Long Term Follow up on Patients with Ph-Abnormal Clones Emerged during Imatinib Therapy.
Authors: Abruzzese Elisabetta , Gozzetti Alessandro , Testoni Nicoletta , Zaccaria Alfonso , Radaelli Franca , Galimberti Sara , Trawinska Malgorzata M. , Mancini Marco , Ferrero Dario , Salvio Michela , Sora’ Federica , Crugnola Monica , Cedrone Michele , Rosti Gianantonio , Amadori Sergio , de Fabritiis Paolo , Baccarani Michele 
Published: October 2019

Abstract
Imatinib (Glivec, Novartis) is a tyrosine kinase specific inhibitor used for the treatment of CML. The occurrence of cytogenetic abnormalities in Ph-negative cells emerging after suppression of the Ph-positive clone has been described; however the origin as well as the biological and clinical significance are unknown. We collected data on 32 patients through the GWP in CML registry. Bone marrow cell segregation, cell culture and morphologic features in a subgroup of these patients were studied to acquire insights into the origin of the Ph-negative clone. Patient characteristics and clinical follow up (up to 49 months) are presented together with hypothesis regarding their biological significance. The emergence of a cytogenetic abnormal clone in Ph-negative cells was evidenced in 32 patients after a median of 16.2 months after starting Imatinib. Median age was 51 years, F:M=18:14, median time from CML diagnosis 35 months. All patients but one have started Imatinib while in chronic phase and were in chronic phase at detection of the abnormal Ph-negative clone. Eight patients were treated with Imatinb at onset. At diagnosis 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 21 complete, 5 major and 6 minor cytogenetic remissions when additional abnormalities were noticed in Ph-negative cells. The clonal cytogenetic abnormalities included +8 in 14 patients, −Y in 5 patients,, three del(20q), two del(5q) and del(7q), one −7, del(13q), t(6;7)(p24;q21), t(2;6)(p25;q23), with one patient presenting with both +8 and +21, and one three clones with +8, double +8, and double +8 and −X. Retrospective analyses of stored pellet using FISH 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 7 patients the abnormal clone was not evidenced in subsequent controls, suggesting the possibility that the abnormalities could be temporary. Two patients that lost response to Glivec were treated with a second generation tyrosine kinase inhibitor Dasatinib (Sprycel, Bristol-Myers Squibb) and, at reduction of Ph+ clone, the del (7q) reappeared in one patient, while the +8 clone of the second patient was not further evidenced. FISH analyses on separated CD34+ and CD34-negative cells evidenced that the abnormal clone was present into the CD34+ compartment suggesting the stem cells involvement. 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. We performed cell culture on a subgroup of 6 patients demonstrating normal growth in five patients and an abnormal growth pattern in one patient with reduced CFU formation affecting BFU-Es, CFU-GM, and colony size microclusters. While a longer follow up observation and laboratory analyses are required, we remark that after >4 years follow up the Ph-negative abnormal clone did not tend in our patients to evolve in MDS/AML, nor it seems to be associated with CML clonal evolution and disease progression.