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Application of droplet digital PCR in minimal residual disease monitoring of rare fusion transcripts and mutations in haematological malignancies.

ABSTRACT: Leukaemia of various subtypes are driven by distinct chromosomal rearrangement or genetic abnormalities. The leukaemogenic fusion transcripts or genetic mutations serve as molecular markers for minimal residual disease (MRD) monitoring. The current study evaluated the applicability of several droplet digital PCR assays for the detection of these targets at RNA and DNA levels (atypical BCR::ABL1 e19a2, e23a2ins52, e13a2ins74, rare types of CBFB::MYH11 (G and I), PCM1::JAK2, KMT2A::ELL2, PICALM::MLLT10 fusion transcripts and CEBPA frame-shift and insertion/duplication mutations) with high sensitivity. The analytical performances were assessed by the limit of blanks, limit of detection, limit of quantification and linear regression. Our data demonstrated serial MRD monitoring for patients at molecular level could become "digitalized", which was deemed important to guide clinicians in treatment decision for better patient care.

SUBMITTER: Ip BBK

PROVIDER: S-EPMC10944481 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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Application of droplet digital PCR in minimal residual disease monitoring of rare fusion transcripts and mutations in haematological malignancies.

Ip Beca B K BBK Wong Anthony T C ATC Law Janet Hei Yin JHY Au Chun Hang CH Ma Shing Yan SY Chim James C S JCS Liang Raymond H S RHS Leung Anskar Y H AYH Wan Thomas S K TSK Ma Edmond S K ESK

Scientific reports 20240316 1

Leukaemia of various subtypes are driven by distinct chromosomal rearrangement or genetic abnormalities. The leukaemogenic fusion transcripts or genetic mutations serve as molecular markers for minimal residual disease (MRD) monitoring. The current study evaluated the applicability of several droplet digital PCR assays for the detection of these targets at RNA and DNA levels (atypical BCR::ABL1 e19a2, e23a2ins52, e13a2ins74, rare types of CBFB::MYH11 (G and I), PCM1::JAK2, KMT2A::ELL2, PICALM::M ...[more]

PMID: 38493200

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Project description:BackgroundAlu repeats, belonging to the Short Interspersed Repetitive Elements (SINEs) class, contain about 25% of CpG sites in the human genome. Alu sequences lie in gene-rich regions, so their methylation is an important transcriptional regulation mechanism. Aberrant Alu methylation has been associated with tumor aggressiveness, and also previously discussed in hematological malignancies, by applying different approaches. Moreover, today different techniques designed to measure global DNA methylation are focused on the methylation level of specific repeat elements. In this work we propose a new method of investigating Alu differential methylation, based on droplet digital PCR (ddPCR) technology.MethodsForty-six patients with hematological neoplasms were included in the study: 30 patients affected by chronic lymphocytic leukemia, 7 patients with myelodysplastic syndromes at intermediate/high risk, according with the International Prognostic Scoring System, and 9 patients with myelomonocytic leukemia. Ten healthy donors were included as controls. Acute promyelocytic leukemia-derived NB4 cell line, either untreated or treated with decitabine (DEC) hypomethylating agent, was also analyzed. DNA samples were investigated for Alu methylation level by digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation, followed by ddPCR.ResultsUsing ddPCR, a significant decrease of the global Alu methylation level in DNA extracted from NB4 cells treated with DEC, as compared to untreated cells, was observed. Moreover, comparing the global Alu methylation levels at diagnosis and after azacytidine (AZA) treatment in MDS patients, a statistically significant decrease of Alu sequences methylation after therapy as compared to diagnosis was evident. We also observed a significant decrease of the Alu methylation level in CLL patients compared to HD, and, finally, for CMML patients, a decrease of Alu sequences methylation was observed in patients harboring the SRSF2 hotspot gene mutation c.284C>D.ConclusionsIn our work, we propose a method to investigate Alu differential methylation based on ddPCR technology. This assay introduces ddPCR as a more sensitive and immediate technique for Alu methylation analysis. To date, this is the first application of ddPCR to study DNA repetitive elements. This approach may be useful to profile patients affected by hematologic malignancies for diagnostic/prognostic purpose.

Project description:BackgroundFebrile haematological patients are at high risk for potential bloodstream infections, the rapid and accurate identification of pathogens is crucial for clinical diagnosis and treatment. Droplet Digital PCR (ddPCR) is a novel and ultra-sensitively molecular technique for the rapid detection of pathogens. We evaluated the ability of ddPCR to identify infectious etiologies to discuss the applicability of ddPCR in the diagnosis and treatment of infections for febrile haematological patients.MethodsThis study enrolled and analyzed 89 ddPCR tests performed on 71 febrile haematological patients. We conducted a comparison between ddPCR results, blood culture (BC), and conventional microbiological testing (CMT). Additionally, we analyzed the correlation between ddPCR results and inflammatory factors, as well as their impact on antimicrobial therapy.ResultsDdPCR detected 113 pathogens in 72 plasma samples, while CMT identified 39 pathogens in 32 plasma samples. The detection rate of bacteria and viruses using ddPCR was significantly higher than that of CMT (p <0.0001). The turnaround time (TAT) for pathogenic diagnosis was significantly shorter with ddPCR compared to CMT (p <0.0001). When we used the CMT as reference standard, the sensitivity and specificity of ddPCR were 93.8%, 26.3%, respectively. We observed a positive correlation between the ddPCR results and CRP, PCT and IL-6, and ddPCR (AUC=0.771) has better diagnostic performance. The anti-infective treatment strategies were adjusted for 30 patients based on the positive ddPCR results, with 86.7% (26/30) of these cases demonstrating effectiveness in the anti-infective treatment.ConclusionDdPCR has the potential to enhance pathogen detection in febrile haematological patients by offering high sensitivity, rapid, precise results, it demonstrates better diagnostic performance compared to inflammatory factors and can contribute to the real-time clinical optimization of antimicrobial regimens, thereby enhancing the efficacy of anti-infective therapy.

Project description:BackgroundThyroid cancer is the most frequent malignancy of the endocrine system, of which papillary thyroid cancer (PTC) is the predominant form with a rapid increasing incidence worldwide. Rearranged during transfection (RET) fusions are common genetic drivers of PTC and the potent RET inhibitor selpercatinib has been recently approved for treating advanced or metastatic RET fusion-positive thyroid cancer. In this study we aimed to develop a droplet digital PCR (ddPCR) system to accurately detect RET fusion in PTC samples.MethodsThe frequency and distribution of RET fusions in PTC were analyzed using genomic data of 402 PTC patients in The Cancer Genome Atlas (TCGA) database. To establish the ddPCR system for detecting CCDC6::RET fusion, a plasmid containing CCDC6::RET infusion fragment was constructed as standard template, the annealing temperature and concentrations of primers and probe were optimized. The analytical performance of ddPCR and quantitative reverse transcription PCR (qRT-PCR) were assessed in standard templates and tissue samples from 112 PTC patients. Sanger sequencing was performed in all the RET fusion-positive samples identified by ddPCR.ResultsRET fusions were observed in 25 (6.2%) of the 402 TCGA samples, and 15 (60%) of the RET fusion-positive patients had the CCDC6::RET fusion. Compared with qRT-PCR, the ddPCR method showed a lower limit of detection (128.0 and 430.7 copies/reaction for ddPCR and qRT-PCR, respectively). When applying the two methods to 112 tissue samples of PTC, eleven (9.8%) CCDC6::RET fusion-positive samples were detected by qRT-PCR, while ddPCR identified 4 additional positive samples (15/112, 13.4%). All the CCDC6::RET fusion-positive cases identified by ddPCR were confirmed by Sanger sequencing except for one case with 0.14 copies/uL of the fusion.ConclusionThe accurate and sensitive ddPCR method reported here is powerful to detection CCDC6::RET fusion in PTC samples, application of this method would benefit more RET fusion-positive patients in the clinic.

Dataset Information

Application of droplet digital PCR in minimal residual disease monitoring of rare fusion transcripts and mutations in haematological malignancies.

Publications

Application of droplet digital PCR in minimal residual disease monitoring of rare fusion transcripts and mutations in haematological malignancies.

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