Abstract A04: Measuring autophagy inhibition as a pharmacodynamic response to hydroxychloroquine in a spontaneous canine lymphoma clinical trial
Authors: Barnard Rebecca A. , Wittenburg Luke A. , Gustafson Daniel L. , Thorburn Andrew , Thamm Douglas H. 
Published: May 2015

Abstract
Background: (Macro) Autophagy is the bulk, lysosomal degradation process of organelles and long-lived cytosolic proteins via sequestration into double membraned vesicles called autophagosomes. In recent years, autophagy inhibition has become an attractive, anti-cancer therapy as numerous studies have provided evidence for autophagy as a mechanism of survival and resistance. The most widely used autophagy inhibitors in clinical trials are chloroquine (CQ) or hydroxychloroquine (HCQ), due to their well-established safety profiles after decades long use as anti-malarial therapeutics. However, since measuring autophagy modulation is difficult in vivo, there is still uncertainty as to whether autophagy inhibition by CQ or HCQ is achievable in tumors. In order to determine the efficacy of HCQ in combination with doxorubicin (DOX) and to obtain measures of pharmacodynamic (PD) response, a clinical trial for canine patients with lymphoma was undertaken. Dogs present a unique model for studying human cancer as they inhabit the same environment and their cancers also arise spontaneously. In addition, canine clinical trials progress more rapidly than human trials, and samples can be collected more readily in dogs.
Material and Methods: After an initial phase I dose escalation study, twelve canine patients presenting with lymphoma were enrolled into the study. Patients received 12.5 mg/kg daily HCQ, orally, and received their first round of 25 mg/m2 DOX treatment 3 days later. This dose is a 20% reduction from the standard 30 mg/m2, as dose-limiting neutropenia was observed at the full DOX dose in the dose escalation study. DOX was delivered IV every 21 days, for five treatments. Whole blood, serum, plasma, lymph node aspirates, and two punch biopsies, were collected prior to HCQ administration and 3 days after HCQ, but before DOX delivery. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and were immediately processed for flow cytometry, Western blot (WB), or electron microscopy (EM) analysis. Lymph node aspirates and PBMCs were stained with an LC3 antibody, a marker for autophagosomes, and analyzed by flow cytometry. Western analysis for LC3 and p62, a selective substrate for autophagy degradation, was performed on PBMCs and punch biopsies. Mass spectrometry analysis was used to determine HCQ levels in the plasma and remaining biopsy.
Results: HCQ was detectable in both plasma and tumor after administration, with tumor levels about 300 times higher than that of plasma. However, HCQ levels varied widely and no correlation was observed between plasma and tumor levels. Patients with at least a 1.5 fold increase in LC3 I or II, as determined by WB, had significantly higher tumor HCQ levels (p=0.028). Increases in LC3 expression, indicative of inhibited autophagy, also correlated to increased p62 expression (p=0.034 Pearson r = 0.745). Yet, there was no correlation of LC3 expression in PBMCs compared with tumor, nor was there a correlation between plasma HCQ levels and LC3 expression. Additionally, there was no correlation between LC3 expression in PBMCs, as determined by flow cytometry, to expression determined by WB. Autophagosomes were visible by EM in the PBMCs after HCQ treatment.
Conclusions: Autophagy inhibition by HCQ was only achieved in those patients with the highest tumor HCQ levels. Flow cytometry does not appear to accurately reflect PD response and it is recommended to use WB or EM analysis instead. Lastly, plasma does not appear to be a good surrogate for tumor PK/PD assessment, and thus, should not be used in lieu of tumor assessment.
Citation Format: Rebecca A. Barnard, Luke A. Wittenburg, Daniel L. Gustafson, Andrew Thorburn, Douglas H. Thamm. Measuring autophagy inhibition as a pharmacodynamic response to hydroxychloroquine in a spontaneous canine lymphoma clinical trial. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A04.