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Quantitation of Minimal Residual Disease in Patients with Chronic Lymphocytic Leukemia Using Locked Nucleic Acid-Modified, Fluorescently Labeled Hybridization Probes and Real-Time PCR Technology

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Abstract

Background: The knowledge of biological characteristics of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) remains sparse. There are no data available on what level of MRD might be ‘safe’ without an overt risk of relapse, or whether any such level exists at all. To address this issue in prospective studies, we have developed a quantitative molecular approach to monitor MRD in CLL, which allows the malignant clone to be traced with far higher sensitivity than possible with the techniques available currently.

Method: To quantify MRD in CLL patients, a novel locked nucleic acid (LNA)-RNA-based quantitative realtime PCR technique was developed. Clone-specific assays were prepared for 62 CLL patients. Thirty patients were followed up molecularly for a median of 250 days (range 69–570 days). All patients were administered chemo/immunotherapy.

Results: In three patients, molecular negativity was achieved, as estimated by LNA-based assays. In one patient, a sustained molecular negativity was established by chemo/immunotherapy and the patient remains molecularly negative (322 days). The LNA-based assay enabled us to evaluate MRD in a reproducible manner with the sensitivity of 10−7.

Conclusion: LNA-RNA-based quantitative real-time PCR is an effective approach for MRD monitoring with the potential for increased sensitivity compared with standard DNA-based assays used for molecular follow-up.

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Acknowledgments

We are indebted to our clinical collaborators, Drs Milada Jankovská and Olga Černá, who were involved in the minimal residual disease monitoring study, for precise clinical monitoring of chronic lymphocytic leukemia patients and for sending their samples for molecular investigations. We would like to thank Andy C. Rawstron for support and counseling in flow cytometry, and Peter Mouritzen, Exiqon, Denmark, for introducing Dr. Peková to the concept of locked nucleic acid technology. The work was supported by a grant from the Ministry of Health of the Czech Republic (Grant NR 9186–30).

The authors have no conflicts of interest that are directly relevant to the content of this study.

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Authors and Affiliations

  1. Laboratory of Molecular Diagnostics, Department of Clinical Biochemistry, Hematology and Immunology, Na Homolce Hospital, Prague 5, 150 30, Czech Republic

    Soňa Peková, Tomáš Kozák, Lucie Tomšiková & Miroslav Průcha

  2. Department of Clinical Hematology, Faculty Hospital Královské Vinohrady and Third Faculty of Medicine, Charles University, Prague, Czech Republic

    Ludmila Bezdîčková & Tomáš Kozák

  3. Department of Clinical Hematology, Second Department of Internal Medicine, University Hospital and Medical School, Hradec Králové, Czech Republic

    Lukáš Smolej & Pavel Zák

  4. Department of Clinical Hematology, Faculty Hospital Motol, Prague, Czech Republic

    Ivana Hochová

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  1. Soňa Peková
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  2. Ludmila Bezdîčková
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  3. Lukáš Smolej
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  4. Tomáš Kozák
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  5. Ivana Hochová
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  6. Pavel Zák
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  7. Lucie Tomšiková
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  8. Miroslav Průcha
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Correspondence to Soňa Peková.

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Peková, S., Bezdîčková, L., Smolej, L. et al. Quantitation of Minimal Residual Disease in Patients with Chronic Lymphocytic Leukemia Using Locked Nucleic Acid-Modified, Fluorescently Labeled Hybridization Probes and Real-Time PCR Technology. Mol Diag Ther 11, 325–335 (2007). https://doi.org/10.1007/BF03256253

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  • DOI: https://doi.org/10.1007/BF03256253

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