Evaluation of Leukemic Stem Cell Persistence in Chronic Myeloid Leukemia (CML) Patients in Complete Molecular Remission Induced by First Line TKI Therapies
Authors: Chomel Jean-Claude , Bonnet Marie Laure , Sorel Nathalie , Sloma Ivan , Proust Alexis , Bennaceur-Griscelli Annelise , Rea Delphine , Uzunov Madalina , Legros Laurence , Ame Shanti , Marfaing-Koka Anne , Guerci Agnès , Rousselot Philippe , Turhan Ali G 
Published: October 2019

Abstract
Abstract 3726
Recent data showed that BCR-ABL expressing leukemic stem cells (LSCs) persist in vivo in patients in complete molecular remission (CMR) according to European Leukemia Net criteria (Chomel et al, Blood 2011, Chu et al, Blood 2011). It has also been established that, in this category of patients (CMR 4.5– 5), Imatinib Mesylate (IM) discontinuation leads to relapse in the majority of cases (60%), especially during the first 6 months. Although very likely, a formal link has not been established between these two phenomena and, in particular, the prevalence of LSC persistence in a cohort of patients treated with TKI as a first line therapy has not been established. To determine the prevalence of LSC persistence in CMR induced by first line TKI therapies, we have evaluated by clonogenic and long-term culture initiating cell (LTC-IC) assays, the presence of BCR-ABL expressing LSCs in marrow samples of 26 patients in CMR for > 2 years. CMR was induced by IM (n=22), Dasatinib (n=3) or Nilotinib (n=1) as a first line treatment. CD34+ cells were isolated from bone marrow aspirates (2–4 ml) using immunomagnetic columns and a clonogenic assay was performed. At week+2, clonogenic progenitors were counted and 20 to 40 individual hematopoietic colonies were plucked from methylcellulose for RNA extraction. In some cases, pooled CFU-Cs were also analyzed (20 pools of 10 hematopoietic colonies). The same CD34+ marrow sample was used to start LTC-IC assays, which were performed in murine MS5 cell feeders. At week+6 the cultures were sacrificed and the number of LTC-IC-derived clonogenic cells was determined. Hematopoietic colonies were plucked as decribed above for CFU-Cs for RNA extraction and BCR-ABL expression was quantified by q-RT-PCR. For 4 patients, the yield of CD34+ cells was not sufficient for stem cell assays (3 pts treated with IM; 1 with Dasatinib). qRT-PCR analyses of CFU-Cs revealed the presence of BCR-ABL expressing cells in 3 patients out of 16 tested so far. In all three patients in CMR induced by IM, this involvement can be estimated to approximately 100–360 leukemic CFU-GM/ml of bone marrow. Concerning LTC-IC assays, 2 patients out of 6 tested so far had evidence of BCR-ABL expressing LTC-IC-derived progenitors, one of these patients having also involvement of the clonogenic compartment. In both patients, there was a significant involvement of LTC-IC-derived progeny estimated to 1000–4000 LTC-IC derived progenitor/ml of bone marrow. In the 4 patients in whom LTC-IC compartment was found to be negative for LSCs, CMR was induced by IM in 3 Pts (IM 400 mg n= 2, IM 800 mg for accelerated phase n=1) and by Dasatinib (100 mg) in 1 Pt. Interestingly, in all q-RT-PCR assays, the amount of BCR-ABL mRNA (evaluated by the BCR-ABL/ABL ratio) in individual CFU-Cs appeared to be low, confirming our previous results (Chomel et al Blood 2012) and suggesting that these cells are not addict to BCR-ABL-TK activity for their survival. In 12 patients, the evaluation of LTC-IC compartment is underway. Overall, These data reveal that in patients in CMR induced by IM as a first line therapy, our strategy could identify two types of stem cell response, with some patients presenting a complete eradication of the leukemic stem cell compartment (within the limits of the sensitivity of the test) and others showing high numbers of BCR-ABL expressing stem cells despite the continuous CMR status on TKI therapy. It remains to be determined if the involvement of LTC-IC-compartment (in 2/6 analyzed so far) by LSC expressing low levels of BCR-ABL can be overcome by long-term TKI therapy in some patients. In addition, it will be of interest to determine if a correlation exists between LSC-positive patients the potential occurence of a molecular relapse upon discontinuation of TKI therapy. From the mechanistic point of view, the persistence of residual marrow LSCs in some CML patients in CMR strongly suggests the presence of active mechanisms of either LSC retention and/or LSC quiescence-promoting interactions within in the hematopoietic niche. Finally, the comparison of the LSC persistence phenomenon in CMR induced by TKI2 versus IM as first line treatment will be of major interest to determine the LSC eradication potential of these drugs.

Disclosures:
Rea: Bristol Myers-Squibb, Novartis, and Teva: Honoraria. Turhan:Novartis, Bristol Myers Squibb: Honoraria, Research Funding.