| 1. |
- Arthur, Cecilia, et al.
(författare)
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Simultaneous Ultra-Sensitive Detection of Structural and Single Nucleotide Variants Using Multiplex Droplet Digital PCR in Liquid Biopsies from Children with Medulloblastoma
- 2023
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Ingår i: Cancers. - : MDPI. - 2072-6694. ; 15:7
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Tidskriftsartikel (refereegranskat)abstract
- Medulloblastoma is one of the most common types of brain tumors in children. During and after treatment with surgery, chemotherapy, and/or radiotherapy, children with this disease are monitored with imaging and cerebrospinal fluid analysis for the detection of tumor cells. These methods are not always sensitive or specific enough to confirm or rule out residual disease. Here, we develop a laboratory test based on the genetic makeup of medulloblastomas in 12 children. We analyze liquid biopsies (cerebrospinal fluid and blood plasma) for specific genetic fragments leaking from the individual tumors and find molecular traces of disease in 75% (9/12) of children overall. None of the children had malignant cells in the cerebrospinal fluid. We propose that this test could open up new technical possibilities to track measurable residual disease in children with medulloblastoma in order to further risk-adapt treatment, but first, larger studies of the approach at standardized time points are warranted.Medulloblastoma is a malignant embryonal tumor of the central nervous system (CNS) that mainly affects infants and children. Prognosis is highly variable, and molecular biomarkers for measurable residual disease (MRD) detection are lacking. Analysis of cell-free DNA (cfDNA) in cerebrospinal fluid (CSF) using broad genomic approaches, such as low-coverage whole-genome sequencing, has shown promising prognostic value. However, more sensitive methods are needed for MRD analysis. Here, we show the technical feasibility of capturing medulloblastoma-associated structural variants and point mutations simultaneously in cfDNA using multiplexed droplet digital PCR (ddPCR). Assay sensitivity was assessed with a dilution series of tumor in normal genomic DNA, and the limit of detection was below 100 pg of input DNA for all assays. False positive rates were zero for structural variant assays. Liquid biopsies (CSF and plasma, n = 47) were analyzed from 12 children with medulloblastoma, all with negative CSF cytology. MRD was detected in 75% (9/12) of patients overall. In CSF samples taken before or within 21 days of surgery, MRD was detected in 88% (7/8) of patients with localized disease and in one patient with the metastasized disease. Our results suggest that this approach could expand the utility of ddPCR and complement broader analyses of cfDNA for MRD detection.
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| 2. |
- Haider, Zahra, et al.
(författare)
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Whole-genome informed circulating tumor DNA analysis by multiplex digital PCR for disease monitoring in B-cell lymphomas : a proof-of-concept study
- 2023
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Ingår i: Frontiers in Oncology. - : Frontiers Media SA. - 2234-943X. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- IntroductionAnalyzing liquid biopsies for tumor-specific aberrations can facilitate detection of measurable residual disease (MRD) during treatment and at follow-up. In this study, we assessed the clinical potential of using whole-genome sequencing (WGS) of lymphomas at diagnosis to identify patient-specific structural (SVs) and single nucleotide variants (SNVs) to enable longitudinal, multi-targeted droplet digital PCR analysis (ddPCR) of cell-free DNA (cfDNA). MethodsIn 9 patients with B-cell lymphoma (diffuse large B-cell lymphoma and follicular lymphoma), comprehensive genomic profiling at diagnosis was performed by 30X WGS of paired tumor and normal specimens. Patient-specific multiplex ddPCR (m-ddPCR) assays were designed for simultaneous detection of multiple SNVs, indels and/or SVs, with a detection sensitivity of 0.0025% for SV assays and 0.02% for SNVs/indel assays. M-ddPCR was applied to analyze cfDNA isolated from serially collected plasma at clinically critical timepoints during primary and/or relapse treatment and at follow-up. ResultsA total of 164 SNVs/indels were identified by WGS including 30 variants known to be functionally relevant in lymphoma pathogenesis. The most frequently mutated genes included KMT2D, PIM1, SOCS1 and BCL2. WGS analysis further identified recurrent SVs including t(14;18)(q32;q21) (IGH::BCL2), and t(6;14)(p25;q32) (IGH::IRF4). Plasma analysis at diagnosis showed positive circulating tumor DNA (ctDNA) levels in 88% of patients and the ctDNA burden correlated with baseline clinical parameters (LDH and sedimentation rate, p-value <0.01). While clearance of ctDNA levels after primary treatment cycle 1 was observed in 3/6 patients, all patients analyzed at final evaluation of primary treatment showed negative ctDNA, hence correlating with PET-CT imaging. One patient with positive ctDNA at interim also displayed detectable ctDNA (average variant allele frequency (VAF) 6.9%) in the follow-up plasma sample collected 2 years after final evaluation of primary treatment and 25 weeks before clinical manifestation of relapse. ConclusionIn summary, we demonstrate that multi-targeted cfDNA analysis, using a combination of SNVs/indels and SVs candidates identified by WGS analysis, provides a sensitive tool for MRD monitoring and can detect lymphoma relapse earlier than clinical manifestation.
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| 3. |
- Sivars, Lars, et al.
(författare)
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Cell-Free Human Papillomavirus DNA Is a Sensitive Biomarker for Prognosis and for Early Detection of Relapse in Locally Advanced Cervical Cancer
- 2024
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Ingår i: Clinical Cancer Research. - : American Association For Cancer Research (AACR). - 1078-0432 .- 1557-3265. ; 30:13, s. 2764-2771
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Tidskriftsartikel (refereegranskat)abstract
- Purpose: Human papillomavirus (HPV) is the cause of the majority of cervical cancer cases and has been showed to be released as cell-free tumor DNA (ctHPV DNA) into the circulation. Here, we analyze if ctHPV DNA could be used as a prognostic biomarker and/or to detect relapse earlier than traditional methods in locally advanced cervical cancer (LACC).Experimental Design: A total of 74 patients with LACC were included; 66of 74 were positive for 13 high-risk HPV types on a bead-based assay of tumor biopsy samples. HPV-type-specific droplet digital PCR assays were developed. Longitudinal plasma samples were then analyzed for the biopsy-verified HPV type for each patient. In total, 418 plasma samples were analyzed. Patients were followed for a median of 37 months. Results were correlated to tumor and clinical characteristics.Results: Of the pretreatment plasma samples, 92.4% were positive for ctHPV DNA. Persistent ctHPV DNA in end-of-treatment, early follow-up (1-2 months after end-of-treatment), or tumor evaluation (3-4 months after end-of-treatment) plasma was correlated with worse progression-free survival (P < 0.001) compared with if ctHPV DNA was not found. The positive predictive value of ctHPV status at early follow-up for predicting disease progression was 87.5%, and the negative predictive value was 89.3%. ctHPV DNA was found in plasma before relapse was diagnosed using radiology in all patients (n = 10) who experienced relapse after complete clinical response to treatment with a median 315 days lead time.Conclusions: ctHPV DNA in follow-up plasma is a promising prognostic biomarker in patients with LACC, useful for analysis of response to therapy and for early detection of relapse.
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