Analysis of the Early Clonal Dynamics in Ibrutinib-Treated Chronic Lymphocytic Leukemia
Authors: Spina Valeria , Diop Fary , Bruscaggin Alessio , De Paoli Lorenzo , Favini Chiara , Stathis Anastasios , Gerber Bernhard , Scarfò Lydia , Zucchetto Antonella , Deambrogi Clara , Clerici Daniela , di Trani Martina , Rinaldi Andrea , Bulian Pietro , Stella Carmelo Carlo , Zaja Francesco , Ghia Paolo , Gregor Michael , Passweg Jakob , Laurenti Luca , Zucca Emanuele , Cavalli Franco , Bertoni Francesco , Gattei Valter , Montillo Marco , Tedeschi Alessandra , Stüssi Georg , Gaidano Gianluca , Rossi Davide 
Published: October 2019

Abstract
Evolution of the chronic lymphocytic leukemia (CLL) clone upon treatment with the BTK-inhibitor ibrutinib has never been systematically assessed in a prospective way, though changes in the tumor genetic composition have been anecdotally reported by retrospective backtracking of mutations acquired in ibrutinib-refractory patients. The IOSI-EMA001 observational study (NCT02827617) enrolls patients treated with ibrutinib in the clinical practice, and aims at prospectively assessing the dynamics of the clonal architecture of high risk CLL upon therapy with ibrutinib by taking advantage of leukemia samples homogeneously collected at pre-specified timepoints during treatment course. Ibrutinib induces a transient lymphocytosis by displacing CLL cells formerly residing in the tissue compartments into the peripheral blood (PB). By analyzing samples collected after two weeks of ibrutinib treatment, here we tracked early genetic changes of the CLL clone occurring at the time of redistribution lymphocytosis. This analysis includes the first 10 patients enrolled in the study (median age=76 years; Binet A=10%, B=60%, C=30%; IGHV unmutated=60%, mutated=20%, NA=20%; 17p deletion=40%). PB samples were collected before ibrutinib treatment start (day -28 to 0) and after two weeks of treatment (week 2, +/-3 days). Tumor genomic DNA was isolated from FACS sorted CLL cells (CD19+/CD5+ elements; >99% purity). T cells were also sorted as source of germline material to confirm the somatic origin of mutations and filter out sequencing noise. The HaloPlex High Sensitivity library preparation protocol and ultra-deep next generation sequencing (coverage ~40.000x) on MiSeq (Illumina) were used to track TP53, BTK and PLCG2 mutations. By uniquely molecular barcoding each DNA library fragment, and by targeting both DNA strands, this approach allowed to reach a sensitivity of 10E-4. The CAPP-seq library preparation protocol and deep next generation sequencing (sensitivity ~5x10E-3) were used to track mutations of a panel of genes known to be recurrently affected in CLL (ASXL1, ATM, BIRC3, BRAF, EGR2, FBXW7, IKZF3, IRF4, KRAS, MAP2K1, MGA, MYD88, NFKBIE, NFKB2, NOTCH1 including 3'UTR, PAX5 enhancer, POT1, RPS15, SAMHD1, SF3B1, TP53, XPO1, ZMYM3). At baseline before treatment start, 33 non-synonymous somatic mutations (allele frequency: 0.65-99%) were identified in 9 of the 10 cases (one patient was devoid of mutations of the above-mentioned genes). The mutational profile was consistent with that expected in a high risk population meeting the current indication for ibrutinib treatment in the clinical practice(TP53=60%; NOTCH1=40%; MGA=30%; ZYMYM3=20%; ATM=10%; BIRC3=10%; EGR2=10%; IRF4=10%; IKZF3=10%; MYD88=10% NFKBIE=10%; SF3B1=10%; XPO1=10%) (Fig. 1A). Before treatment start, high sensitive ultra deep next generation sequencing did not disclose any non-synonymous somatic mutation of BTK and PLCG2 in CLL cells circulating in the blood. The genetic composition of the circulating leukemia clone did not significantly change after two weeks of treatment despite redistribution of CLL cells in the blood compartment (median lymphocyte count at baseline 50.9x10E9/L vs 77.5x10E9/L at week 2) (Fig 1B). Minor genetic changes between baseline and week 2 included the appearance of a small clone mutated in SAMHD1 and the disappearance of a small clone mutated in BIRC3 in 2 different patients. At week 2, no mutations of BTK and PLCG2 became evident in the circulating compartment. With the limitation of the current sample size, these data suggest that redistribution lymphocytosis does not mobilize CLL high risk clones harboring mutations of clinical relevance into the blood compartment.
Figure 1 Figure 1.

Disclosures
Rossi: Gilead: Honoraria, Research Funding; Janssen: Honoraria; AbbVie: Honoraria.