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MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR

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Lymphoma

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1956))

Abstract

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL) and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. MRD diagnostics performed by real-time quantitative PCR (RQ-PCR) is still the gold standard and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma with comparable sensitivity, accuracy, and reproducibility.

The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy chain gene (IGHV). Chromosomal translocations like the t(14;18) translocation in FL and t(11;14) translocation in MCL can be used as MRD target in selected lymphoma subtypes. In patients with B-cell chronic lymphocytic leukemia, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10−4 shall be achieved.

MRD diagnostics targeting the IGHV gene is complex and requires extensive knowledge and experience because the junctional regions of each lymphoma have to be identified before the patient-specific RQ-PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation (SHM) within the rearranged IG heavy variable (IGHV) gene occurring as during B-cell development of germinal center and post-germinal center lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. RQ-PCR-based methods can reach a good sensitivity (≤10−4). This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IGHV gene rearrangement and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results.

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Notes

  1. 1.

    ddPCR should be repeated on more replicates (≥  6, based on the sample availability) to verify if the results are reproducible.

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Acknowledgment

We gratefully acknowledge Anne-Wiebke Kruse for her excellent technical support.

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Authors and Affiliations

  1. Second Medical Department, University Hospital Schleswig-Holstein, Kiel, Germany

    Christiane Pott, Monika Brüggemann, Matthias Ritgen & Michael Kneba

  2. Department of Immunology, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands

    Vincent H. J. van der Velden

  3. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands

    Jacques J. M. van Dongen

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  1. Christiane Pott
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  2. Monika Brüggemann
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  4. Vincent H. J. van der Velden
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Correspondence to Christiane Pott .

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  1. Institute of Cell Biology (Cancer Research), Medical School, University of Duisburg-Essen, Nordrhein-Westfalen, Essen, Germany

    Ralf Küppers

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Pott, C., Brüggemann, M., Ritgen, M., van der Velden, V.H.J., van Dongen, J.J.M., Kneba, M. (2019). MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR. In: Küppers, R. (eds) Lymphoma. Methods in Molecular Biology, vol 1956. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9151-8_9

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