Comparison between automated FISH analysis and RQ-PCR as monitoring tools for minimal residual disease (MRD) in CML.

Comparison between automated FISH analysis and RQ-PCR as monitoring tools for minimal residual disease (MRD) in CML. G. Calabrese^1,2,3, D. Fantasia¹, F. Pompetti⁴, R. DiGianfilippo³, D. Romagno¹, E. Morizio¹, P. Guanciali-Franchi^1,3, M. Alfonsi^1,3, C. Nuzzi³, R. DiLorenzo⁵, A. Iacone⁴, G. Fioritoni⁵, G. Palka^1,3 1) Scienze Biomed/Genetica Medica, Univ G D'Annunzio, Chieti, Italy; 2) Center for Ageing, DAnnunzio Foundation, Chieti, Italy; 3) Servizio di Genetica Umana; 4) Dip. Med Trasfusionale; 5) Dip. Ematologia, Ospedale di Pescara, Italy.

To compare RQ-PCR method, to date the gold standard approach for MRD monitoring in CML, with a novel FISH analysis approach based on a fully automated FISH slide scanner and image analyzer (Duet BioView, Israel), we investigated 51 CML patients in clinical and cytogenetic remission following imatinib therapy (IM; 44 patients), or hematopoietic stem cell transplantation (7 patients). Ninety samples, 75 bone marrow aspirates and 15 peripheral blood withdrawals, were tested with both RQ-PCR using TaqMan protocol (Applied BioSystems, USA), and FISH slide scanner by scoring 1600-4500 cells for BCR-ABL rearrangement using a dual-fusion FISH probe (Kreatech, DK). Leukemic cell levels in the samples were arbitrarily grouped in 3 classes: 1%; 0.99%-0.04%; and <0.04% of scored cells. FISH/RQ-PCR concordance was 100% for class 1%, 96% for class 0.99%-0.04%, and 84% for class <0.04% leukemic cells. Samples with FISH/RQ-PCR discordant results showed leukemic cells as evidenced by FISH close to the detection limit of FISH procedure (i.e. 0.04%) with <0.01% Bcr-Abl transcript level and molecular remission in the following 18 months of treatment. In 2 patients FISH unravelled 3/1900 (0.16%), and 8/3800 (0.21%) leukemic cells carrying 2 copies of BCR-ABL fusion, i.e. double Ph, which were undistinguishable from those with a single copy of BCR-ABL rearrangement as by RQ-PCR. IM dose escalation (800mg/day) resulted in disappearance of double BCR-ABL leukemic cells, which are still absent 30 and 38 months from high-dose therapy start, respectively. In conclusion, automated FISH results largely overlapped with RQ-PCR data. Furthermore, double Ph-positive cells could also be early recognized by automated FISH analysis allowing appropriate therapy protocol modification.