Functional Evidence from Deuterated Water Labeling That the Bruton Tyrosine Kinase Inhibitor Ibrutinib Blocks Leukemia Cell Proliferation and Trafficking and Promotes Leukemia Cell Death in Patients with Chronic Lymphocytic Leukemia and small Lymphocytic Lymphoma
Authors: Burger Jan A. , Li Kelvin , Keating Michael , Sivina Mariela , Ferrajoli Alessandra , Jalayer Annette , Huang Xuelin , Kantarjian Hagop M. , Wierda William G. , O'Brien Susan , Hellerstein Marc , Turner Scott , Emson Claire , Chen Shih-Shih , Yan Xiao-Jie , Chiorazzi Nicholas 
Published: October 2019

Abstract
BACKGROUND: In patients with chronic lymphocytic leukemia (CLL), the Bruton tyrosine kinase(BTK) inhibitor ibrutinib induces a rapid reduction in lymphadenopathy, accompanied by a transient lymphocytosis. Continuation of therapy leads to a reduction in peripheral blood disease burden with achievement of objective responses in a majority of patients over time. Inhibition of B cell receptor (BCR) and chemokine receptor signaling are considered the mechanisms for redistribution lymphocytosis and CLL cell death. However, these mechanisms of action have not been formally documented directly in patients. To address this, before initiating therapy with ibrutinib, we labeled the DNA of proliferating CLL cells with deuterium (2H) by asking patients to drink deuterated water (2H2O) and then determined the effects of ibrutinib on leukemia cell kinetics (proliferation and death rates) and mobilization of cells from lymphoid tissues (trafficking) after ibrutinib administration.
METHODS: 30 previously untreated CLL/SLL patients were enrolled between December 2012 and June 2013 at MD Anderson Cancer Center. Patients required treatment per iwCLL guidelines, had adequate hematopoietic (platelets > 50,000/μL, ANC > 750/μL) and organ functions. Patients drank 50 mL of 70% 2H2O 3 times a day for 5 days, followed by 60 mL daily for a total of 4 weeks (“labeling phase”). After a 6-12 week “washout phase”, patients started once-daily ibrutinib 420 mg continuously on 28 day cycles. Study objectives were to determine the impact on CLL cell proliferation and death rates, before and after ibrutinib, and on CLL cell trafficking after ibrutinib.
PATIENT CHARACTERISTICS: Median age of the 30 patients was 64 years (range 48–78) with 19 males. 16 patients exhibited early stage (Rai stage 0-2), and 14 advanced stage (Rai stage 3-4) disease. 17 patients expressed unmutated IGHVs, 11 patients had mutated IGHVs, and 2 had inconclusive IGHV results. 8 were CD38+ and 15 ZAP-70 + by immunohistochemistry. FISH cytogenetics revealed 11 patients with del13q, 6 with trisomy 12, 3 with del17p or TP53 mutation, 4 with del11q, and 6 without abnormalities. Median β2 microglobulin level was 2.8 mg/L (1.6 - 7.4).
RESPONSE TO THERAPY: At a median follow up of 13 months, 28 of 30 patients continued on therapy without disease progression. All patients were evaluable for response assessment per 2008 IWCLL guidelines: 28 (93%) achieved partial remission, 1 (3%) complete remission, and 1 (3%) had stable disease, yielding an ORR of 97%. Two patients came off study at 184 and 480 days, respectively.
CLL CELL KINETICS AND TRAFFICKING: The average CLL cell birth rate determined before ibrutinib therapy was 0.42% per day (range 0.32 – 1.42%). After initiating ibrutinib, there was a rapid increase in absolute lymphocyte count in the blood in 26 of 30 patients; this contained previously labeled CLL cells rather than newly-divided unlabeled cells. Over time, the proportion of circulating CLL cells that were labeled did not decrease appreciably despite a concomitant fall in circulating lymphocytes, indicating that newly divided cells were not entering the circulation and strongly suggesting that ibrutinib had a major inhibitory effect on leukemia cell proliferation. In 3 of the 4 patients in whom there was a rapid influx of unlabeled cells with the onset of ibrutinib treatment, maintenance of circulating labeled cells concomitant with a significant fall in circulating lymphocytes suggested proliferation of CLL cells was inhibited with extended treatment. Prior to ibrutinib therapy, the measured proliferation rate of CLL cells was 0.42 % per day. Because after ibrutinib therapy both cell proliferation and exiting from the blood were markedly reduced, the elimination rate should equal the death rate. Notably, with treatment the elimination rate of CLL cells was much faster than before ibrutinib, revealing a “true” death rate of 1.48 % of the clone per day.
CONCLUSIONS: Using 2H-labelling of dividing leukemic cells, we have shown for the first time directly in CLL patients that ibrutinib significantly inhibits leukemia cell proliferation, leads to an efflux of CLL cells from tissues into the blood, and promotes a remarkably high death rate of CLL cells in an apparently indirect manner, likely due to the interruption of survival signals from the BCR and other receptors that are engaged in lymphoid tissues.

Disclosures
Burger: Pharmacyclics, Inc.: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Li:KineMed, Inc.: Employment, Equity Ownership. O'Brien:Pharmacyclics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Hellerstein:KineMed Inc: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Turner:KineMed, Inc.: Employment, Equity Ownership. Emson:KineMed, Inc.: Employment, Equity Ownership. Chiorazzi:Pharmacyclics, Inc.: Research Funding; KineMed: Equity Ownership.