Molecular analysis of hematopoietic colonies derived from chronic myeloid leukemia patients: interphase fluorescence in situ hybridization compared with RT-PCR

Autor: A.P. Theijsmeijer, Gert J. Ossenkoppele, S.F.T. Thijsen, M. M. A. C. Langenhuijsen, J.W. van Oostveen, Gerrit-Jan Schuurhuis
Rok vydání: 1997
Předmět:
Adult
Male
Cancer Research
DNA
Complementary

Myeloid
Fusion Proteins
bcr-abl

Biology
Polymerase Chain Reaction
Sensitivity and Specificity
law.invention
Fusion gene
Bone Marrow
law
Leukemia
Myelogenous
Chronic
BCR-ABL Positive

hemic and lymphatic diseases
Granulocyte Colony-Stimulating Factor
medicine
Humans
RNA
Messenger

RNA
Neoplasm

Interphase
In Situ Hybridization
Fluorescence

Tumor Stem Cell Assay
Polymerase chain reaction
medicine.diagnostic_test
Bone Marrow Purging
Myeloid leukemia
Hematology
Middle Aged
Hematopoietic Stem Cells
Molecular biology
Bone marrow purging
Clone Cells
Neoplasm Proteins
Reverse transcription polymerase chain reaction
medicine.anatomical_structure
Real-time polymerase chain reaction
Oncology
Leukemia
Myeloid
Chronic-Phase

Female
Fluorescence in situ hybridization
Zdroj: Leukemia. 11:301-305
ISSN: 1476-5551
0887-6924
DOI: 10.1038/sj.leu.2400563
Popis: 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.
Databáze: OpenAIRE