Genomic quantitative real-time PCR proves residual disease positivity in more than 30% samples with negative mRNA-based qRT-PCR in Chronic Myeloid Leukemia
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https://doi.org/10.18632/oncoscience.65
Ilaria S. Pagani1,2, Orietta Spinelli3, Elia Mattarucchi1, Cristina Pirrone1, Diana Pigni1, Elisabetta Amelotti1, Silvia Lilliu3, Chiara Boroni3, Tamara Intermesoli3, Ursula Giussani4, Luigi Caimi5, Federica Bolda6, Renata Baffelli6, Eleonora Candi2, Francesco Pasquali1, Francesco Lo Curto1, Arnalda Lanfranchi6, Fulvio Porta6, Alessandro Rambaldi3 and Giovanni Porta1
1 Department of Experimental and Clinical Medicine, Insubria University, Varese, Italy
2 Department of Experimental Medicine and Surgery, Tor Vergata University, Rome, Italy
3 Hematology laboratory, USC Hematology, Papa Giovanni XXIII Hospital, Bergamo, Italy
4 Laboratory of Medical Genetics, Papa Giovanni XXIII Hospital, Bergamo, Italy
5 Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
6 Laboratory of chemical-clinical analysis, Section of Hematology and blood coagulation, Stem Cells laboratory, Spedali Civili of Brescia, Brescia, Italy
Correspondence:
Giovanni Porta , email:
Correspondence:
Ilaria Stefania Pagani, email:
Keywords: chronic myeloid leukemia, minimal residual disease, stop imatinib, leukemic stem cells, DNA Q-PCR
Received: July 6, 2014 Accepted: July 23, 2014 Published: July 23, 2014
Abstract
Imatinib mesylate (IM) is the first line therapy against Chronic Myeloid Leukemia, effectively prolonging overall survival. Because discontinuation of treatment is associated with relapse, IM is required indefinitely to maintain operational cure. To assess minimal residual disease, cytogenetic analysis is insensitive in a high background of normal lymphocytes. The qRT-PCR provides highly sensitive detection of BCR-ABL1 transcripts, but mRNA levels are not directly related to the number of leukemic cells, and undetectable results are difficult to interpret. We developed a sensitive approach to detect the number of leukemic cells by a genomic DNA (gDNA) Q-PCR assay based on the break-point sequence, with a formula to calculate the number of Ph-positive cells. We monitored 8 CML patients treated with IM for more than 8 years. We tested each samples by patient specific gDNA Q-PCR in parallel by the conventional techniques. In all samples positive for chimeric transcripts we showed corresponding chimeric gDNA by Q-PCR, and in 32.8% (42/128) of samples with undetectable levels of mRNA we detected the persistence of leukemic cells.
The gDNA Q-PCR assay could be a new diagnostic tool used in parallel to conventional techniques to support the clinician’s decision to vary or to STOP IM therapy.
