Abstract 565: A single institutional experience with droplet digital polymerase chain reaction (ddPCR) liquid biopsy for therapeutic decision in avanced solid tumors

2021 ◽  
Author(s):  
Andre Marcio Murad
Keyword(s):  
Polymerase Chain Reaction ◽  
Solid Tumors ◽  
Liquid Biopsy ◽  
Therapeutic Decision ◽  
Chain Reaction ◽  
Institutional Experience ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

A single institution experience with droplet digital polymerase chain reaction (dd-PCR) liquid biopsy (LB) for therapeutic decision in advanced solid tumors.

2021 ◽  
Vol 39 (15_suppl) ◽  
pp. 3038-3038
Author(s):  
Andre Marcio Murad ◽  
Juliana Garcia Carneiro ◽  
José Claudio Casali-da-Rocha
Keyword(s):  
Polymerase Chain Reaction ◽  
Targeted Therapy ◽  
Solid Tumors ◽  
Braf V600e ◽  
Advanced Solid Tumors ◽  
Chain Reaction ◽  
Secondary Resistance ◽  
Dna And Rna ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

3038 Background: Droplet digital polymerase chain reaction (ddPCR) is a promising method for analyzing minor amounts of cell-free circulating free nucleic acid (DNA and RNA) due to its high sensitivity, low cost, and fast reading, since it dispenses bioinformatics, making it an appropriate alternative to new generation sequencing (NGS) for the detection of biomarkers to guide molecularly targeted cancer therapy. The assay covers main actionable hotspot alterations across many actionable genes: EGFR(mutations), ALK (fusion, mutations), ROS1(fusion), BRAF(mutations), KRAS (mutations), NRAS(mutations), PIK3CA (mutations), ERBB2(CNV- copy number variation), ESR1(mutations), KIT(CNV) and PDGFRA(CNV). Therefore, our genes can be used in panels, both in therapeutically applied genotyping and in detecting molecular responses as well as in secondary resistance to tumors such as NSCLC, breast, cervix. rectal, GIST, melanoma and pancreas. Methods: dd-PCR was performed using the QX200 system (BIO-RAD, Hercules). The extraction of DNA and RNA was done using the magnetic beads technique of Thermofisher's MagMAX Kit. All samples were tested in duplicate. Up to now, 108 metastatic cancer patients were tested: NSCLC - 28 (26%), breast - 22 (20%), colorectal - 22(20%), melanoma - 18(17%), pancreas - 8(7%), ovarian - 6(6%), salivary glands - 2 (4%) and GIST 2(4%). Results: Significant genomic alterations were detected in 38(35%) patients: 10 (9%) mutations in the KRAS G12V gene (all in colorectal cancer), 10 (9%) ERBB2 amplification (breast cancer), 4 (3.65%) EGFR L858R mutations (NSCLC), 4 (3.5%) EGFR del19 mutations (NSLC), 4 (3.5%) EGFR T790M mutations (NSCLC), 4 (3.5%) BRAF-V600E mutations (colon and melanoma), 2 (1.8%) ALK-EML4 fusion (NSCLC). The MAF (Mutant Allele Fraction) ranged from 0.9% to 24%. In all cases, the results were decisive for the indication or change of a targeted therapy. Median turnaround time was 36 hours and the average cost of the panels was around 500 USD (median of 4 genes per panel). Conclusions: Our results suggest that dd-PCR is a highly sensitive method and could be used for a routine laboratory detection of the important genomic variations to determine the targeted therapy in patients with varied advanced solid tumors.

A preliminary single institution experience with droplet digital polymerase chain reaction (dd-PCR) liquid biopsy (LB) for therapeutic decision in advanced solid tumors: Personal precision oncology clinic and genetic diagnostics, Brazil.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14613-e14613
Author(s):  
Andre Marcio Murad ◽  
Jose Claudio Casali Rocha
Keyword(s):  
Polymerase Chain Reaction ◽  
Targeted Therapy ◽  
Solid Tumors ◽  
Genetic Diagnostics ◽  
Egfr Mutations ◽  
Precision Oncology ◽  
Advanced Solid Tumors ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

e14613 Background: Droplet digital polymerase chain reaction (dd-PCR) is a promising method for analyzing minor amounts of cell-free circulating free nucleic acid (DNA and RNA) due to its high sensitivity, low cost, and fast reading, since it dispenses bioinformatics, making it an appropriate alternative to new generation sequencing (NGS) for the detection of biomarkers to guide molecularly targeted cancer therapy. The assay covers main actionable hotspot alterations across many actionable genes: EGFR(mutations), ALK (fusion, mutations), ROS1(fusion), BRAF(mutations), KRAS (mutations), NRAS(mutations), ERBB2(CNV), ESR1(mutations), KIT(CNV) and PDGFRA(CNV). Methods: We analyzed so far 38 patients with advanced NSCLC: 13(35%), breast: 9(24%), colorectal: 6(16%), pancreatic: 4(10%), ovary: 2(5%), salivary gland: 2(5%), melanoma 1(2.5%), and GIST: 1 (2.5%). ddPCR was performed using the QX200 system (BIO-RAD, Hercules). All samples were tested in duplicate. All assays were performed at Personal Precision Genetic Diagnostics. Belo Horizonte, MG. Brazil. Results: We detected meaningful genomic alterations in 7(18.5%) patients: 4(10.5%) KRAS G12V mutations (all in colorectal cancer), 1(2.6%) EGFR Del19 mutation, 1 EGFR L858R mutation(2.6%), both in NSCLC and 1(2.6%) ERBB2 amplification (in breast cancer). MAF (Mutant Allele Fraction) varied from 0.9% to 24%. In all cases the results were decisive for the indication or the change of a targeted therapy. Conclusions: Our preliminary results suggest that dd-PCR is a highly sensitive method and could be used for a routine laboratory detection of the important genomic variations to determine the targeted therapy in patients with varied advanced solid tumors.

A single institution experience with droplet digital polymerase chain reaction (dd-PCR) liquid biopsy (LB) for therapeutic decision in advanced solid tumors.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15562-e15562
Author(s):  
Andre Marcio Murad ◽  
José Claudio Casali-da-Rocha ◽  
Juliana Garcia Carneiro
Keyword(s):  
Polymerase Chain Reaction ◽  
Targeted Therapy ◽  
Solid Tumors ◽  
Braf V600e ◽  
Advanced Solid Tumors ◽  
Chain Reaction ◽  
Secondary Resistance ◽  
Dna And Rna ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

e15562 Background: Droplet digital polymerase chain reaction (ddPCR) is a promising method for analyzing minor amounts of cell-free circulating free nucleic acid (DNA and RNA) due to its high sensitivity, low cost, and fast reading, since it dispenses bioinformatics, making it an appropriate alternative to new generation sequencing (NGS) for the detection of biomarkers to guide molecularly targeted cancer therapy. The assay covers main actionable hotspot alterations across many actionable genes: EGF(mutations), ALK (fusion, mutations), ROS1(fusion), BRAF(mutations), KRAS (mutations), NRAS(mutations), PIK3CA (mutations), ERBB2(CNV- copy number variation), ESR1(mutations), KIT(CNV) and PDGFRA(CNV). Therefore, our genes can be used in panels, both in therapeutically applied genotyping and in detecting molecular responses as well as in secondary resistance to tumors such as NSCLC, breast, cervix. rectal, GIST, melanoma and pancreas. Methods: dd-PCR was performed using the QX200 system (BIO-RAD, Hercules). The extraction of DNA and RNA was done using the magnetic beads technique of Thermofisher's MagMAX Kit. All samples were tested in duplicate. Up to now, 54 metastatic cancer patients were tested: NSCLC - 14 (26%), breast - 11 (20%), colorectal - 11 (20%), melanoma - 9(17%), pancreas - 4(7%), ovarian - 3(6%) and salivary glands - 2 (4%). Results: Significant genomic alterations were detected in 19 (35%) patients: 5 (9%) mutations in the KRAS G12V gene (all in colorectal cancer), 5 (9%) ERBB2 amplification (breast cancer), 2 (3.65%) EGFR L858R mutations (NSCLC), 2 (3.5%) EGFR del19 mutations (NSLC), 2 (3.5%) EGFR T790M mutations (NSCLC), 2 (3.5%) BRAF-V600E mutations (colon and melanoma), 1 (1.8%) ALK-EML4 fusion (NSCLC). The MAF (Mutant Allele Fraction) ranged from 0.9% to 24%. In all cases, the results were decisive for the indication or change of a targeted therapy. Median turnaround time was 36 hours and the average cost of the panels was around 500 USD (median of 4 genes per panel). Conclusions: Our results suggest that dd-PCR is a highly sensitive method and could be used for a routine laboratory detection of the important genomic variations to determine the targeted therapy in patients with varied advanced solid tumors.

scholarly journals Digital Droplet PCR for SARS-CoV-2 Resolves Borderline Cases

2021 ◽  
Author(s):  
Jing Xu ◽  
Timothy Kirtek ◽  
Yan Xu ◽  
Hui Zheng ◽  
Huiyu Yao ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
False Negative ◽  
Accurate Method ◽  
Viral Envelope ◽  
Chain Reaction ◽  
Borderline Cases ◽  
Droplet Pcr ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Abstract Objectives The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR—in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results. Methods We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer’s specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription–polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated. Results The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate. Conclusions The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.

Comparison of Droplet Digital Polymerase Chain Reaction (ddPCR) and Real-Time Quantitative Polymerase Chain Reaction (qPCR) in Detecting Neonatal Invasive Fungal Infections

2021 ◽  
Vol 11 (3) ◽  
pp. 373-379
Author(s):  
Huitao Li ◽  
Xueyu Chen ◽  
Xiaomei Qiu ◽  
Weimin Huang ◽  
Chuanzhong Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Methods ◽  
Chain Reaction ◽  
Fungal Isolation ◽  
Blood Samples ◽  
Fungal Strains ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Invasive fungal infection (IFI) is the leading cause of death in neonatal patients, yet the diagnosis of IFI remains a major challenge. At present, most IFI laboratory diagnostic methods are based on classical, but limited, methods such as fungal isolation and culture and histopathological examination. Recently, quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) technology have been adopted to quantify nucleic-acid identification. In this study, we established qPCR and ddPCR assays for IFI diagnosis and quantification. qPCR and ddPCR were carried out using identical primers and probe for the amplification of 18S rRNA. Assay results for three fungal strains were positive, whereas ten non-fungal strains had negative results, indicating 100% specificity for both ddPCR and qPCR methods. Genomic DNA of Candida albicans was tested after a serial dilution to compare the sensitivity of the two PCR methods. The limit of detection of ddPCR was 3.2 copies/L, which was a ten-fold increase compared with that of the qPCR method (32 copies/L). Blood samples from 127 patients with high-risk factors and clinical symptoms for IFI were collected from a NICU in Shenzhen, China, and analyzed using qPCR and ddPCR. Thirty-four blood samples from neonates had a proven or probable diagnosis of IFI, and 25 of these were positive by qPCR, whereas 30 were positive by ddPCR. Among the 93 blood samples from neonates who had a possible IFI or no IFI, 24 were positive using qPCR, and 7 were positive using ddPCR. In conclusion, ddPCR is a rapid and accurate pan-fungal detection method and provides a promising prospect for IFI clinical screening.

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