title: Combined use of reverse transcriptase polymerase chain reaction and flow cytometry to study minimal residual disease in Philadelphia positive acute lymphoblastic leukemia.
abstract: OBJECTIVE
  The Philadelphia chromosome in acute lymphoblastic leukemia (Ph+ ALL) is associated with a poor prognosis given the high frequency of chemoresistance and leukemia relapse. Minimal residual disease (MRD) detection before cytogenetic and hematologic relapse could be useful in early therapy. The most suitable methods for detecting MRD in Ph+ ALL are flow cytometry (FC) and reverse transcriptase polymerase chain reaction (RT-PCR). However, since both techniques carry the risk of false-negative results the combined use of these two techniques could overcome this problem.
  
  
  METHODS
  We report our experience using this approach in 47 bone marrow samples obtained from 10 Ph+ ALL patients. Twenty-seven marrow aspirates were taken from patients in clinical remission (CR). The samples were considered positive for MRD by FC when two conditions were met: 1) detection of an abnormal B-cell differentiation pattern and 2) presence of more than 1x10(-3) cells coexpressing CD22/CD34/CD45 or CD66/CD34/CD10. After FC analysis, RNA was purified using standard methods.
  
  
  RESULTS
  FC was positive in 23/27 samples in CR (sensitivity 85%). RT-PCR was successfully performed in 23 samples in CR. RT-PCR was positive in 18/23 samples (sensitivity 78%). There were 5 samples with discordant results. FC was positive in 3 samples with a negative RT-PCR and FC was negative in 2 samples with a positive RT. All the 10 patients relapsed and only 1 is currently alive after an allogeneic stem cell transplantation (alloSCT). The median (range) time from MRD detection to relapse in patients treated with chemotherapy was 42 (39-71) days.
  
  
  CONCLUSIONS
  These data suggest that RT-PCR may be negative despite the presence of neoplastic cells identified by their immunophenotypic traits. We conclude that immunologic and molecular techniques can be used in tandem for monitoring MRD in Ph+ ALL.
authors.affiliation: Hematology Division, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Muñoz
authors.forename: O
authors.initials: O
authors.lastname: López
authors.forename: R
authors.initials: R
authors.lastname: Martino
authors.forename: S
authors.initials: S
authors.lastname: Brunet
authors.forename: M
authors.initials: M
authors.lastname: Bellido
authors.forename: E
authors.initials: E
authors.lastname: Rubiol
authors.forename: J
authors.initials: J
authors.lastname: Sierra
authors.forename: J F
authors.initials: JF
authors.lastname: Nomdedéu
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D015703:Antigens, CD / Q000032:analysis; D001854:Bone Marrow Cells / Q000276:immunology; D005188:False Negative Reactions; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D016044:Fusion Proteins, bcr-abl / Q000235:genetics / Q000276:immunology; D006801:Humans; D016130:Immunophenotyping; D008297:Male; D008875:Middle Aged; D018365:Neoplasm, Residual / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology; D012313:RNA / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D015703:Antigens, CD; D012313:RNA; D016044:Fusion Proteins, bcr-abl