scholarly journals Determination of MYD88L265P mutation fraction in IgM monoclonal gammopathies

2021 ◽  
Author(s):  
TINA BAGRATUNI ◽  
Athina N Markou ◽  
Dimitrios Patseas ◽  
Nefeli Mavrianou-Koutsoukou ◽  
Foteini Aktypi ◽  
...  
Keyword(s):  
Cost Effective ◽  
Pcr Assay ◽  
Cell Free Dna ◽  
Monoclonal Gammopathies ◽  
Specific Pcr ◽  
Free Dna ◽  
Highly Sensitive ◽  
Sequential Samples ◽  
Tumor Load ◽  
Allele Specific

We here describe a novel method for the detection of MYD88L265P mutation using a competitive allele specific PCR (Cast-PCR) assay. This assay has a sensitivity of 1x10-3, is applicable in reactions containing very low amounts of DNA (as low as 20 pg) and allowed the detection of MYD88L265P somatic mutation in both tumor derived DNA (tDNA) and cell free DNA (cfDNA). In addition, using Cast-PCR assay we were able to determine the mutation allele fraction (MAF) in each tested sample. We then analyzed baseline tDNA and cfDNA samples from 163 patients (53 with IgM-MGUS and 110 with WM, of which 54 were asymptomatic and 56 symptomatic) and also in sequential samples of 37 patients. MAF in both cfDNA and tDNA was higher among patients with symptomatic compared to asymptomatic WM and in those with asymptomatic WM compared to IgM-MGUS patients. In addition, the evaluation of sequential samples showed that MAF decreased after treatment while increased in patients who relapsed or progressed to symptomatic WM. Thus, Cast-PCR is a highly-sensitive, cost-effective diagnostic tool for MYD88L265P detection, applicable in both tumor and cell free DNA samples which also provides a quantitative evaluation of the tumor load in patients with IgM monoclonal gammopathies.

scholarly journals A novel ultra-sensitive method for the detection of FGFR3 mutations in urine of bladder cancer patients – Design of the Urodiag® PCR Kit for surveillance of patients with non-muscle-invasive bladder cancer (NMIBC)

2020 ◽  
Author(s):  
Jean-Pierre ROPERCH ◽  
Claude HENNION
Keyword(s):  
Bladder Cancer ◽  
High Risk ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Wild Type ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Multiplex Pcr Assay ◽  
Allele Specific ◽  
Fgfr3 Mutations

Abstract Background We have recently developed a highly accurate urine-based test, named Urodiag ® , associating FGFR3 mutation and DNA methylation assays for recurrence surveillance in patients with low-, intermediate-, and high-risk NMIBC. Previously, the detection of four FGFR3 mutations (G372C, R248C, S249C and Y375C) required amplification steps and PCR products were analyzed by capillary electrophoresis (Allele Specific-PCR, AS-PCR), which was expensive and time-consuming. Here, we present the development a novel ultra-sensitive multiplex PCR assay as called “Mutated Allele Specific Oligonucleotide-PCR (MASO-PCR)”, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in voided urine. Methods Comparative clinical performances of MASO-PCR and AS-PCR technologies were performed from 263 urine DNA samples (87 FGFR3 mutated and 176 FGFR3 wild-type). In the development of Urodiag ® PCR Kit, we studied the stability and reproducibility of each all-in-one PCR master mix (single reaction mixture including all the necessary PCR components) for MASO-PCR and QM-MSPCR (Quantitative Multiplex Methylation-Specific PCR to co-amplify SEPTIN9 , HS3ST2 and SLIT2 methylated genes) assays. Results Complete concordance (100%) was observed between the MASO-PCR and AS-PCR results. Each PCR master mix displayed excellent reproducibility and stability after 12 months of storage at -20°C, with intra-assay standard deviations lower than 0.3 Ct and coefficient of variations (CV) lower than 1%. The limit of detection (LoD) of MASO-PCR was 5% mutant detection in a 95% of wild-type background. The limit of quantification (LoQ) of QM-MSPCR was 10 pg of bisulfite-converted DNA. Conclusions We developed and clinically validated the MASO-PCR assay, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine. We also designed the Urodiag ® PCR Kit, which includes the MASO-PCR and QM-MSPCR assays. Adapted to routine clinical laboratory (simplicity, accuracy), the kit will be a great help to urologists for recurrence surveillance in patients at low-, intermediate- and high-risk NMIBC. Reducing the number of unnecessary cystoscopies, it will have extremely beneficial effects for patients (painless) and for the healthcare systems (low cost).

scholarly journals Development and Application of a Tri-allelic PCR Assay for ScreeningVgsc-L1014FKdrMutations Associated with Pyrethroid and Organochlorine Resistance in the MosquitoCulex quinquefasciatus

2019 ◽  
Author(s):  
Walter Fabricio Silva Martins ◽  
Bárbara Natieli Silva Pereira ◽  
Ana Thayse Vieira Alves ◽  
Annabel Murphy ◽  
Paulo Geovani Silva Martins ◽  
...  
Keyword(s):  
Allelic Variation ◽  
High Specificity ◽  
Cost Effective ◽  
Diagnostic Assay ◽  
Pcr Assay ◽  
Public Health Importance ◽  
Tropical Regions ◽  
Vector Borne Diseases ◽  
Specific Pcr ◽  
Allele Specific

AbstractBackgroundCulex quinquefasciatus,has a widespread distribution across tropical and sub-tropical regions, and plays an important role in the transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF) and multiple arboviruses. Increased resistance to insecticides threatens the efficacy and sustainability of insecticide-based anti-vector interventions which mitigate the burden of mosquito transmitted diseases in endemic regions. InC. quinquefasciatustwo non-synonymous voltage gated sodium channel (Vgsc) variants, both resulting in a leucine to phenylalanine change at codon 1014, are associated with resistance to pyrethroids and DDT. This tri-allelic variation has compromised the ability to perform high-throughput single-assay screening. To facilitate the detection and monitoring of theVgsc-1014 locus in field-caught mosquitoes, an Engineered-Tail Allele-Specific-PCR (ETAS-PCR) diagnostic assay was developed and applied to wild mosquitoes from Brazil, Tanzania and Uganda.ResultsThis new cost-effective, single-tube assay was compared to two, well-established, genotyping approaches – pyrosequencing and TaqMan. The ETAS-PCR assay showed high specificity for discriminating the three alleles atVgsc-L1014F, with genotyping results strongly correlated with 98.64% and 100% against pyrosequencing and TaqMan, respectively.ConclusionsOur results support the utility of the ETAS-PCR/Vgsc-1014 diagnostic assay, which stands as an effective alternative for genotyping tri-allelic variants.

scholarly journals Development and validation of an allele-specific PCR assay for genotyping a promoter and exonic single nucleotide polymorphisms of MGMT gene

2018 ◽  
Vol 5 (2) ◽  
pp. e92
Author(s):  
Anuj Kumar Tyagi ◽  
Mary Boudal Khoshbeen ◽  
Patricia Huezo-Diaz Curtis ◽  
Chakradhara Rao S. Uppugunduri ◽  
Marc Ansari
Keyword(s):  
Dna Methyltransferase ◽  
Cost Effective ◽  
Nucleotide Polymorphisms ◽  
Pcr Assay ◽  
Mgmt Gene ◽  
Methylguanine Dna Methyltransferase ◽  
Specific Pcr ◽  
Allele Specific ◽  
Specific Pcr Assay ◽  
Allele Specific Pcr

DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) specifically remove the methyl/alkyl group from the O6-position of guanine and restore the guanine to its normal form without causing DNA strand breaks. Relationship between MGMT activity and resistance to alkylating therapeutic agents is well established. Non-availability of simple, cost-effective and efficient methods of genotyping may hinder investigations on genotype-phenotype associations. No simple genotyping procedures such as allele-discrimination Taqman Assays were available for two genetic variations in MGMT gene that had previously demonstrated to be affecting its function and expression.These two variants were included to genotype in a clinical study (Clinicaltrail.gov ID: NCT01257854). Hence, the present study is aimed at developing,   validating a rapid and simple allele-specific PCR method that genotypes exonic variant rs2308321 (c.520A>G) and a promoter variant rs113813075 (c.-459C>A) with standard PCR instruments.Web-based allele-specific (AS) primer design application called web-based allele-specific primer was used to design primers. Genomic DNA of lymphoblastoid cell line obtained from the Coriell repository with known genotypes were used to standardize the genotyping procedure. The PCR products were analyzed by 3% Agarose gel electrophoresis and by DNA Screen Tape assay with the Agilent 4200 TapeStation. The allele-specific PCR assay described here is a suitable strategy for efficient and reliable genotyping for difficult variants. This method offers cost-effective strategy for genotyping in clinical cohort studies provided positive controls established by Sanger sequencing are available for the variant. 

scholarly journals Targeted treatment of brainstem neurohistiocytosis guided by urinary cell-free DNA

2016 ◽  
Vol 4 (1) ◽  
pp. e299 ◽  
Author(s):  
David Hunt ◽  
Paul Milne ◽  
Peter Fernandes ◽  
Venetia Bigley ◽  
Matthew Collin
Keyword(s):  
Braf Inhibitor ◽  
Targeted Treatment ◽  
Observation Study ◽  
Cell Free Dna ◽  
Single Observation ◽  
Specific Pcr ◽  
Free Dna ◽  
Allele Specific ◽  
A Cell ◽  
Allele Specific Pcr

Objective:To identify a treatment-responsive BRAFV600E mutation in brainstem neurohistiocytosis, where no lesional tissue was readily obtainable, using a cell-free DNA approach.Methods:Cell-free DNA was extracted from urine and allele-specific PCR for the BRAFV600E mutation was performed. Response to conventional treatment (corticosteroids and interferon) and targeted treatment with a BRAF inhibitor was assessed by clinical evaluation, gadolinium-enhanced MRI brain scan, and serial testing of urinary cell-free DNA for mutant alleles.Results:BRAFV600E mutation could be readily identified in urinary cell-free DNA at an allele frequency of 4.2%. Treatment of Erdheim-Chester disease with corticosteroids and interferon was ineffective and associated with disease progression. Treatment with BRAF inhibitors was associated with clinical improvement and near-complete radiologic remission. Following 6 months of BRAF inhibitor therapy, no enhancing lesions could be detected in the brain and mutant alleles were cleared from the urine.Conclusions:Analysis of urinary cell-free DNA using allele-specific PCR for BRAFV600E mutations allows rapid noninvasive identification of a highly treatment-responsive pathway, leading to clinical and radiologic remission of disease. Our case demonstrates that this assay may have a particular role in challenging neurohistiocytosis cases, where attempts at obtaining lesional tissue have failed or are not feasible.Classification of evidence:This study provides Class IV evidence. This is a single observation study without controls.

scholarly journals A novel ultra-sensitive urine based FGFR3 mutation assay for diagnosis and recurrence detection of non-muscle-invasive bladder cancer (NMIBC) – Design of the Urodiag® Kit

2020 ◽  
Author(s):  
Jean-Pierre ROPERCH ◽  
Claude HENNION
Keyword(s):  
High Risk ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Wild Type ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Multiplex Pcr Assay ◽  
Fgfr3 Mutation ◽  
Allele Specific ◽  
Fgfr3 Mutations

Abstract Background We have recently developed a highly accurate urine-based test, named Urodiag ® , associating FGFR3 mutation and DNA methylation assays for recurrence surveillance in patients with low-, intermediate-, and high-risk NMIBC. Previously, the detection of four FGFR3 mutations (G372C, R248C, S249C and Y375C) required the amplification steps and the PCR products were analyzed by capillary electrophoresis (Allele Specific-PCR, AS-PCR), which was expensive and time-consuming. Here, we present the development a novel ultra-sensitive multiplex PCR assay as called “Mutated Allele Specific Oligonucleotide-PCR (MASO-PCR)”, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in voided urine. Methods Comparative clinical performances of MASO-PCR and AS-PCR technologies were performed from 263 urine DNA samples (87 FGFR3 mutated and 176 FGFR3 wild type). In the development of Urodiag ® Kit, we studied the stability and reproducibility of each all-in-one PCR master mix (single reaction mixture including all the necessary PCR components) for MASO-PCR and QM-MSPCR (Quantitative Multiplex Methylation-Specific PCR to co-amplify SEPTIN9, HS3ST2 and SLIT2 methylated genes) assays. Results Complete concordance (100%) was observed between the MASO-PCR and AS-PCR results. Each PCR master mix displayed excellent reproducibility and stability after 12 months of storage at -20°C, with intra-assay standard deviations lower than 0.3 Ct and coefficient of variations (CV) lower than 1%. The limit of detection (LoD) of MASO-PCR was 5% mutant detection in a 95% of wild-type background. The limit of quantification (LoQ) of QM-MSPCR was 10 pg of bisulfite-converted DNA. Conclusions We developed and clinically validated the MASO-PCR assay, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine. We also designed the Urodiag ® Kit, which includes the MASO-PCR and QM-MSPCR assays. Adapted to routine clinical laboratory (simplicity, accuracy), the kit will be a great help to urologists for recurrence surveillance in patients at low-, intermediate- and high-risk NMIBC. Reducing the number of unnecessary cystoscopies, it will have extremely beneficial effects for patients (painless) and for the healthcare systems (low cost).

scholarly journals A novel ultra-sensitive method for the detection of FGFR3 mutations in urine of bladder cancer patients – Design of the Urodiag® PCR Kit for surveillance of patients with non-muscle-invasive bladder cancer (NMIBC)

2020 ◽  
Author(s):  
Jean-Pierre ROPERCH ◽  
Claude HENNION
Keyword(s):  
Bladder Cancer ◽  
High Risk ◽  
Clinical Laboratory ◽  
Limit Of Detection ◽  
Cost Effective ◽  
Wild Type ◽  
Pcr Assay ◽  
Specific Pcr ◽  
Multiplex Pcr Assay ◽  
Allele Specific

Abstract Background We have recently developed a highly accurate urine-based test, named Urodiag®, associating FGFR3 mutation and DNA methylation assays for recurrence surveillance in patients with low-, intermediate-, and high-risk NMIBC. Previously, the detection of four FGFR3 mutations (G372C, R248C, S249C and Y375C) required amplification steps and PCR products were analyzed by capillary electrophoresis (Allele Specific-PCR, AS-PCR), which was expensive and time-consuming. Here, we present the development a novel ultra-sensitive multiplex PCR assay as called “Mutated Allele Specific Oligonucleotide-PCR (MASO-PCR)”, generating a cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in voided urine.Methods Comparative clinical performances of MASO-PCR and AS-PCR technologies were performed from 263 urine DNA samples (87 FGFR3 mutated and 176 FGFR3 wild-type). In the development of Urodiag® PCR Kit, we studied the stability and reproducibility of each all-in-one PCR master mix (single reaction mixture including all the necessary PCR components) for MASO-PCR and QM-MSPCR (Quantitative Multiplex Methylation-Specific PCR to co-amplify SEPTIN9, HS3ST2 and SLIT2 methylated genes) assays.Results Complete concordance (100%) was observed between the MASO-PCR and AS-PCR results. Each PCR master mix displayed excellent reproducibility and stability after 12 months of storage at -20°C, with intra-assay standard deviations lower than 0.3 Ct and coefficient of variations (CV) lower than 1%. The limit of detection (LoD) of MASO-PCR was 5% mutant detection in a 95% of wild-type background. The limit of quantification (LoQ) of QM-MSPCR was 10 pg of bisulfite-converted DNA. Conclusions We developed and clinically validated the MASO-PCR assay, generating cost-effective, simple, fast and clinically applicable assay for the detection of FGFR3 mutations in urine. We also designed the Urodiag® PCR Kit, which includes the MASO-PCR and QM-MSPCR assays. Adapted to routine clinical laboratory (simplicity, accuracy), the kit will be a great help to urologists for recurrence surveillance in patients at low-, intermediate- and high-risk NMIBC. Reducing the number of unnecessary cystoscopies, it will have extremely beneficial effects for patients (painless) and for the healthcare systems (low cost).

Use of Circulating Cell-Free DNA to Guide Precision Medicine in Patients with Colorectal Cancer

2021 ◽  
Vol 72 (1) ◽  
pp. 399-413
Author(s):  
Van K. Morris ◽  
John H. Strickler
Keyword(s):  
Colorectal Cancer ◽  
Precision Medicine ◽  
Residual Disease ◽  
Cost Effective ◽  
Patient Specific ◽  
Blood Draw ◽  
Cell Free Dna ◽  
Free Dna ◽  
Metastatic Setting ◽  
Diagnostic Technology

Patient-specific biomarkers form the foundation of precision medicine strategies. To realize the promise of precision medicine in patients with colorectal cancer (CRC), access to cost-effective, convenient, and safe assays is critical. Improvements in diagnostic technology have enabled ultrasensitive and specific assays to identify cell-free DNA (cfDNA) from a routine blood draw. Clinicians are already employing these minimally invasive assays to identify drivers of therapeutic resistance and measure genomic heterogeneity, particularly when tumor tissue is difficult to access or serial sampling is necessary. As cfDNA diagnostic technology continues to improve, more innovative applications are anticipated. In this review, we focus on four clinical applications for cfDNA analysis in the management of CRC: detecting minimal residual disease, monitoring treatment response in the metastatic setting, identifying drivers of treatment sensitivity and resistance, and guiding therapeutic strategies to overcome resistance.

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