Efficacy and Safety of Hydroxychloroquine Sulphate In Chronic Lymphocytic Leukemia: Clinical Trial Experience In Untreated Patients
Authors: Chen Shih-Shih , Kaufman Matthew , Caramanica Angelica , Damle Rajendra N , Garofalo Peter , Ly Patricia , Janson Dale , Allen Steven L. , Kolitz Jonathan , Quill Linda , Rai Kanti R. , Chiorazzi Nicholas 
Published: October 2019

Abstract
Abstract 1392

Background:
Hydroxychloroquine (HCQ) has been used safely for many years for treatment of malaria, systemic lupus erythematosus and rheumatoid arthritis. It is known to have exerted immunomodulatory actions by affecting antigen presentation by macrophages and dendritic cells, DNA/RNA synthesis, and apoptosis in lymphocytes and endothelial cells. All of these actions can be attributed at least partially to the inactivation of endosomal acid proteases.
CLL is the abnormal proliferation of a B lymphocyte clone. Based on the unique structural characteristics of the B-cell antigen receptor (BCR), at least some CLL cases probably results from ongoing antigen selection and stimulation. RNA and/or DNA from infection or cell apoptosis are candidate stimulants of this process. Binding of RNA and DNA to toll-like receptors (TLR 7 and 9) in endosomes is dependent on an acidic pH and can be prevented by HCQ due to its de-acidifiying effect. HCQ can also directly induce apoptosis in CLL cells through caspase 3 activation in vitro. We therefore hypothesized that administration of HCQ early in the disease would protect CLL patients from disease progression by eliminating these stimulatory TLR 7 and 9 signals.

Methods:
22 CLL patients with no urgent need for treatment were enrolled in the clinical trial. Patients took HCQ 400 mg/day for 52 weeks. Clinical response was measured every 4 weeks by history, physical exam, and absolute lymphocyte counts. Additional blood samples were taken every 8 weeks for companion laboratory studies. Statistical analyses used include Fisher's Exact Test, Slopes analysis and Repeated measures analysis of variance on the log-transformed absolute lymphocyte counts (ALCs).

Results:
In total, 8 IGHV unmutated (U-CLL) and 14 mutated (M-CLL) patients were enrolled; 3 U-CLL and 10 M-CLL completed the full year of HCQ treatment. Five U-CLL patients were dropped from the study due to disease progression requiring CLL therapy. One M-CLL patient left for personal reasons. Three M-CLL patients are still continuing on HCQ therapy.
Patient outcomes can be separated into three patterns based on ALC changes: 7 patients (6 M-CLL and 1 U-CLL) had stable counts throughout the trial; 12 patients (8 M-CLL and 4 U-CLL) had a fall in ALCs after treatment; and 3 patients (all U-CLL) developed increased ALCs. The response to HCQ in CLL is associated with Ig V mutation status of patients (P<.03). With some U-CLL patients having a worsening of disease; overall U-CLL patients have worse outcome than M-CLL (P<.045). Nevertheless in most instances, U-CLL patients did respond to HCQ with stabilization or fall in ALC initially, although this was followed at an average of 18–22 weeks with an increase in ALCs and in some instances a need for therapy.
Maximum HCQ blood concentrations in healthy individuals receiving 400 mg/day HCQ orally are reported to range from 67–221 ng/ml. In vitro assay using samples collected at trial initiation indicated that direct CLL cytotoxicity occurred at levels ≥10 μg/ml; this value was similar for U- and M-CLL clones. Using a non-cytotoxic concentration of 1 μg/ml of HCQ, in vitro TLR9 ligand ODN2006 enhanced expression of TLR9 was diminished. The proliferation and activation of both U- and M-CLL was blocked completely, although concentrations of 10–100 ng/ml had a superior blocking effect on M-CLL than U-CLL clones.
In vivo administration of HCQ blocked CLL cell proliferation initiated by ODN2006. In comparison with samples taken before HCQ administration, patient samples obtained after trial initiation responded to ODN2006 poorly. Interestingly, the type of CLL cell response to ODN2006 closely correlated with patient's ALC change. The least blocking of HCQ on cell proliferation was again observed in U-CLL patients, especially with the samples taken from the timepoint at which elevated ALCs were observed.

Conclusion:
In this clinical trial, oral administration of HCQ (400 mg/day) stabilized or decreased ALCs in the initial 18–22 weeks (of a 46–52 week regimen) in 91% of patients (14 M-CLL and 6-U-CLL). Subsequently, rising ALCs were observed in most of the U-CLL cases (n=4), and ALCs in M-CLL patients remain stabilized. Our in vitro evaluation suggests that an increased dose of HCQ might target TLR9 signaling better and thus improve outcome especially in U-CLL cases. Another possibility that warrants further study is that HCQ may have several actions on CLL cells leading to worse outcome of the disease.

Disclosures:
No relevant conflicts of interest to declare.