scholarly journals Optimal Molecular Methods in Detecting p190BCR-ABLFusion Variants in Hematologic Malignancies: A Case Report and Review of the Literature

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Rebecca J. Sonu ◽  
Brian A. Jonas ◽  
Denis M. Dwyre ◽  
Jeffrey P. Gregg ◽  
Hooman H. Rashidi
Keyword(s):  
Targeted Therapy ◽  
Kinase Inhibitors ◽  
Rare Variants ◽  
Lymphoblastic Leukemia ◽  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Hematologic Malignancies ◽  
Molecular Diagnostic ◽  
Diagnostic Pcr ◽  
Negative Results

Patients with BCR-ABL1 positive hematologic malignancies and Philadelphia-like B-lymphoblastic leukemia (B-ALL) are potential candidates for targeted therapy with tyrosine kinase inhibitors (TKI). Before TKIs, patients with B-ALL had a much worse prognosis and current treatments with targeted TKI therapy have improved outcomes. Thus, the detection of BCR-ABL1 is crucial and a false negative BCR-ABL1 result may adversely affect patient care. We report a case of a 76-year-old male with a new diagnosis of B-ALL who was initially found to be BCR-ABL1 negative by quantitative polymerase chain reaction (PCR). A concurrent qualitative PCR was performed which detected a positive BCR-ABL1 result that was confirmed by a next generation sequencing (NGS) based assay and identified as the rare fusion variant e1a3 of p190BCR-ABL. Based on this result, the patient was placed on dasatinib as a targeted therapy. In the era of molecular diagnostic medicine and targeted therapy, it is essential to have an understanding of the limitations of molecular assays and to follow a comprehensive diagnostic approach in order to detect common abnormalities and rare variants. Incorporating NGS methods in an algorithmic manner into the standard diagnostic PCR-based approach for BCR-ABL1 will aid in minimizing false negative results.

scholarly journals Most but not all laboratories can detect the recently emerged Neisseria gonorrhoeae porA mutants – results from the QCMD 2013 N. gonorrhoeae external quality assessment programme

2014 ◽  
Vol 19 (8) ◽  
Author(s):  
D Luijt ◽  
C Di Lorenzo ◽  
A M van Loon ◽  
M Unemo
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Quality Assessment ◽  
External Quality Assessment ◽  
False Negative ◽  
Molecular Diagnostic ◽  
External Quality ◽  
Diagnostic Assays ◽  
Assessment Programme ◽  
Negative Results ◽  
False Negative Results

We describe the results of the Quality Control for Molecular Diagnostics 2013 Neisseria gonorrhoeae external quality assessment programme that included an N. gonorrhoeae strain harbouring an N. meningitidis porA gene which causes false-negative results in molecular diagnostic assays targeting the gonococcal porA pseudogene. Enhanced awareness of the international transmission of such gonococcal strains is needed to avoid false-negative results in both in-house and commercial molecular diagnostic assays used in laboratories worldwide, but particularly in Europe.

scholarly journals Detection of LytA Genes in Streptococcus pneumoniae Isolated from sputum pneumonia patients

Biomedika ◽  
2020 ◽  
Vol 13 (1) ◽  
pp. 23-30
Author(s):  
Mustika Sari Hutabarat ◽  
Firdaus Hamid ◽  
Irawaty Djaharuddin ◽  
Alfian Zainuddin ◽  
Rossana Agus ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
False Negative ◽  
Pneumococcal Infection ◽  
Diagnostic Methods ◽  
Molecular Diagnostic ◽  
Clinical Samples ◽  
Biochemical Tests ◽  
Negative Results ◽  
False Negative Results ◽  
Polymerase Chain

Streptococcus pneumoniae (pneumococcus) is a Gram-positive facultative anaerobic bacterium that is a major cause of morbidity and mortality worldwide. But the lack of reporting of disease by this bacterium in Indonesia, one of the causes is because the diagnosis of pneumococcal infection is often clinically not typical and conventional methods which are still the standard gold method often give false-negative results. So the purpose of this study was to evaluate the performance of culture and molecular diagnostic methods using the Polymerase Chain Reaction (PCR) technique in detecting Streptococcus pneumoniae in sputum clinical samples using the Autolysin (LytA) gene which is a virulence factor of this bacterium. 57 isolates from 60 samples were confirmed as Streptococcus sp through microscopic identification, culture, and biochemical tests. Then the sensitivity test with an optochin test of 9 (9%) compared the results descriptively with the PCR technique using the Autolysin A (LytA) gene which was obtained more sensitive by 15 (25%).

NUP214-ABL1 in adult T-ALL: the GMALL study group experience

Blood ◽  
2006 ◽  
Vol 108 (10) ◽  
pp. 3556-3559 ◽  
Author(s):  
Thomas Burmeister ◽  
Nicola Gökbuget ◽  
Richard Reinhardt ◽  
Harald Rieder ◽  
Dieter Hoelzer ◽  
...  
Keyword(s):  
T Cell ◽  
Kinase Inhibitors ◽  
Fusion Gene ◽  
Lymphoblastic Leukemia ◽  
Quantitative Polymerase Chain Reaction ◽  
Prognostic Impact ◽  
Significant Difference ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Pcr Method ◽  
Polymerase Chain

Abstract The NUP214-ABL1 fusion gene in T-cell acute lymphoblastic leukemia (T-ALL) has recently been identified as a possible target for imatinib and related tyrosine kinase inhibitors, but exact data regarding the prognostic impact and frequency of the several putative NUP214-ABL1 mRNA transcripts are still missing. We investigated 279 adult patients with T-ALL treated within the framework of the GMALL 5/93 and 6/99 therapy trials for NUP214-ABL1 by using a novel multiplex real-time, quantitative polymerase chain reaction (PCR). Eleven (3.9%) patients were NUP214-ABL1 positive, and 5 different transcripts were observed; 8 patients had a thymic immunophenotype, 1 had an early T-cell immunophenotype, and 2 had a mature T-cell immunophenotype. NUP214-ABL1-positive and -negative patients did not differ significantly in their major clinical features. In contrast to previous reports suggesting an adverse clinical course for NUP214-ABL1-positive patients, no significant difference in overall survival was observed. Based on the results, we have established and tested a novel PCR method for simplified detection of the NUP214-ABL1 fusion gene.

scholarly journals Detection of MRD may predict the outcome of patients with Philadelphia chromosome–positive ALL treated with tyrosine kinase inhibitors plus chemotherapy

Blood ◽  
2013 ◽  
Vol 122 (7) ◽  
pp. 1214-1221 ◽  
Author(s):  
Farhad Ravandi ◽  
Jeffrey L. Jorgensen ◽  
Deborah A. Thomas ◽  
Susan O’Brien ◽  
Rebecca Garris ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Quantitative Polymerase Chain Reaction ◽  
Philadelphia Chromosome ◽  
Multiparameter Flow Cytometry ◽  
Molecular Response ◽  
Cell Transplant ◽  
Treatment Intensification ◽  
Philadelphia Chromosome Positive

Abstract From 2001 to 2011, 122 patients with newly diagnosed Philadelphia chromosome–positive acute lymphoblastic leukemia were treated with chemotherapy + imatinib (n = 54) or + dasatinib (n = 68). One hundred fifteen (94%) achieved complete remission (CR) including 101 patients who achieved it with only 1 induction course and had at least 1 minimal residual disease (MRD) assessment; 25 patients underwent an allogeneic stem cell transplant in first CR and were excluded, leaving 76 patients as the subject of this report. MRD monitoring by multiparameter flow cytometry (MFC) and real-time quantitative polymerase chain reaction (PCR) was performed at the end of induction and at ∼3-month intervals thereafter. Median age was 54 years (range, 21-84 years). There was no difference in survival by achievement of at least a major molecular response (MMR; BCR-ABL/ABL < 0.1%) at CR (P = .22). Patients achieving MMR at 3, 6, 9, and 12 months had a better survival (P = .02, .04, .05, and .01, respectively). Negative MFC at CR did not predict for improved survival (P = .2). At 3 and 12 months, negative MRD by MFC was associated with improved survival (P = .04 and .001). MRD monitoring by PCR and MFC identifies patients who benefit from treatment intensification in first CR.

scholarly journals Can We Use Targeted Next-Generation Sequencing an Alternative Method to the Conventional Tests in Haematological Malignancies?

2020 ◽  
Author(s):  
Emine Ikbal Atli ◽  
Hakan Gurkan ◽  
Engin Atli ◽  
Hakki Onur Kirkizlar ◽  
Sinem Yalcintepe ◽  
...  
Keyword(s):  
False Negative ◽  
Molecular Cytogenetics ◽  
Hematologic Malignancies ◽  
Turnaround Time ◽  
Diagnostic Methods ◽  
Myeloid Malignancies ◽  
Targeted Next Generation Sequencing ◽  
Negative Results ◽  
Golden Standard ◽  
Molecular Cytogenetic Techniques

Abstract Introduction: Advanced diagnostic methods give a huge advantage for identification of the abnormalities in myeloid malignancies. Researchers tried to show the potential importance of genetic tests both before the onset of the disease and during the remission. Large testing panels prevents false negative results in myeloid malignancies. But the important question is how can be merged with conventional cytogenetic and molecular cytogenetic techniques together with NGS technologies. Methods: In this paper, we draw an algorithm for evaluation of the malignancies. In order to evaluation of genetic abnormities we performed cytogenetics, molecular cytogenetics and NGS testing panels in hematologic malignancies. In this study, we analyzed 132 patients which are referred to Medical Genetics Laboratory within different type of hematologic malignancies. We highlighted possible algorithm for cytogenetically normal cases.Results: We analyzed cytogenetically normal patients by using NGS 141 gene panel and we detected two or more pathogenic variations in 20 out of 132 patients.Conclusions: Despite of long turnaround time conventional techniques is still golden standard for myeloid malignancies but sometimes cryptic gene fusions or complex abnormalities cannot easily be identified by conventional techniques that conditions advanced technologies are recommended.

scholarly journals Pediatric acute lymphoblastic leukemia

Haematologica ◽  
2020 ◽  
Vol 105 (11) ◽  
pp. 2524-2539
Author(s):  
Hiroto Inaba ◽  
Charles G. Mullighan
Keyword(s):  
Targeted Therapy ◽  
Acute Lymphoblastic Leukemia ◽  
Risk Stratification ◽  
Kinase Inhibitors ◽  
Lymphoblastic Leukemia ◽  
Molecular Taxonomy ◽  
Philadelphia Chromosome ◽  
Experimental Models ◽  
T Cell Therapy ◽  
Molecular Alterations

The last decade has witnessed great advances in our understanding of the genetic and biological basis of childhood acute lymphoblastic leukemia (ALL), the development of experimental models to probe mechanisms and evaluate new therapies, and the development of more efficacious treatment stratification. Genomic analyses have revolutionized our understanding of the molecular taxonomy of ALL, and these advances have led the push to implement genome and transcriptome characterization in the clinical management of ALL to facilitate more accurate risk-stratification and, in some cases, targeted therapy. Although mutation- or pathway-directed targeted therapy (e.g., using tyrosine kinase inhibitors to treat Philadelphia chromosome [Ph]-positive and Phlike B-cell-ALL) is currently available for only a minority of children with ALL, many of the newly identified molecular alterations have led to the exploration of approaches targeting deregulated cell pathways. The efficacy of cellular or humoral immunotherapy has been demonstrated with the success of chimeric antigen receptor T-cell therapy and the bispecific engager blinatumomab in treating advanced disease. This review describes key advances in our understanding of the biology of ALL and optimal approaches to risk-stratification and therapy, and it suggests key areas for basic and clinical research.

scholarly journals Porcine reproductive and respiratory syndrome virus

2012 ◽  
Vol 24 (5) ◽  
pp. 855-866 ◽  
Author(s):  
Kerstin Wernike ◽  
Paolo Bonilauri ◽  
Malte Dauber ◽  
Jane Errington ◽  
Neil LeBlanc ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Respiratory Syndrome Virus ◽  
Analytical Sensitivity ◽  
Negative Results ◽  
False Negative Results ◽  
Ring Trial ◽  
Polymerase Chain ◽  
Commercial Kits ◽  
The Individual

To compare the real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays used for the diagnosis of Porcine reproductive and respiratory syndrome virus (PRRSV), a Europe-wide interlaboratory ring trial was conducted. A variety of PRRSV strains including North American (NA) and European (EU) genotype isolates were analyzed by the participants. Great differences regarding qualitative diagnostics as well as analytical sensitivity were observed between the individual RT-qPCR systems, especially when investigating strains from the EU genotype. None of the assays or commercial kits used in the ring trial could identify all different PRRSV strains with an optimal analytical and diagnostic sensitivity. The genetic variability of the PRRSV strains, which is supposed to hinder the diagnostic of the RT-PCR because of mutations at the primer binding sites, was also confirmed by sequencing and subsequent phylogenetic analysis. In summary, a major problem in PRRSV diagnostics by RT-qPCR is false-negative results. To achieve maximum safety in the molecular diagnosis of PRRSV, the combined usage of different assays or kits is highly recommended.

scholarly journals Frequent deletion in the methylthioadenosine phosphorylase gene in T- cell acute lymphoblastic leukemia: strategies for enzyme-targeted therapy

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 3083-3090 ◽  
Author(s):  
A Batova ◽  
MB Diccianni ◽  
T Nobori ◽  
T Vu ◽  
J Yu ◽  
...  
Keyword(s):  
Targeted Therapy ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Polymerase Chain Reaction Amplification ◽  
Lymphoblastic Leukemia ◽  
Quantitative Polymerase Chain Reaction ◽  
Break Point ◽  
Suppressor Gene ◽  
Methylthioadenosine Phosphorylase ◽  
The Status

Methylthioadenosine phosphorylase (MTAP), an enzyme essential for the salvage of adenine and methionine, is deficient in a variety of cancers, including acute lymphoblastic leukemia (ALL). Because the MTAP gene is located adjacent to the tumor-suppressor gene p16 on chromosome 9p21 and more than 60% of T-cell ALL (T-ALL) patients have deletion in the p16 gene, we examined the status of the MTAP gene in T-ALL patients. Quantitative polymerase chain reaction amplification of exon 8 of MTAP showed a deletion in 16 of 48 (33.3%) patients at diagnosis and in 13 of 33 (39.4%) patients at relapse. Southern blot analysis showed that, in addition to deletion of the entire MTAP gene, a common break point was between exons 4 and 5, resulting in deletion of exons 5 through 8. The finding of frequent deficiency of MTAP in T-ALL offers the possibility of an enzyme targeted therapy for T-ALL. MTAP(-) T-ALL-derived cell line, CEM cells were very sensitive to methionine deprivation, with cell viability at 50% of control as early as 48 hours after methionine deprivation. In contrast, methionine deprivation had little effect on the viability of normal lymphocytes or on their proliferative response to phytohemagglutinin. Alanosine, an inhibitor of AMP synthesis, inhibited the growth of both MTAP(+) (Molt-4 and Molt-16) and MTAP(-) (CEM and HSB2) cell lines. However, the addition of methylthioadenosine, the substrate of MTAP, protected the MTAP(+) cells but not the MTAP(-) cells from alanosine toxicity. These findings suggest the possibility of targeting MTAP for selective therapy of T-ALL.

scholarly journals Exogenous Controls Increase Negative Call Veracity in Multiplexed, Quantitative PCR Assays for Phakopsora pachyrhizi

Plant Disease ◽  
2011 ◽  
Vol 95 (3) ◽  
pp. 343-352 ◽  
Author(s):  
James S. Haudenshield ◽  
Glen L. Hartman
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
False Negative ◽  
Arbitrary Sequence ◽  
Phakopsora Pachyrhizi ◽  
Synthetic Dna ◽  
Negative Results ◽  
Detection And Identification ◽  
False Negative Results ◽  
Pcr Assays ◽  
Rust Pathogens

Quantitative polymerase chain reaction (Q-PCR) utilizing specific primer sequences and a fluorogenic, 5′-exonuclease linear hydrolysis probe is well established as a detection and identification method for Phakopsora pachyrhizi and P. meibomiae, two rust pathogens of soybean. Because of the extreme sensitivity of Q-PCR, the DNA of single urediniospores of these fungi can be detected from total DNA extracts of environmental samples. However, some DNA preparations unpredictably contain PCR inhibitors that increase the frequency of false negatives indistinguishable from true negatives. Three synthetic DNA molecules of arbitrary sequence were constructed as multiplexed internal controls (ICs) to cull false-negative results by producing a positive signal to validate the PCR process within each individual reaction. The first two, PpaIC and PmeIC, are a single-stranded oligonucleotide flanked by sequences complementary to the primers of either the P. pachyrhizi or P. meibomiae primary assay but hybridizing to a unique fluorogenic probe; the third contains unique primer- and probe-binding sequences, and was prepared as a cloned DNA fragment in a linearized plasmid. These ICs neither qualitatively nor quantitatively affected their primary assays. PpaIC and PmeIC were shown to successfully identify false-negative reactions resulting from endogenous or exogenous inhibitors, and can be readily adapted to function in a variety of diagnostic Q-PCR assays; the plasmid was found to successfully validate true negatives in similar Q-PCR assays for other soybean pathogens, as well as to function as a tracer molecule during DNA extraction and recovery.

scholarly journals Mutations in Animal SARS-CoV-2 Induce Mismatches with the Diagnostic PCR Assays

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 371
Author(s):  
Ahmed Elaswad ◽  
Mohamed Fawzy
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Animal Species ◽  
False Negative ◽  
Bioinformatic Analysis ◽  
Diagnostic Pcr ◽  
Animal Host ◽  
Negative Results ◽  
False Negative Results ◽  
Pcr Assays

Recently, the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was detected in several animal species. After transmission to animals, the virus accumulates mutations in its genome as adaptation to the new animal host progresses. Therefore, we investigated whether these mutations result in mismatches with the diagnostic PCR assays and suggested proper modifications to the oligo sequences accordingly. A comprehensive bioinformatic analysis was conducted using 28 diagnostic PCR assays and 793 publicly available SARS-CoV-2 genomes isolated from animals. Sixteen out of the investigated 28 PCR assays displayed at least one mismatch with their targets at the 0.5% threshold. Mismatches were detected in seven, two, two, and six assays targeting the ORF1ab, spike, envelope, and nucleocapsid genes, respectively. Several of these mismatches, such as the deletions and mismatches at the 3’ end of the primer or probe, are expected to negatively affect the diagnostic PCR assays resulting in false-negative results. The modifications to the oligo sequences should result in stronger template binding by the oligos, better sensitivity of the assays, and higher confidence in the result. It is necessary to monitor the targets of diagnostic PCR assays for any future mutations that may occur as the virus continues to evolve in animals.

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