaction mix was also tested with RT-qPCR and gave excellent reaction efficiency (between 90% and 100%) with the optimized MUT/WT duplexed assay thus enabling the direct comparison of the two platforms from the same reaction mix and thermal cycling protocol. ddPCR gave a marked improvement in sensitivity (>30-fold) for mutation abundance using a mixture of purified MUT and WT RNA and increased precision (>10 fold, p<0.05 for both inter- and intra-assay variability) versus RT-qPCR from patient samples to accurately identify residual mutant viral population during recovery.


PubMed | University of Western Ontario, University of Alberta and Digital Biology Center
Type: Journal Article | Journal: PloS one | Year: 2016

The rapid adoption of gene editing tools such as CRISPRs and TALENs for research and eventually therapeutics necessitates assays that can rapidly detect and quantitate the desired alterations. Currently, the most commonly used assay employs mismatch nucleases T7E1 or Surveyor that recognize and cleave heteroduplexed DNA amplicons containing mismatched base-pairs. However, this assay is prone to false positives due to cancer-associated mutations and/or SNPs and requires large amounts of starting material. Here we describe a powerful alternative wherein droplet digital PCR (ddPCR) can be used to decipher homozygous from heterozygous mutations with superior levels of both precision and sensitivity. We use this assay to detect knockout inducing alterations to stem cell associated proteins, NODAL and SFRP1, generated using either TALENs or an all-in-one CRISPR/Cas plasmid that we have modified for one-step cloning and blue/white screening of transformants. Moreover, we highlight how ddPCR can be used to assess the efficiency of varying TALEN-based strategies. Collectively, this work highlights how ddPCR-based screening can be paired with CRISPR and TALEN technologies to enable sensitive, specific, and streamlined approaches to gene editing and validation.


PubMed | Childrens Hospital Oakland Research Institute, Digital Biology Center, University of California at Los Angeles and University of California at Berkeley
Type: Journal Article | Journal: Science translational medicine | Year: 2016

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34

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