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 real-time 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.

• MeSH
• antigeny CD38 genetika   metabolismus   MeSH
• chronická lymfatická leukemie diagnóza   genetika   MeSH
• diagnostické techniky molekulární metody   MeSH
• hybridizace nukleových kyselin metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• nádorový supresorový protein p53 genetika   metabolismus   MeSH
• polymerázová řetězová reakce metody   MeSH
• prospektivní studie MeSH
• protein-tyrosinkináza ZAP-70 genetika   metabolismus   MeSH
• průtoková cytometrie MeSH
• reprodukovatelnost výsledků MeSH
• reziduální nádor diagnóza   genetika   MeSH
• senioři MeSH
• senzitivita a specificita MeSH
• těžké řetězce imunoglobulinů genetika   metabolismus   MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny CD38 MeSH
• nádorový supresorový protein p53 MeSH
• protein-tyrosinkináza ZAP-70 MeSH
• těžké řetězce imunoglobulinů MeSH

BACKGROUND: Patients with chronic lymphocytic leukemia (CLL) can relapse even after aggressive therapy and autografts. It is commonly assumed that to prevent relapse the level of minimal residual disease (MRD) should be as low as possible. To evaluate MRD, highly sensitive quantitative assays are needed. AIM: The aim of the study was to develop a robust and sensitive method for detection of the clonal immunoglobulin heavy-chain variable (IgV(H)) rearrangement in CLL and to introduce a highly sensitive and specific methodology for MRD monitoring in patients with CLL who undergo intensive treatment. METHODS: As a prerequisite for MRD detection, touch-down reverse transcriptase (RT)-PCR using degenerate primers were used for the diagnostic identification of (H) gene rearrangement(s). For quantitative MRD detection in 18 patients, we employed a real-time RT-PCR assay (RQ-PCR) making use of patient-specific primers and the cost-saving Sybr-Green reporter dye (SG). For precise calibration of RQ-PCR, patient-specific IgV(H) sequences were cloned. RESULTS: Touch-down RT-PCR with degenerate primers allowed the successful detection of IgV(H) clonal rearrangement(s) in 252 of 257 (98.1%) diagnostic samples. Biallelic rearrangements were found in 27 of 252 (10.7%) cases. Degenerate primers used for the identification of clonal expansion at diagnosis were not sensitive enough for MRD detection. In contrast, our RQ-PCR assay using patient-specific primers and SG reached the sensitivity of 10(-)(6). We demonstrated MRD in each patient tested, including four of four patients in complete remission following autologous hematopoietic stem cell transplantation (HSCT) and three of three following allogeneic 'mini'-HSCT. Increments in MRD might herald relapse; aggressive chemotherapy could induce molecular remission. CONCLUSIONS: Our touch-down RT-PCR has higher efficiency to detect clonal IgV(H) rearrangements including the biallelic ones. MRD quantitation of IgV(H) expression using SG-based RQ-PCR represents a highly specific, sensitive, and economic alternative to the current quantitative methods.

• MeSH
• benzothiazoly MeSH
• chinoliny MeSH
• chronická lymfatická leukemie diagnóza   MeSH
• diaminy MeSH
• lidé MeSH
• organické látky analýza   chemie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí metody   MeSH
• přestavba genů pro těžké řetězce B-lymfocytů genetika   MeSH
• reziduální nádor MeSH
• RNA nádorová analýza   chemie   MeSH
• senzitivita a specificita MeSH
• těžké řetězce imunoglobulinů genetika   MeSH
• variabilní oblast imunoglobulinu genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• benzothiazoly MeSH
• chinoliny MeSH
• diaminy MeSH
• organické látky MeSH
• RNA nádorová MeSH
• SYBR Green I MeSH Prohlížeč
• těžké řetězce imunoglobulinů MeSH
• variabilní oblast imunoglobulinu MeSH