Ultrasensitive Quantitation of Genomic Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms

Wu, David; Kanaan, Sami B; Penewit, Kelsi; Waalkes, Adam; Urselli, Francesca; Nelson, J. Lee; Radich, Jerald P.; Salipante, Stephen J.

doi:10.1016/j.jmoldx.2021.10.005

Cited by 3 publications

(5 citation statements)

References 40 publications

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“…These assays targeted HLA polymorphisms on chromosome 6 as well as other chromosomes. 26 , 27 , 28 , 29 , 30 , 31 , 32 , 34 , 35 …”

Section: Resultsmentioning

confidence: 99%

“…In a separate study, our panel was benchmarked against a next-generation sequencing (NGS)-based chimerism technique, with an LOD comparable with ours, and showed significant agreement with the results. 32 …”

Section: Discussionmentioning

confidence: 99%

“…Donor–recipient genetic mismatches were used to measure chimerism. Our panel of highly sensitive, polymorphism-specific TaqMan qPCR assays consisted of 40 assays targeting polymorphism in HLA (n = 23) and non-HLA regions (n = 17) 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ( supplemental Table 1 ). Part of the genomic regions and molecular assay oligonucleotides are available in the patent US 10 604 805 B2.…”

Section: Methodsmentioning

confidence: 99%

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Ultrasensitive chimerism enhances measurable residual disease testing after allogeneic hematopoietic cell transplantation

et al. 2023

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Increasing mixed chimerism (reemerging recipient cells) after allogeneic hematopoietic cell transplantation (allo-HCT) can indicate relapse, the leading factor determining mortality in blood malignancies. Most clinical chimerism tests have limited sensitivity and are primarily designed to monitor engraftment. We developed a panel of qPCR assays using TaqMan chemistry capable of quantifying chimerism on the order of 1-in-a-million. At such analytic sensitivity, we hypothesized it could inform on relapse risk. As a proof-of-concept, we applied our panel on a retrospective cohort of acute leukemia patients with known outcomes post-allo-HCT. Recipient cells in bone marrow aspirates (BMA) remained detectable in 97.8% of tested samples. Absolute recipient chimerism proportions and rates at which these proportions increased in BMA in the first 540 days post-allo-HCT were associated with relapse. Detectable MRD (measurable residual disease) by flow cytometry in BMA post-allo-HCT showed limited correlation with relapse. This correlation noticeably strengthened when combined with increased recipient chimerism in BMA, demonstrating the ability of our ultrasensitive chimerism assay to augment MRD data. Our technology reveals an underappreciated usefulness of clinical chimerism. Used side-by-side with MRD assays, it promises to improve identification of patients with the highest risk of disease reoccurrence for a chance for early intervention.

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“…These assays targeted HLA polymorphisms on chromosome 6 as well as other chromosomes. 26 , 27 , 28 , 29 , 30 , 31 , 32 , 34 , 35 …”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Ultrasensitive chimerism enhances measurable residual disease testing after allogeneic hematopoietic cell transplantation

et al. 2023

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“…The main strengths of our study include our large sample size of 118 pregnancies, our access to maternal and fetal biomaterial and, finally, our established and validated 23 , 27 , 28 , 29 , 30 , 31 PCR approach to detecting and quantifying nucleated cells of fetal origin. A final strength of our study is that none of the pregnancies included had a history of trauma, and our sampling was performed prior to labor and before cesarean section, ensuring that the cells of fetal origin detected were unlikely to have resulted from fetal‐maternal cell leakage or bleeding.…”

Section: Discussionmentioning

confidence: 99%

Markers of placental function correlate with prevalence and quantity of nucleated fetal cells in maternal circulation in normotensive term pregnancies

Fjeldstad

Jacobsen

Johnsen

et al. 2023

Acta Obstet Gynecol Scand

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Introduction Transplacental fetal cell transfer results in the engraftment of fetal‐origin cells in the pregnant woman's body, a phenomenon termed fetal microchimerism. Increased fetal microchimerism measured decades postpartum is implicated in maternal inflammatory disease. Understanding which factors cause increased fetal microchimerism is therefore important. During pregnancy, circulating fetal microchimerism and placental dysfunction increase with increasing gestational age, particularly towards term. Placental dysfunction is reflected by changes in circulating placenta‐associated markers, specifically placental growth factor (PlGF), decreased by several 100 pg/mL, soluble fms‐like tyrosine kinase‐1 (sFlt‐1), increased by several 1000 pg/mL, and the sFlt‐1/PlGF ratio, increased by several 10 (pg/mL)/(pg/mL). We investigated whether such alterations in placenta‐associated markers correlate with an increase in circulating fetal‐origin cells. Material and methods We included 118 normotensive, clinically uncomplicated pregnancies (gestational age 37+1 up to 42+2 weeks‘ gestation) pre‐delivery. PlGF and sFlt‐1 (pg/mL) were measured by Elecsys® Immunoassays. We extracted DNA from maternal and fetal samples and genotyped four human leukocyte antigen loci and 17 other autosomal loci. Paternally inherited, unique fetal alleles served as polymerase chain reaction (PCR) targets for detecting fetal‐origin cells in maternal buffy coat. Fetal‐origin cell prevalence was assessed by logistic regression, and quantity by negative binomial regression. Statistical exposures included gestational age (weeks), PlGF (100 pg/mL), sFlt‐1 (1000 pg/mL) and the sFlt‐1/PlGF ratio (10 (pg/mL)/(pg/mL)). Regression models were adjusted for clinical confounders and PCR‐related competing exposures. Results Gestational age was positively correlated with fetal‐origin cell quantity (DRR = 2.2, P = 0.003) and PlGF was negatively correlated with fetal‐origin cell prevalence (odds ratio [OR]100 = 0.6, P = 0.003) and quantity (DRR100 = 0.7, P = 0.001). The sFlt‐1 and the sFlt‐1/PlGF ratios were positively correlated with fetal‐origin cell prevalence (OR1000 = 1.3, P = 0.014 and OR10 = 1.2, P = 0.038, respectively), but not quantity (DRR1000 = 1.1, P = 0.600; DRR10 = 1.1, P = 0.112, respectively). Conclusions Our results suggest that placental dysfunction as evidenced by placenta‐associated marker changes, may increase fetal cell transfer. The magnitudes of change tested were based on ranges in PlGF, sFlt‐1 and the sFlt‐1/PlGF ratio previously demonstrated in pregnancies near and post‐term, lending clinical significance to our findings. Our results were statistically significant after adjusting for confounders including gestational age, supporting our novel hypothesis that underlying placental dysfunction potentially is a driver of increased fetal microchimerism.

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“…A major limitation is the low sensitivity, as reappearance of 1-5% recipient DNA in PB is often past the window of an impending relapse (23). New methods based on real-time quantitative PCR (RQ-PCR) techniques have been developed over the past decades, using amplification of highly polymorphic targets such as single-nucleotide polymorphisms (SNP) or insertion-deletions (In/del) and, more recently, the application of digital PCR and Next Generation Sequencing (NGS) platforms (24)(25)(26)(27)(28). Clinical studies have shown that analysis of chimerism by RQ-PCR is a more precise and thus early predictor of relapse compared to standard chimerism analysis (29)(30)(31)(32)(33)(34)(35)(36)(37)(38).…”

Section: Introductionmentioning

confidence: 99%

Highly-sensitive chimerism analysis in blood after allogeneic hematopoietic cell transplantation in childhood leukemia: Results from the Nordic Microchimerism Study

Haugaard

Madsen²,

Masmas³

et al. 2023

Front. Hematol.

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Analysis of chimerism in blood post‐HCT using STR‐PCR is routinely applied in parallel with quantification of MRD to predict relapse of leukemia. Real time quantitative PCR (RQ-PCR) chimerism is 10‐ to 100‐fold more sensitive, but clinical studies in children are sparse. In a prospective multicenter study, we analyzed increasing mixed chimerism (IMC) in blood samples following transplantation for leukemia in 64 children. IMC was defined as a minimum increase of either 0.1% or 0.05% recipient DNA between two samples or a ≥10-fold increase. Samples closer than 30 days to diagnosis of relapse were omitted. The risk of relapse was higher in children with IMC of both 0.1% and 0.05% compared to children without IMC (27.8 (95% CI 4.4-175.8; P<.001), and 18.4 (95% CI 2.8-120.5; P=0.002), respectively). From the date of IMC, the 3-year CI of relapse or MRD-positivity was 26.7% (CI 9.4-47.0) and 18.5% (6.4-35.3) for IMC ≥ 0.1% (n=27) and ≥ 0.05% (n= 40), respectively. In the subset of children without an IMC ≥ 0.1% or ≥ 0.05%, CI of relapse or molecular relapse were 16.7% (5.0 -34.1) and 10.8% (3.4 -23.3), respectively. In all cases with a relapse undetectable by IMC, MRD remained undetectable prior to relapse and standard chimerism negative. In a landmark analysis, neither an IMC ≥ 0.1% nor ≥ 0.05% prior to 90 days post‐HCT was significantly associated with an increased relapse incidence. These results indicate that the serial monitoring of RQ‐PCR chimerism in peripheral blood post-HCT may be a valuable supplement to the minimal residual disease analysis for an early detection of relapse in acute childhood leukemia.

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Ultrasensitive Quantitation of Genomic Chimerism by Single-Molecule Molecular Inversion Probe Capture and High-Throughput Sequencing of Copy Number Deletion Polymorphisms

Cited by 3 publications

References 40 publications

Ultrasensitive chimerism enhances measurable residual disease testing after allogeneic hematopoietic cell transplantation

Ultrasensitive chimerism enhances measurable residual disease testing after allogeneic hematopoietic cell transplantation

Markers of placental function correlate with prevalence and quantity of nucleated fetal cells in maternal circulation in normotensive term pregnancies

Highly-sensitive chimerism analysis in blood after allogeneic hematopoietic cell transplantation in childhood leukemia: Results from the Nordic Microchimerism Study

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