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1.
PLoS One ; 18(4): e0284538, 2023.
Article in English | MEDLINE | ID: mdl-37083935

ABSTRACT

AIMS: qPCR, is widely used for quantifying minimal residual disease (MRD) and is conventionally performed according to guidelines proposed by the EuroMRD consortium. However it often fails when quantifying MRD levels below 10-4. By contrast, HAT-PCR, a recent modification designed to minimise false-positive results, can quantify MRD down to 10-6. METHODS: The factors leading to failure of conventional qPCR to quantify low levels of MRD were studied by analysing PCR reagents, protocol and primers and by testing for inhibition by adding primers to a plasmid amplification system. Complementary primers, ending in either G/C or A/T, were used to determine the effect of the 3' end of a primer. RESULTS: Inhibition of conventional PCR resulted from interaction of primers with genomic DNA leading to exponential amplification of nonspecific amplicons. It was observed with approximately half of the EuroMRD J primers tested. Inhibition by a primer was significantly related to primer Tm and G/C content and was absent when extension at the 3' end was blocked. Nonspecificity and inhibition were decreased or abolished by increasing the annealing temperature and inhibition was decreased by increasing the concentration of polymerase. Primers terminating with G/C produced significantly more nonspecificity and inhibition than primers terminating with A/T. HAT-PCR produced minimal nonspecificity and no inhibition. CONCLUSIONS: Inhibition of the PCR may result from the presence of genomic DNA and resultant exponential amplification of nonspecific amplicons. Factors contributing to the phenomenon include suboptimal annealing temperature, suboptimal primer design, and suboptimal polymerase concentration. Optimisation of these factors, as in HAT-PCR, enables sensitive quantification of MRD. PCR assays are increasingly used for sensitive detection of other rare targets against a background of genomic DNA and such assays may benefit from similar improvement in PCR design.


Subject(s)
DNA , Nucleic Acid Amplification Techniques , Polymerase Chain Reaction/methods , DNA/genetics , DNA Primers/genetics , Genomics
2.
J Mol Diagn ; 24(6): 632-641, 2022 06.
Article in English | MEDLINE | ID: mdl-35430373

ABSTRACT

PCR is widely used to measure minimal residual disease (MRD) in lymphoid neoplasms, but its sensitivity is limited. High Adenine/Thymine PCR and High Annealing Temperature PCR (HAT-PCR) is a modified PCR designed to minimize nonspecificity and hence increase sensitivity. It was evaluated in the laboratory and the clinic, using samples from 58 patients. Of these patients, 57 were adolescents or young adults who were participating in the Australasian Leukemia and Lymphoma Group ALL06 trial in which MRD was measured in blood principally by HAT-PCR and in marrow by conventional PCR. HAT-PCR produced significantly less nonspecificity than conventional PCR, and its limit of detection was <10-6 in 90% of patients. In 196 samples, an excellent correlation was found between blood and marrow MRD. Variable partitioning of leukemic cells between blood and marrow was observed. Measurement of MRD in blood by HAT-PCR was noninferior to measurement of MRD in marrow by conventional PCR, in terms of both detecting disease and predicting clinical outcome. At a median follow-up of 3 years and for MRD levels in blood at the end of consolidation treatment, an MRD level of >10-4 cells/L significantly predicted relapse and mortality, whereas undetectable MRD significantly predicted relapse-free survival and overall survival. HAT-PCR is a simple, quick, cheap and sensitive method for measurement of MRD, and its adoption for MRD in blood may be clinically useful.


Subject(s)
Bone Marrow , Adolescent , Bone Marrow/pathology , Humans , Neoplasm, Residual/diagnosis , Neoplasm, Residual/genetics , Polymerase Chain Reaction , Recurrence , Young Adult
3.
PLoS One ; 12(10): e0185556, 2017.
Article in English | MEDLINE | ID: mdl-28973007

ABSTRACT

INTRODUCTION: The level of minimal residual disease (MRD) in marrow predicts outcome and guides treatment in childhood acute lymphoblastic leukemia (ALL) but accurate prediction depends on accurate measurement. METHODS: Forty-one children with ALL were studied at the end of induction. Two samples were obtained from each iliac spine and each sample was assayed twice. Assay, sample and side-to-side variation were quantified by analysis of variance and presumptively incorrect decisions related to high-risk disease were determined using the result from each MRD assay, the mean MRD in the patient as the measure of the true value, and each of 3 different MRD cut-off levels which have been used for making decisions on treatment. RESULTS: Variation between assays, samples and sides each differed significantly from zero and the overall standard deviation for a single MRD estimation was 0.60 logs. Multifocal residual disease seemed to be at least partly responsible for the variation between samples. Decision errors occurred at a frequency of 13-14% when the mean patient MRD was between 10-2 and 10-5. Decision errors were observed only for an MRD result within 1 log of the cut-off value used for assessing high risk. Depending on the cut-off used, 31-40% of MRD results were within 1 log of the cut-off value and 21-16% of such results would have resulted in a decision error. CONCLUSION: When the result obtained for the level of MRD is within 1 log of the cut-off value used for making decisions, variation in the assay and/or sampling may result in a misleading assessment of the true level of marrow MRD. This may lead to an incorrect decision on treatment.


Subject(s)
Neoplasm, Residual , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Adolescent , Child , Child, Preschool , Female , Humans , Infant , Male
5.
Biotechniques ; 49(6): 893-7, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21143211

ABSTRACT

Optimal accuracy of quantitative PCR (qPCR) requires correction for integrity of the target sequence. Here we combine the mathematics of the Poisson distribution and exponential amplification to show that the frequency of lesions per base (which prevent PCR amplification) can be derived from the slope of the regression line between cycle threshold (Ct) and amplicon length. We found that the amplifiable fraction (AF) of a target can be determined from this frequency and the target length. Experimental results from this method correlated with both the magnitude of a damaging agent and with other measures of DNA damage. Applying the method to a reference sequence, we determined the values for lesions/base in control samples, as well as in the AFs of the target sequence in qPCR samples collected from leukemic patients. The AFs used to calculate the final qPCR result were generally >0.5, but were <0.2 in a few samples, indicating significant degradation. We conclude that DNA damage is not always predictable; quantifying the DNA integrity of a sample and determining the AF of a specific qPCR target will improve the accuracy of qPCR and aid in the interpretation of negative results.


Subject(s)
DNA/chemistry , Polymerase Chain Reaction/methods , DNA/genetics , DNA/metabolism , DNA Damage , Humans , Models, Genetic , Poisson Distribution , Regression Analysis
6.
J Mol Diagn ; 11(3): 201-10, 2009 May.
Article in English | MEDLINE | ID: mdl-19324989

ABSTRACT

A sensitive and specific quantitative real-time polymerase chain reaction method, involving three rounds of amplification with two allele-specific oligonucleotide primers directed against an rearrangement, was developed to quantify minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (ALL). For a single sample containing 10 microg of good quality DNA, MRD was quantifiable down to approximately 10(-6), which is at least 1 log more sensitive than current methods. Nonspecific amplification was rarely observed. The standard deviation of laboratory estimations was 0.32 log units at moderate or high levels of MRD, but increased markedly as the level of MRD and the number of intact marker gene rearrangements in the sample fell. In 23 children with ALL studied after induction therapy, the mean MRD level was 1.6 x 10(-5) and levels ranged from 1.5 x 10(-2) to less than 10(-7). Comparisons with the conventional one-round quantitative polymerase chain reaction method on 29 samples from another 24 children who received treatment resulted in concordant results for 22 samples and discordant results for seven samples. The sensitivity and specificity of the method are due to the use of nested polymerase chain reaction, one segment-specific and two allele-specific oligonucleotide primers, and the use of a large amount of good quality DNA. This method may improve MRD-based decisions on treatment for ALL patients, and the principles should be applicable to DNA-based MRD measurements in other disorders.


Subject(s)
Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Child , DNA, Neoplasm/analysis , Fluorescence , Humans , Neoplasm, Residual , Polymerase Chain Reaction , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Sensitivity and Specificity
7.
J Mol Diagn ; 11(3): 194-200, 2009 May.
Article in English | MEDLINE | ID: mdl-19324994

ABSTRACT

Molecular markers for minimal residual disease in B-lineage acute lymphoblastic leukemia were identified by determining, at the time of diagnosis, the repertoire of rearrangements of the immunoglobulin heavy chain (IGH) gene using segment-specific variable (V), diversity (D), and junctional (J) primers in two different studies that involved a total study population of 75 children and 18 adults. This strategy, termed repertoire analysis, was compared with the conventional strategy of identifying markers using family-specific V, D, and J primers for a variety of antigen receptor genes. Repertoire analysis detected significantly more markers for the major leukemic clone than did the conventional strategy, and one or more IgH rearrangements that were suitable for monitoring the major clone were detected in 96% of children and 94% of adults. Repertoire analysis also detected significantly more IGH markers for minor clones. Some minor clones were quite large and a proportion of them would not be able to be detected by a minimal residual disease test directed to the marker for the major clone. IGH repertoire analysis at diagnosis has potential advantages for the identification of molecular markers for the quantification of minimal residual disease in acute lymphoblastic leukemia cases. An IGH marker enables very sensitive quantification of the major leukemic clone, and the detection of minor clones may enable early identification of additional patients who are prone to relapse.


Subject(s)
Cell Lineage , Gene Rearrangement, B-Lymphocyte , Immunoglobulin Heavy Chains/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Adult , Child , Cooperative Behavior , DNA, Neoplasm/genetics , Genetic Markers , Genome, Human/genetics , Humans , Neoplasm, Residual/genetics
8.
J Neurosci Nurs ; 40(2): 78-84, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18481737

ABSTRACT

The incidence of primary malignant brain tumors has remained stable over the past 10 years, with median survival reported as 12 months. Once the patient has been diagnosed, providing care for him or her is primarily performed by family members. Although previous research has documented the stress, depression, anxiety, and burden associated with caregiving, when these conditions occur is not known. The purpose of this study was to explore the caregiver perspective of providing care while the patient was receiving chemotherapy as initial treatment for the disease. Using phenomenological techniques, data were obtained from semistructured interviews with family caregivers and self-disclosed demographic data. Each interview occurred while the patient was receiving treatment; all patients were within 6 months of initial diagnosis. Interview data were analyzed using Colaizzi's method, which allowed themes universal to the participants to be uncovered. Interview data from 10 participants provided saturation and identified three themes: (a) the diagnosis of a brain tumor is a shock; (b) immediate family role changes occur; and (c) there are psychosocial effects for the caregiver, his or her family, and the person with the brain tumor.


Subject(s)
Adaptation, Psychological , Attitude to Health , Brain Neoplasms , Caregivers/psychology , Cost of Illness , Family/psychology , Adult , Anxiety/psychology , Brain Neoplasms/diagnosis , Brain Neoplasms/drug therapy , Brain Neoplasms/psychology , Caregivers/education , Depression/psychology , Female , Humans , Life Change Events , Male , Nursing Methodology Research , Qualitative Research , Role , Stress, Psychological/psychology , Surveys and Questionnaires , United States
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