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Combined use of reverse transcriptase polymerase chain reaction and flow cytometry to study minimal residual disease in Philadelphia positive acute lymphoblastic leukemia.
Haematologica. 2000 Jul; 85(7):704-10.H

Abstract

BACKGROUND AND OBJECTIVES

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.

DESIGN AND 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.

INTERPRETATION AND 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+Show Affiliations

Hematology Division, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

10897122

Citation

Muñoz, L, et al. "Combined Use of Reverse Transcriptase Polymerase Chain Reaction and Flow Cytometry to Study Minimal Residual Disease in Philadelphia Positive Acute Lymphoblastic Leukemia." Haematologica, vol. 85, no. 7, 2000, pp. 704-10.
Muñoz L, López O, Martino R, et al. Combined use of reverse transcriptase polymerase chain reaction and flow cytometry to study minimal residual disease in Philadelphia positive acute lymphoblastic leukemia. Haematologica. 2000;85(7):704-10.
Muñoz, L., López, O., Martino, R., Brunet, S., Bellido, M., Rubiol, E., Sierra, J., & Nomdedéu, J. F. (2000). Combined use of reverse transcriptase polymerase chain reaction and flow cytometry to study minimal residual disease in Philadelphia positive acute lymphoblastic leukemia. Haematologica, 85(7), 704-10.
Muñoz L, et al. Combined Use of Reverse Transcriptase Polymerase Chain Reaction and Flow Cytometry to Study Minimal Residual Disease in Philadelphia Positive Acute Lymphoblastic Leukemia. Haematologica. 2000;85(7):704-10. PubMed PMID: 10897122.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Combined use of reverse transcriptase polymerase chain reaction and flow cytometry to study minimal residual disease in Philadelphia positive acute lymphoblastic leukemia. AU - Muñoz,L, AU - López,O, AU - Martino,R, AU - Brunet,S, AU - Bellido,M, AU - Rubiol,E, AU - Sierra,J, AU - Nomdedéu,J F, PY - 2000/7/18/pubmed PY - 2000/10/21/medline PY - 2000/7/18/entrez SP - 704 EP - 10 JF - Haematologica JO - Haematologica VL - 85 IS - 7 N2 - BACKGROUND AND OBJECTIVES: 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. DESIGN AND 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. INTERPRETATION AND 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. SN - 0390-6078 UR - https://www.unboundmedicine.com/medline/citation/10897122/Combined_use_of_reverse_transcriptase_polymerase_chain_reaction_and_flow_cytometry_to_study_minimal_residual_disease_in_Philadelphia_positive_acute_lymphoblastic_leukemia_ L2 - http://www.diseaseinfosearch.org/result/188 DB - PRIME DP - Unbound Medicine ER -