Genetic Screening Test to Detect Translocations in Acute Leukemias by Use of Targeted Locus Amplification

Alimohamed, Mohamed Zahir; Johansson, Lennart; Boer, Eddy N. de; Splinter, Erik; Klous, Petra; Yılmaz, Mehmet; Bosga, Anneke; Min, Max van; Mulder, André B.; Vellenga, Edo; Sinke, Richard J.; Sijmons, Rolf H.; Berg, Eva van den; Sikkema‐Raddatz, Birgit

doi:10.1373/clinchem.2017.286047

Cited by 3 publications

(5 citation statements)

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“…Considering these recurrent regions with copy number aberrations, it would be interesting to explore a multiplex approach of TLA/TLC for neuroblastoma, targeting several regions often carrying amplifications. This approach has shown its potential for detection of translocations in acute leukemia ( 26 ).…”

Section: Discussionmentioning

confidence: 99%

“…TLA/TLC is a technique that uses crosslinking of physically proximal sequences to selectively amplify and sequence regions of >100 kb surrounding specific primer or probe binding sites without prior detailed locus information. TLA can be applied to cells, while TLC is optimized for formalin fixed paraffin embedded (FFPE) material (24)(25)(26)(27). The breakpoint sequence revealed by the TLA/TLC technique can be used to design an assay that targets the patient-specific breakpoint.…”

Section: Introductionmentioning

confidence: 99%

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Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors

et al. 2023

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BackgroundLiquid biopsies combine minimally invasive sample collection with sensitive detection of residual disease. Pediatric malignancies harbor tumor-driving copy number alterations or fusion genes, rather than recurrent point mutations. These regions contain tumor-specific DNA breakpoint sequences. We investigated the feasibility to use these breakpoints to design patient-specific markers to detect tumor-derived cell-free DNA (cfDNA) in plasma from patients with pediatric solid tumors.Materials and methodsRegions of interest (ROI) were identified through standard clinical diagnostic pipelines, using SNP array for CNAs, and FISH or RT-qPCR for fusion genes. Using targeted locus amplification (TLA) on tumor organoids grown from tumor material or targeted locus capture (TLC) on FFPE material, ROI-specific primers and probes were designed, which were used to design droplet digital PCR (ddPCR) assays. cfDNA from patient plasma at diagnosis and during therapy was analyzed.ResultsTLA was performed on material from 2 rhabdomyosarcoma, 1 Ewing sarcoma and 3 neuroblastoma. FFPE-TLC was performed on 8 neuroblastoma tumors. For all patients, at least one patient-specific ddPCR was successfully designed and in all diagnostic plasma samples the patient-specific markers were detected. In the rhabdomyosarcoma and Ewing sarcoma patients, all samples after start of therapy were negative. In neuroblastoma patients, presence of patient-specific markers in cfDNA tracked tumor burden, decreasing during induction therapy, disappearing at complete remission and re-appearing at relapse.ConclusionWe demonstrate the feasibility to determine tumor-specific breakpoints using TLA/TLC in different pediatric solid tumors and use these for analysis of cfDNA from plasma. Considering the high prevalence of CNAs and fusion genes in pediatric solid tumors, this approach holds great promise and deserves further study in a larger cohort with standardized plasma sampling protocols.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors

et al. 2023

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“…Since its first publication, TLA approach has been employed in different contexts such as transgene detection, vector design, and novel SNVs identification, thus resulting in a promising technology also for onco-hematology [24][25][26]. In this context, the application of a multiplex TLA, as a marker screening tool, showed promising results in acute leukemia through detection of cryptic rearrangements and multiple (un)known translocated genes involved in leukemia pathogenesis [27,28].…”

Section: Introductionmentioning

confidence: 99%

Targeted Locus Amplification as Marker Screening Approach to Detect Immunoglobulin (IG) Translocations in B-Cell Non-Hodgkin Lymphomas

Genuardi

Alessandria

Civita

et al. 2022

Methods in Molecular Biology

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Although MRD monitoring by the classic polymerase chain reaction (PCR) approach is a powerful outcome predictor, about 20% of mantle cell lymphoma (MCL) and 50% of follicular lymphoma (FL) patients still lack a molecular marker and are thus resulting not eligible for MRD monitoring. Targeted locus amplification (TLA), a new NGS technology, has been revealed as a feasible marker screening approach able to identify uncommon B-cell leukemia/lymphoma 1 (BCL1) and B-cell leukemia/lymphoma 2 (BCL2) rearrangements in MCL and FL cases defined as having “no marker” by the classic PCR approach.

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“…TLA is a strategy to selectively amplify and sequence regions of >100 kb around a preselected primer pair by cross-linking of physically proximal genomic sequences and is highly suitable for the detection of (balanced) chromosomal rearrangements in leukaemia samples. [8][9][10] Combined with next-generation sequencing, the TLA technique directly reveals genomic breakpoints that can be used to design genomic targets for MRD. 8 In the present study, we aimed to determine the applicability of genomic breakpoints from leukaemia-specific FG and deletions (FG/DEL) for MRD.…”

Section: Introductionmentioning

confidence: 99%

“…In a subset of cases, a suitable FG has already been identified by standard karyotyping, fluorescent in situ hybridisation (FISH) or RNA sequencing, which allows a targeted approach for genomic breakpoint sequencing, like targeted locus amplification (TLA). TLA is a strategy to selectively amplify and sequence regions of >100 kb around a preselected primer pair by cross‐linking of physically proximal genomic sequences and is highly suitable for the detection of (balanced) chromosomal rearrangements in leukaemia samples 8–10 . Combined with next‐generation sequencing, the TLA technique directly reveals genomic breakpoints that can be used to design genomic targets for MRD 8 …”

Section: Introductionmentioning

confidence: 99%

Minimal residual disease (MRD) detection in acute lymphoblastic leukaemia based on fusion genes and genomic deletions: towards MRD for all

et al. 2021

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Minimal residual disease (MRD) diagnostics are implemented in most clinical protocols for patients with acute lymphoblastic leukaemia (ALL) and are mostly performed using rearranged immunoglobulin (IG) and/or T-cell receptor (TR) gene rearrangements as molecular polymerase chain reaction targets. Unfortunately, in 5-10% of patients no or no sensitive IG/TR targets are available, and patients therefore cannot be stratified appropriately. In the present study, we used fusion genes and genomic deletions as alternative MRD targets in these patients, which retrospectively revealed appropriate MDR stratification in 79% of patients with no (sensitive) IG/TR target, and a different risk group stratification in more than half of the cases.

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Genetic Screening Test to Detect Translocations in Acute Leukemias by Use of Targeted Locus Amplification

Abstract: Multiplex TLA shows promising results as an acute leukemia screening test. It can detect cryptic and other translocations in selected genes. Further optimization may make this assay suitable for diagnostic use.

Cited by 3 publications

References 28 publications

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors

Targeted locus amplification to develop robust patient-specific assays for liquid biopsies in pediatric solid tumors

Targeted Locus Amplification as Marker Screening Approach to Detect Immunoglobulin (IG) Translocations in B-Cell Non-Hodgkin Lymphomas

Minimal residual disease (MRD) detection in acute lymphoblastic leukaemia based on fusion genes and genomic deletions: towards MRD for all

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