title: Touch-down reverse transcriptase-PCR detection of IgV(H) rearrangement and Sybr-Green-based real-time RT-PCR quantitation of minimal residual disease in patients with chronic lymphocytic leukemia.
abstract: 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.
  
  
  OBJECTIVE
  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.
authors.affiliation: Institute of Hematology and Blood Transfusion, Prague, Czech RepublicInstitute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic. pekova@img.cas.cz
authors.forename: Sona
authors.initials: S
authors.lastname: Peková
authors.forename: Jana
authors.initials: J
authors.lastname: Marková
authors.forename: Petr
authors.initials: P
authors.lastname: Pajer
authors.forename: Michal
authors.initials: M
authors.lastname: Dvorák
authors.forename: Petr
authors.initials: P
authors.lastname: Cetkovský
authors.forename: Jirí
authors.initials: J
authors.lastname: Schwarz
mesh_terms: D052160:Benzothiazoles; D003959:Diamines; D005260:Female; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis*; D008297:Male; D018365:Neoplasm, Residual; D009930:Organic Chemicals / Q000032:analysis / Q000737:chemistry; D011804:Quinolines; D012334:RNA, Neoplasm / Q000032:analysis / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D009930:Organic Chemicals; D011804:Quinolines; D012334:RNA, Neoplasm; C098022:SYBR Green I