Autophagy inhibition to augment mTOR inhibition: A phase I/II trial of everolimus and hydroxychloroquine in patients with previously treated renal cell carcinoma

Abstract

Purpose

Everolimus inhibits the mechanistic target of rapamycin (mTOR), activating cytoprotective autophagy. Hydroxychloroquine (HCQ) inhibits autophagy. Based on preclinical data demonstrating synergistic cytotoxicity when mTOR inhibitors are combined with an autophagy inhibitor, we launched a clinical trial of combined everolimus and HCQ, to determine its safety and activity in patients with clear cell renal carcinoma (ccRCC).

Experimental Design

Three centers conducted a phase I/II trial of everolimus 10 mg daily and HCQ in patients with advanced ccRCC. The objectives were to determine the maximum tolerated dose of HCQ with daily everolimus, and to estimate the rate of 6 month progression-free survival (PFS) in ccRCC patients receiving everolimus/HCQ after 1–3 prior treatment regimens. Correlative studies to identify patient subpopulations that achieved the most benefit included population pharmacokinetics, measurement of autophagosomes by electron microscopy and next generation tumor sequencing.

Results

No DLT was observed in the phase I trial. The recommended phase II dose of HCQ 600 mg bid with everolimus was identified. Disease control (Stable disease (SD) + partial response (PR)) occurred in 22/33 (67%) evaluable patients. Partial response was observed in 2/33 patients (6%). PFS ≥6 months was achieved in 15/33 (45%) of patients who achieved disease control.

Conclusion

Combined HCQ 600mg twice daily with 10 mg daily everolimus was tolerable. The primary endpoint of >40% 6 month PFS rate was met. HCQ is a tolerable autophagy inhibitor in future RCC or other trials.

Introduction

Everolimus inhibits the mechanistic target of rapamycin (mTOR), blocking a key downstream effector of growth factor signaling. Inhibition of mTOR causes a rapid decline in protein translation, including key glucose and amino acid transporters, resulting in abrogation of extracellular nutrient uptake. Thus, through inhibition of mTOR, everolimus is a potent initiator of metabolic stress for cancer cells (1).

A phase III randomized controlled trial (2) of everolimus 10 mg daily versus placebo in patients with advanced clear cell renal carcinoma (ccRCC), who progressed on an approved tyrosine kinase inhibitor (TKI) demonstrated a median PFS of 4.01 months for everolimus treated compared to 1.87 months for placebo treated patients (hazard ratio 0.30, 95% CI 0.22–0.40, p<0·0001)(2). Consequently, everolimus was commercially approved for the treatment of ccRCC after failure of treatment with sunitinib or sorafenib. Although safe and well tolerated, the 1% response rate and 4-month median PFS with everolimus reflects modest activity of this mTOR inhibitor on RCC as a single agent (2).

Autophagy is an intracellular process characterized by the formation of autophagic vesicles (AV), which sequester cytoplasmic contents and target them for degradation in lysosomes. This process is activated by metabolic stress and also by most cancer therapies (3, 4). Autophagy is used by cancer cells to remove damaged organelles and recycle macromolecules that serve as an internal reservoir of fuel in the face of a crisis of nutrient availability. Inhibition of mTOR is one of the most potent inducers of autophagy. Therapeutic activation of autophagy may be a key resistance mechanism to mTOR inhibition with everolimus. Our initial in vivo studies demonstrate that inhibition of therapy-induced autophagy with chloroquine derivatives enhances cell death and tumor regression in a mouse lymphoma model (5). Further, combining agents that target mTOR signaling with HCQ results in enhanced cell death compared to each single agent alone in multiple cancer types (6, 7). Synergistic cell death was observed when the rapamycin analog temsirolimus was combined with HCQ in renal cell carcinoma cell lines and in an orthotopic mouse model of renal cell carcinoma (8). Additionally, we demonstrated the safety and preliminary activity of combining temsirolimus and HCQ in patients with solid tumors (9). In this phase Ib study the highest FDA-approved dose of HCQ 600 mg po bid was combined safely with temsirolimus. We hypothesized that combining mTOR and autophagy inhibition would be most effective in a disease such as ccRCC, where the mTOR inhibitor everolimus has single agent activity. Based on this hypothesis, we conducted a multi-institution open labelled phase I/II trial of everolimus in combination with HCQ in patients with advanced ccRCC.

Materials and Methods

Patients

Three centers (University of Pennsylvania Abramson Cancer Center, the University of Pittsburgh, and Rutgers Cancer Institute of New Jersey) enrolled patients with histological evidence of metastatic renal cell carcinoma. For the phase I portion of the trial, patients could have any number of prior therapies and any renal cell carcinoma histology, but in the phase II portion, patients were required to have renal cell carcinoma with some clear cell features and have previously received 1–3 prior regimens, including a vascular endothelial growth factor receptor-tyrosine kinase inhibitor (VEGF-TKI).

This study was conducted in accordance with the U.S. Common Rule and received institutional review board approval at each respective center with informed written consent obtained from each subject. All patients had at least one measurable site of disease per RECIST 1.1 criteria (10), as well as normal organ function and an ECOG performance status of 0–2. Also patients had fasting serum cholesterol ≤300 mg/dL or ≤7.75 mmol/L and fasting triglycerides ≤ 2.5 × ULN, due to anticipated disruption of the glucose and lipid axis from everolimus.

For the phase I portion of the trial, patients received 10 mg everolimus for one week followed by the addition of 400mg (dose level 1) or 600mg (dose level 2) HCQ twice daily beginning one week later. These doses continued for the duration of therapy. Cycle 1 included a one-week run-in of everolimus alone in order to obtain pharmacodynamic (PD) markers. In subsequent cycles, both everolimus and HCQ were given without interruption. Cycle length for the first cycle for all patients was 35 days. All subsequent cycles were 28 days of therapy. Imaging was obtained every 2 cycles for assessment of response. Response was assessed by RECIST 1.1 criteria (10). Toxicity was assessed using the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Additional blood samples were obtained for pharmacokinetic and biomarker analyses described below. In addition, available archival tissue was obtained for predictive biomarker studies.

Definition of Dose Limiting Toxicity (DLT)

DLTs were defined by toxicity occurring during the first 5 weeks of this study in the phase I portion of the study. A DLT was any non-hematologic AE of Grade 3 or higher that was at least possibly treatment-related with the exception of nausea and vomiting not treated with optimal anti-emetic therapy. For hematologic toxicity, a DLT was defined as grade 4 neutropenia lasting more than 7 days or febrile neutropenia or platelet count less than 25,000/mm³.

Any DLT that caused a patient to miss > 28 consecutive days of HCQ resulted in the patient being taken off treatment. A DLT was defined as a grade 3 or 4 toxicity considered at least possibly related to HCQ. Known toxicities specific to everolimus such as rash were not considered dose limiting unless the treating physician considered the toxicity to be exacerbated by HCQ. Toxicities attributable to HCQ included but were not limited to nausea, vomiting, diarrhea, and visual field deficit.

In the phase I portion of the study, the target DLT rate was ≤ 33%. The MTD was defined as a) the dose producing DLT in 2 out of 6 patients, or b) the dose level below the dose which produced DLT in ≥ 2 out of 3 patients, or in ≥ 3 out of 6 patients. Patients were evaluable for a DLT if they finished 5 weeks of combined therapies. Patients removed from study due to clinical progression prior to the 5 week period in the phase I portion were replaced. Patients were evaluable for response if they completed 90% of their expected dose of HCQ for the 8 weeks. Patients were evaluable toxicity if they had received at least one dose of HCQ.

Dose Escalation Rules

If a DLT was observed in 1 patient per cohort the cohort was expanded to 6. If a DLT occurred in 2 or more patients per cohort, then the cohort one dose below was the declared MTD provided that at least 6 patients had been treated at that level with no more than one third having DLTs. No intra-patient dose escalation was allowed.

Correlative methods

HCQ population pharmacokinetics:

This was conducted as previously described (14). Briefly, whole blood was collected in tubes containing sodium heparin, and stored at −70 °C until analysis. Whole blood concentrations of HCQ were measured using high-performance liquid chromatography with tandem mass spectrometry detection. Sample aliquots containing 500 ng of internal standard (IS) (d4-HCQ) were vortexed with acetonitrile/methanol, then centrifuged. An aliquot of the supernatant was withdrawn, dried under nitrogen gas, then reconstituted with mobile phase and 10 uL injected onto a Kinetex 50 × 3 mm 2.6 um 100A HPLC column (Phenomenex, Torrance, CA). Samples were eluted with a gradient mobile phase of 0.1% formic acid in acetonitrile and water using a 1200 Series Agilent HPLC system with an API 4000™ (AB SCIEX, Foster City, CA) mass spectrometer and electrospray interface operated in positive mode with multiple reaction monitoring detection. The capillary voltage was 4000 V with a source temperature of 500 °C. Mass spectrometer parameters were adjusted to maximize the intensity of the [M + H]⁺ ions in quadrupole 1 and the m/z transition ions of HCQ (337.275 → 248.152) and IS (341.150 → 252.035) in quadropole 3.

The mass spectrometers were controlled by AB SCIEX Analyst® software (Version 1.6.1) and data collection and analyses were conducted with the same software. Standard curves were constructed by plotting the analyte to IS ratio vs. the known concentration of HCQ (x) in each sample. Standard curves were fit by linear regression with weighting by 1/x. Samples were assayed in duplicate; samples for which the percent difference exceeded 15% were reanalyzed and samples for which concentrations exceeded the range values for the calibration curve were diluted appropriately and reanalyzed. The calibration curve was linear from 1 to 7500 ng/mL with correlation coefficients ranging from 0.9990- to 0.9999. The lower limit of quantitation was 1.0 ng/mL. The correlation coefficients for both inter- and intra-day variability were < 5.6% for each concentration (15 ng/mL, 150 ng/mL, 1500 ng/mL, and 5000 ng/mL) studied. The mean accuracy for inter- and intra-day evaluations was between 97.2 and 102%.

Electron microscopy:

Serial PBMC were collected from patients before treatment after one cycle of treatment and after 2 cycles of treatment. Fixation and analysis by EM was conducted as previously described (11)

DNA sequencing:

DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue and sequenced via the 147 gene UCM OncoPlus panel, as previously described (12). Briefly, DNA was fragmented (Covaris, Woburn, MA) and used for library preparation using the KAPA HTP Library Preparation Kit (Kapa Biosystems, Wilmington, MA). Libraries were quantified via QPCR (Kapa Biosystems), pooled and captured using a custom-designed SeqCap EZ capture panel (Roche, Indianapolis, IN), supplemented with select xGen Lockdown Probes (IDT, Coralville, IA). Amplified post-capture libraries were sequenced via HiSeq 2500 (Illumina, San Diego, CA) with rapid run v2 reagents to generate 2 × 101 base pair sequencing reads. After demultiplexing, data was analyzed via custom bioinformatics pipelines, as previously described (12).

Statistical Analysis

The primary objective for the phase I portion of the trial was to determine the maximum tolerated dose of HCQ when administered with daily everolimus in patients with advanced RCC. The primary phase II objective was to estimate the rate of 6 month PFS in RCC patients receiving everolimus and HCQ who have had between 1 and 3 prior treatment regimens for advanced disease. Secondary objectives were (1) to estimate the response rate of this combination, (2) to measure evidence of autophagy inhibition by EM to characterize significant associations between baseline genetic mutations and outcome. Based on a Simon two-stage design for a single arm phase II trial if greater than 5 patients were progression-free at 6 months, an additional 15 patients would be enrolled to complete this single arm phase II trial. With alpha=0.05 and 80% power with a total of 35 patients, if 14 or more patients were progression free (out of 35), the study would be considered a success and the regimen worthy of further investigation. This two-stage designed was based on Kaplan-Meier survival curves indicating 6-month PFS of roughly 40% in RECORD-1, METEOR and CHECKMATE 025 (2, 13, 14). Under this design, the probability of stopping the study in the first stage is 58% if the true 6 month PFS rate was 26% or less and the probability was 5% when the true 6 month PFs rate was 46% or more. For each gene, the binary variable was created; the presence or absence of the gene mutations was coded as 1 and 0, respectively. The heatmap and hierarchical clustering analysis was done by R package gplots heatmap.2. The order of the genes and the samples were reorganized by the hierarchical clustering. Kaplan-Meier curves were computed and an exact log-rank test was used to compare survival curves.

Results

Patient characteristics

Thirty-eight patients from the 3 participating centers were enrolled in this phase I/II trial from 2/9/12 until 1/16/17. Complete demographics for these patients are provided in Table 1. An additional 2 patients were screen failures and did not receive treatment with study agents. The majority of patients were Caucasian males with metastatic clear cell renal cell carcinoma who had received at least 2 prior therapies. Three patients were enrolled in dose level 1 (oral everolimus 10 mg daily plus oral HCQ 400mg twice daily) and 3 patients were enrolled at dose level 2 (oral everolimus 10 mg daily plus oral HCQ 600mg twice daily). Because no dose limiting toxicities occurred, the phase II portion proceeded at dose level 2. Patients represented a refractory treatment population: 14 patients had received at least 1 prior regimen of therapy for their metastatic RCC disease, 14 patients had received 2 prior lines of therapy, and 9 patients had received at least 3 prior regimens.

Table 1.

Patient Characteristics (%)

Gender	Male	28 (73%)
	Female	10 (27%)
Race	Caucasian	34 (89%)
	African American	3 (8%)
	Other	1 (3%)
Age	Median	65
	Range	44–82
Number of prior therapies	1	14 (37%)
	2	15 (39%)
	3	8 (21%)
	4	1 (3%)
Prior Therapies
	Sunitinib	20 (54%)
	Pazopanib	16 (43%)
	Interleukin-2	2 (5%)
	Axitinib	6 (16%)
	Sorafenib	2 (5%)
	Bevacizumab	2 (5%)
	Other	7 (19%)
ECOG PS	0	26 (68%)
	1	12 (32%)
Hemoglobin	< 12.0 g/dL	12 (32%)
Neutrophils	>7.4×109/L	1 (3%)
Platelets	>400×109 /L	3 (8%)
Calcium	>10.2 mg/dL	6 (16%)
Heng Score	0	15 (41%)
	1	12 (32%)
	2	7 (19%)
	3	1 (3%)
	4	2 (5%)

Safety and Identification of Recommended Phase 2 Dose

No DLTs were observed in the phase I portion of the study. HCQ 600 mg po twice daily in combination with standard dose everolimus 10 mg daily was identified as the recommended phase 2 dose (RP2D) and further evaluated for safety in the expansion cohort. Among 38 patients evaluable for adverse events in both phase I and phase II, the most common Grade 1–2 AEs attributable to treatment were nausea, fatigue, anemia, diarrhea, and rash (Table 2). All grade 3–4 AEs occurred at a rate of <10%. No Grade 5 adverse events were observed. Importantly the dose limiting toxicities of everolimus were not significantly worsened by the addition of HCQ.

Table 2.

Adverse Events >5% (N=38)

Adverse Event	Grade 1–2	Grade 3–4
Nausea	16 (42%)	2 (5%)
Fatigue	15 (39%)	3 (8%)
Anemia	14 (37%)	3 (8%)
Diarrhea	13 (34%)	0
Rash	12 (32%)	1 (3%)
Elevated AST	11 (29%)	0
Anorexia	8 (21%)	2 (5%)
Elevated creatinine	8 (21%)	0
Elevated triglycerides	8 (21%)	2 (5%)
Thrombocytopenia	8 (21%)	0
Elevated ALT	6 (16%)	0
Headache	6 (16%)	0
Hyperkalemia	6 (16%)	0
Mucositis	6 (16%)	0
Dry skin	5 (13%)	0
Vomitting	5 (13%)	0
Dry mouth	4 (11%)	0
Dysgeusia	4 (11%)	0
Edema	4 (11%)	1 (3%)
Elevated cholesterol	4 (11%)	0
Hypoalbuminemia	4 (11%)	0
Pruritus	4 (11%)	0
Weight loss	4 (11%)	1 (3%)
Blurred vision	3 (8%)	0
Constipation	3 (8%)	0
Hyperglycemia	3 (8%)	2 (5%)
Hypokalemia	3 (8%)	0
Hyponatremia	3 (8%)	1 (3%)
Neutropenia	3 (8%)	2 (5%)

Primary Efficacy Results

The primary endpoint was to estimate the rate of 6 month PFS in ccRCC patients receiving everolimus and HCQ who have had between 1 and 3 prior treatment regimens for advanced disease. The median PFS and best overall response rate were the secondary clinical endpoints. When 15 patients had enrolled, a total of 5 patients achieved at least stable disease exceeding 6 months, and therefore met the desired endpoint, so the trial proceeded to the second stage. Of 38 patients treated on study, 33 were assessable for efficacy. Four patients withdrew from the trial for intolerable side effects that did not meet the definition of Grade 3 or higher AEs (including body-aches or nausea), and one patient deteriorated due to rapid disease progression within the first cycle of treatment. Thirty-three patients received a median of 5 treatment cycles. Among these patients, 22/33 (67%) experienced either partial response (2 PR) or stable disease (20 SD) more than 3 months, as their best response per RECIST v1.1. Of the 22 patients who achieved stable disease or PR, 15 exceeded 6 months (Fig 1A). The range was 7 to 21 months. Eleven patients developed disease progression at the time of first disease assessment (9 weeks). The median PFS was estimated to be 6.3 months (Figure 1B).

Figure 1. Duration of benefit on everolimus and HCQ (n=33).

(A) Swimmer’s plot of time on study for each patient (B) Kaplan-Meier survival curves for progression-free survival (PFS)

Hydroxychloroquine Pharmacokinetics (PK)

We have previously performed population PK studies of HCQ in combination with other cancer drugs in numerous phase I studies (9, 11, 13, 14). These studies have all used a limited blood sampling approach (one blood draw at the time of treatment visits) apropriate for a compound that has a long half-life. PK studies of HCQ in rheumatoid arthritis patients have been performed with early intensive sampling following drug administration and showed a multiphasic exponential decline in HCQ blood concentrations(15, 16). In this study, we therefore included early intensive sampling (pre-dose, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours and 6 hours after dosing) in a small number of patients, providing concentration-time data during the absorption phase of HCQ dosing. We performed population pharmacokinetic (PK) analysis using 163 non-baseline blood samples from 27 patients collected over a period up to 616 days (average, 143; median, 89). The population model PK parameters do not specifically represent steady-state values, as they were determined from multiple repeated single doses taken by individual patients during their period of participation in the study. The model that best described the disposition of HCQ blood concentrations was a 2-compartment model with first-order absorption with no lag time. No covariate interactions were identified that significantly improved the model. A nondiagonal fit was superior to a diagonal fit based on −2(LL). The final model was as follows: first order absorption rate constant (Ka) = typical value (tv)Ka * exp(nKa); apparent volume of distribution in central compartment (V/F) = tvV * exp(nV)/F; apparent volume of distribution in peripheral compartment (V2/F) = tvV2 * exp(nV2)/F; apparent oral clearance (Cl/F) = tvCl * exp(nCl)/F; intercompartmental clearance (Q) = tvQ * exp(nQ); lag time (tLag) = tvTlag = exp(nTlag). Visual inspection of conditional weighted residuals (CWRES) versus individual predicted values (IPRED) plots suggested that an additive error model was appropriate for intra-individual error (residual error). The first-order conditional maximum likelihood estimation, extended-least squares first-order conditional estimation (FOCE-ELS) method was used for modeling. A visual predictive check with 200 replicates was performed to assess the model performance. A total of 1000 bootstrap runs were performed to provide estimates of the precision of parameter estimates and the 95% confidence intervals for the pharmacokinetic parameters (Table 3). Figure 2 shows the individual predicted concentrations vs. the individual observed concentrations from the population PK model.

Table 3.

HCQ population pharmacokinetic parameters

Parameter	Model estimate	Bootstrap Estimate	Stderr	CV%	2.5%−97.5% CI
Ka (h)	0.93	1.00	0.048	5.11	0.84–1.02
V/F (L)	599.89	599.80	20.30	3.38	559.75–640.03
V2/F (L)	3604.83	3598.91	13.28	0.37	3578.57–3631.09
Cl/F (L/h)	7.98	8.00	0.11	1.38	7.76–8.20
Q (L/h)	14.98	15.02	0.05	0.33	14.89–15.08
Stdev	38.91	43.10	4.63	11.91	29.75–48.08
Stderr, standard error; CV, coefficient of variation; CI, confidence interval; Ka, absorption rate constant; L, liters; h, hours; V/F, apparent central volume of distribution; V2/F, apparent peripheral volume of distribution; Cl/F, apparent oral clearance; Q, intercompartmental clearance; Stdev, standard deviation

Figure 2.

Individual predicted HCQ whole blood concentrations versus observed

Evidence of autophagy inhibition:

Serial peripheral blood mononuclear cells were collected in a limited number of patients and fixed for quantitative electron microscopy as previously described (Suppl. Fig. 1A) (13). Amongst 3 patients treated with HCQ 400 mg and 8 patients treated with HCQ 600 mg on the phase II portion, there were no significant drug-induced changes in autophagic vesicles observed in serially collected PBMC, either after one week of everolimus alone or after one month combined everolimus and HCQ. Although there was a trend of therapy-induced accumulation of vesicles in a minority of individual patients, there was no correlation between increased mean vesicle count/cell and response or PFS. (Suppl. Fig. 1B).

Genetic determinants of PFS:

To determine genetic determinants of PFS, genomic DNA from archival tumor tissue was subjected to 147 gene panel massively parallel sequencing panel. Out of 33 evaluable patients 29 patients had viable archival tumor for DNA and RNA sequencing analysis. DNA sequencing was successful in 22/29 samples, with the main reason for unsuccessful DNA sequencing being failed library preparation or inadequate DNA content. Hierarchical clustering was performed on known pathogenic mutations identified in the 147 gene panel (Suppl. Fig. 2A). Among the 22 patient, 6 patients had a potentially pathogenic mutated gene in the PI3K/mTOR pathway. Two of the six mutations would likely not result in constitutively activated mTORC1 signaling: PIK3CA^F791I, and PTEN^L928M. The remaining 4 mutations, mTOR^G2223L(17), TSC1^L462K which produces a truncating frameshift(18), FBXW7^G233A which produces a truncating frameshift(19) and PIK3CB^D1067H(20). These four gene mutations are predicted to result in constitutively activated mTORC1 signaling, and respond to mTOR inhibitors in vitro(17–21). There was a significant difference between the PFS survival curves of the patients that harbored these 4 pathogenic PI3K/mTOR mutations compared to the 18 patients that did not have these mutations (p=0.033). The median PFS for the 4 patients that harbored these pathogenic PI3K/mTOR mutations and the 18 patients without a pathogenic PI3K/mTOR mutation was 55 days and 145 days, respectively (Suppl. Fig 2B; Table 4).

Table 4.

Patients with mTOR activating mutations

Patient number	PFS, days	Pathogenic Mutations
01–015	43	mTORQ2223K; PBRM1E916*; VHL166*
03–006	56	TSC1 L463fs , IDH2 I2304H , MLH1 D567Q , TP53 R175H ,VHL V155M
01–003	145	FBXW7G233Afs; KDRT771M; PBRM1K231Dfs
03–007	55	PIK3CBD1067H PBRM1M713D; VHLW117G; NBNQ326*

Discussion

The combination of everolimus and HCQ was well tolerated with only everolimus-related toxicities observed. In this phase I/II study the grade 3–4 adverse event rate was < 10%. Although our observed 6-month PFS (15/33=43%) met the primary endpoint, it was close to the observed results with everolimus alone in the 3 phase III studies (2, 13,14). On the other hand, our median PFS was 6.3 months, which is longer than the median everolimus PFS for RECORD-1 of 4 months; METEOR- 3.8 months; and CHECKMATE 025– 4.6 months (2,13,14). During the course of this clinical trial, cabozantanib, nivolumab, and the combination of lenvatinib and everolimus have all become commercially available for treatment of metastatic renal cell carcinoma following treatment with a vascular endothelial growth factor receptor 2 tyrosine kinase inhibitor (19–21). The combination of lenvatinib plus everolimus demonstrated a survival advantage in a randomized phase III study compared to either agent alone. The median PFS of this regimen was 14.6 months making it a very attractive combination treatment for previously treated advanced renal cell carcinoma (24). However, 71% of patients treated with lenvatinib and everolimus developed grade 3 or higher AEs on the lenvatinib/everolimus arm of this trial (24). HCQ did not significantly worsen the toxicity of everolimus in the present study. Therefore, while further development of the everolimus and HCQ combination studied here is not warranted in advanced RCC,, the addition of HCQ to the potent lenvatinib plus everolimus combination might further control disease progression without excess toxicity. Consideration of adding HCQ to other RCC combinations is also provocative.

The HCQ population pharmacokinetic parameters are comparable with previously reported analyses in patients with advanced cancer receiving oral HCQ, where there is large interpatient variability in HCQ pharmacokinetics(9, 11, 13, 14). HCQ is distributed widely in body tissues as reflected by its large apparent volume of distribution, particularly of the peripheral compartment. HCQ has a long half-life, which can be attributed to extensive distribution into tissues and partitioning into red blood cells(15). Since HCQ is not extensively bound to plasma proteins, alterations in protein binding are unlikely to affect its disposition. Factors identified as having an effect on HCQ disposition have not been extensively studied, but include body weight (14, 25). We did not find weight to have a significant effect on HCQ parameter estimates in this analysis.

Pharmacokinetic studies with early sampling following drug administration show a multiphasic exponential decline in HCQ blood concentrations. [5,7] We performed extensive sampling in a small number of patients after the first dose and at steady state, providing concentration-time data during the absorption phase of HCQ dosing. Thus, we found that a 2 compartment model best fit the data. A lag time did not significantly improve the model fit, and thus it was not incorporated into the final population pharmacokinetic model.

Efficacy and toxicity are unlikely to be linked to differences in HCQ pharmacokinetics. Although higher HCQ doses and exposure have been associated with increases in markers of autophagy in PBMC, relationships between blood HCQ concentrations with efficacy in limiting tumor progression or toxicities have not been consistently observed in combination anticancer studies and are generally absent with HCQ monotherapy.

Our analysis of a limited set of serial PBMC did not reveal a significant accumulation of autophagic vesicles in this study, despite both everolimus and HCQ-being well accepted autophagy modulators, and the maximal dose of HCQ allowed by the FDA being achieved. This is in contrast to what was observed with a phase I trial of temsirolimus and HCQ in solid tumors (9), in which 600 mg bid HCQ there was a significant accumulation of autophagic vesicles in the PBMCs of patients treated on the combination regimen. The sample size of this analysis was limited in the current study, and a larger sample size may have found a significant therapy-associated change in vesicle accumulation. Another explanation for this difference is that pharmacodynamic studies of everolimus in mice rats and humans have demonstrated that measureable changes in phosphoS6 kinase, the downstream effector of mTOR signaling, following treatment with everolimus are much more pronounced in tumor tissue than in PBMC. In contrast, temsirolimus treatment produces sustained mTOR inhibition in PBMC (26). Importantly, significant accumulation of autophagic vesicles has only been detected after one cycle of combined therapy across trials, and never with single agent therapy (11, 13, 14), suggesting that the vesicle accumulation observed in previous studies was reflecting combined autophagy induction and inhibition. If an agent is less effective at pathway blockade in PBMC, this may explain why the combination of everolimus and HCQ did not produce a significant accumulation of autophagic vesicles. This suggests that the EM assay used here may not be sensitive or specific enough to detect autophagy induction or inhibition well in PBMC, or that the time-points chosen are too late to detect these changes. Alternatively, the use of PBMC may be broadly problematic as a surrogate tissue to study autophagy dynamics, and as previous experience suggests, therapy induced changes in autophagy may be occurring in a more striking fashion within the tumor tissue (9).

Who are the patients likely to respond to mTOR inhibitor combinations? In a previous study, analysis of a limited panel of cancer cell lines found that cell lines that harbor activating mutations in the mTOR kinase domain were more sensitive to rapamycin analogues than cell lines which harbored other mTOR pathway mutations(21). Clinical correlative studies in renal cell carcinoma have found that mutations in mTOR, TSC1, and TSC2 are found in higher frequency in patients who respond to rapamycin analogs (17,18). Unexpectedly, in this trial, next generation sequencing identified a number of mutations predicted to activate mTOR signaling in patients with the shortest PFS. This finding suggests that somatic mutations that have been suggested as predictive markers for rapamycin analogues fail to predict a combination involving a rapamycin analog and a lysosomal inhibitor. It could be that HCQ somehow limits the efficacy of everolimus in the presence of mTOR pathway mutations. Alternatively, this association between mTOR pathway activating mutations and shortened PFS could be confounded by other genomic or epigenetic alterations that were not tested for in our study. Finally, only 22 patients were sequenced in this study, therefore our findings could be limited by small sample size.

In summary, the combination of HCQ 600mg and everolimus 10 mg daily was safe and prolonged stable disease was seen in a subset of patients. Activating mutations in the mTOR signaling pathway were associated with shorter PFS with this regimen.

Translational relevance.

The anti-malarial drug, hydroxychloroquine, inhibits autophagy and can be safely combined with everolimus. Some patients with metastatic clear cell renal cancer on this trial had prolonged responses to the combination. Contrary to other publications, we did not find a correlation with mutations in the PI3 kinase pathways and overall response. Hydroxychloroquine can be combined with other agents in renal or other solid cancers to augment autophagy inhibition as an anti-cancer mechanism.