title: 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.
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.
  
  
  METHODS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Clinical Biochemistry, Hematology and Immunology, Na Homolce Hospital, Prague, Czech Republic. sona.pekova@homolka.cz
authors.forename: Sona
authors.initials: S
authors.lastname: Peková
authors.forename: Ludmila
authors.initials: L
authors.lastname: Bezdícková
authors.forename: Lukás
authors.initials: L
authors.lastname: Smolej
authors.forename: Tomás
authors.initials: T
authors.lastname: Kozák
authors.forename: Ivana
authors.initials: I
authors.lastname: Hochová
authors.forename: Pavel
authors.initials: P
authors.lastname: Zák
authors.forename: Lucie
authors.initials: L
authors.lastname: Tomsíková
authors.forename: Miroslav
authors.initials: M
authors.lastname: Průcha
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000235:genetics / Q000378:metabolism; D000368:Aged; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics*; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011446:Prospective Studies; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000235:genetics / Q000378:metabolism
chemical_list: D007143:Immunoglobulin Heavy Chains; D016159:Tumor Suppressor Protein p53; D051746:ZAP-70 Protein-Tyrosine Kinase; D051997:ADP-ribosyl Cyclase 1