title: Genomic quantitative real-time PCR proves residual disease positivity in more than 30% samples with negative mRNA-based qRT-PCR in Chronic Myeloid Leukemia.
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.
authors.affiliation: Department of Experimental and Clinical Medicine, Insubria University, Varese, Italy ; Department of Experimental Medicine and Surgery, Tor Vergata University, Rome, Italy.
authors.forename: Ilaria S
authors.initials: IS
authors.lastname: Pagani
authors.affiliation: Hematology laboratory, USC Hematology, Papa Giovanni XXIII Hospital, Bergamo, Italy.
authors.forename: Orietta
authors.initials: O
authors.lastname: Spinelli
authors.affiliation: Department of Experimental and Clinical Medicine, Insubria University, Varese, Italy.
authors.forename: Elia
authors.initials: E
authors.lastname: Mattarucchi
authors.affiliation: Department of Experimental and Clinical Medicine, Insubria University, Varese, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Pirrone
authors.affiliation: Department of Experimental and Clinical Medicine, Insubria University, Varese, Italy.
authors.forename: Diana
authors.initials: D
authors.lastname: Pigni
authors.affiliation: Department of Experimental and Clinical Medicine, Insubria University, Varese, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Amelotti
authors.affiliation: Hematology laboratory, USC Hematology, Papa Giovanni XXIII Hospital, Bergamo, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Lilliu
authors.affiliation: Hematology laboratory, USC Hematology, Papa Giovanni XXIII Hospital, Bergamo, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Boroni
authors.affiliation: Hematology laboratory, USC Hematology, Papa Giovanni XXIII Hospital, Bergamo, Italy.
authors.forename: Tamara
authors.initials: T
authors.lastname: Intermesoli
authors.affiliation: Laboratory of Medical Genetics, Papa Giovanni XXIII Hospital, Bergamo, Italy.
authors.forename: Ursula
authors.initials: U
authors.lastname: Giussani
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Caimi
authors.affiliation: Laboratory of chemical-clinical analysis, Section of Hematology and blood coagulation, Stem Cells laboratory, Spedali Civili of Brescia, Brescia, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Bolda
authors.affiliation: Laboratory of chemical-clinical analysis, Section of Hematology and blood coagulation, Stem Cells laboratory, Spedali Civili of Brescia, Brescia, Italy.
authors.forename: Renata
authors.initials: R
authors.lastname: Baffelli
authors.affiliation: Department of Experimental Medicine and Surgery, Tor Vergata University, Rome, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Candi
authors.affiliation: Department of Experimental and Clinical Medicine, Insubria University, Varese, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Pasquali
authors.affiliation: Department of Experimental and Clinical Medicine, Insubria University, Varese, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Lo Curto
authors.affiliation: Laboratory of chemical-clinical analysis, Section of Hematology and blood coagulation, Stem Cells laboratory, Spedali Civili of Brescia, Brescia, Italy.
authors.forename: Arnalda
authors.initials: A
authors.lastname: Lanfranchi
authors.affiliation: Laboratory of chemical-clinical analysis, Section of Hematology and blood coagulation, Stem Cells laboratory, Spedali Civili of Brescia, Brescia, Italy.
authors.forename: Fulvio
authors.initials: F
authors.lastname: Porta
authors.affiliation: Hematology laboratory, USC Hematology, Papa Giovanni XXIII Hospital, Bergamo, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Rambaldi
authors.affiliation: Department of Experimental and Clinical Medicine, Insubria University, Varese, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Porta
keywords: DNA Q-PCR; chronic myeloid leukemia; leukemic stem cells; minimal residual disease; stop imatinib