title: Molecular analysis of hematopoietic colonies derived from chronic myeloid leukemia patients: interphase fluorescence in situ hybridization compared with RT-PCR.
abstract: In this study we compared interphase fluorescence in situ hybridization (I-FISH) with reverse transcription polymerase chain reaction (RT-PCR) for the molecular analysis of hematopoietic colonies derived from patients with chronic myeloid leukemia (CML). Molecular analysis of individual colonies is often performed to monitor purging efficacy in CML. We harvested individual colony-forming unit granulocyte-macrophage (CFU-GM) colonies. One half was analyzed with I-FISH, for the presence of bcr-abl fusion gene. The other half was analyzed with RT-PCR for the presence of the bcr-abl mRNA. We wanted to address the following questions: (1) is the bcr-abl gene always expressed in CFU-GM colonies and (2) which technique has to be preferred to analyze individual CFU-GM colonies? In total, 133 colonies, derived from six CML patients, could be analyzed both with I-FISH and RT-PCR. We found a positive correlation in 89% of the cases: 118 colonies showed the same results with both techniques. However, 15 of the 106 I-FISH-positive colonies were negative in the RT-PCR. Serial analysis of the cDNA derived from 22 colonies showed in each round of amplification 21-29% RT-PCR-negative but I-FISH-positive colonies. However, all I-FISH-positive colonies showed at least one positive RT-PCR, either in the first, second or third round of amplification. These results indicate that the bcr-abl gene is probably always transcriptionally active in CFU-GM colonies. Reliable analysis with RT-PCR is possible but likely to generate false negative results. We conclude that: (1) I-FISH offers a reliable alternative to RT-PCR for analyzing individual hematopoietic colonies and (2) results obtained with RT-PCR should only be interpreted with caution.
authors.affiliation: Department of Hematology, Free University Hospital, Amsterdam, The Netherlands.
authors.forename: S F
authors.initials: SF
authors.lastname: Thijsen
authors.forename: G J
authors.initials: GJ
authors.lastname: Schuurhuis
authors.forename: J W
authors.initials: JW
authors.lastname: van Oostveen
authors.forename: A P
authors.initials: AP
authors.lastname: Theijsmeijer
authors.forename: M M
authors.initials: MM
authors.lastname: Langenhuijsen
authors.forename: G J
authors.initials: GJ
authors.lastname: Ossenkoppele
mesh_terms: D000328:Adult; D001853:Bone Marrow / Q000473:pathology*; D016465:Bone Marrow Purging; D002999:Clone Cells / Q000378:metabolism / Q000473:pathology; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D016044:Fusion Proteins, bcr-abl / Q000032:analysis / Q000235:genetics; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007399:Interphase; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics / Q000473:pathology*; D015466:Leukemia, Myeloid, Chronic-Phase / Q000235:genetics / Q000473:pathology*; D008297:Male; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012334:RNA, Neoplasm / Q000096:biosynthesis / Q000235:genetics; D012680:Sensitivity and Specificity; D014410:Tumor Stem Cell Assay
chemical_list: D018076:DNA, Complementary; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D016179:Granulocyte Colony-Stimulating Factor; D016044:Fusion Proteins, bcr-abl