Ph-Abnormal Clones Emerged during Imatinib Therapy: Clinical Report and Clonal Analyses on 23 Patients from GIMEMA Working Party (GWP) in CML Registry.

Abstract

Imatinib mesylate (Glivec, Novartis) is a tyrosine kinase specific inhibitor that kills BCR-ABL cells in vitro and in vivo. Cytogenetic abnormalities in Ph-negative cells emerging after treatment-induced suppression of the neoplastic clone have been described. A registry through the GWP in CML has been set and data on 23 patients collected. To acquire insights into the origin of the Ph-negative clone as well as the evolution of the coexisting Ph− and Ph+ cell populations, we have analyzed bone marrow cell segregation, cell culture and morphologic features. Patients characteristics and 28 months follow up are presented. The emergence of a cytogenetic abnormal clone in Ph-negative cells was evidenced in 23 patients after a median of 14.5 months after starting Imatinib. Median age was 51 years, median time from diagnosis 36 months. All patients started Imatinib while in chronic phase and none of the patients had ever presented accelerated or blastic phase. Five patients were treated with Imatinb at onset. Cytogenetics at diagnosis was characterized by the presence of Ph chromosome, except for one patient which presented with normal karyotype, but BCR-ABL B3A2 transcript. No additional abnormalities were evidenced except for one patient which presented with the Ph and a dup(1q)(q11q21). All patients achieved a good response to Glivec with 16 complete, 4 major and 3 minor cytogenetic remissions when additional abnormalities were noticed in Ph-negative cells. The clonal cytogenetic abnormalities included +8 in 13 patients, -Y in 2 patients, one −7, del(5q), del(7q), del(13q), t(6;7)(p24;q21), t(2;6)(p25;q23), and one patient presenting with both +8 and +21. The patient with dup(1q) maintained the abnormality while clearing the marrow from Ph positive cells (constitutional karyotype was normal). Retrospective analyses of stored pellet using FISH in patients presenting +8, −7, or −Y, did not evidence abnormalities in previous samples. Patients that lost cytogenetic response showed that the percentage of the Ph+ cells inversely correlated to the abnormal clone. In 5 patients the abnormal clone was not evidenced in subsequent controls, suggesting the possibility that the abnormalities could be temporary. We performed cell culture on a subgroup of patients demonstrating normal growth in four patients and an abnormal growth in one patient with reduced CFU formation affecting BFU-Es, CFU-GM, and colony size microclusters. FISH analyses on separated CD34+ and CD34-negative cells evidenced that the abnormal clone segregated into the CD34+ compartment suggesting the stem cells involvement. FISH on cultured cells did not demonstrate a growth advantage for Ph+ cells or for the new clone. Bone marrow biopsies presented with reduced cellularity, normal differential and mild dysplastic signs as documented in patients responding to Imatinib. No increased angiogenesis was evidenced. While a longer follow up observation and laboratory analyses are required, we remark that after >2 years follow up the Ph-negative abnormal clone did not tend in our patients to evolve in MDS, nor it seems to be associated with CML clonal evolution and disease progression. Hypothesis regarding the biological significance of these abnormalities are formulated.