Introduction
Arginine is a conditional/semi-essential amino acid involved in the synthesis of a wide range of proteins, a myriad of metabolic pathways as well as being a signing molecule in various cellular pathways [
1]. Arginine is also intimately involved in T cell cellular metabolism, T cell receptor (TCR) expression and T cell functionalities [
2]. In preclinical studies, arginine deprivation could induce tumour regression in a group of cancers designated as arginine auxotrophic cancers which include melanoma, prostate cancer, acute myeloid leukaemia and hepatocellular carcinoma (HCC) [
3,
4]. For selection of cancers for arginine deprivation therapy, it has been postulated that tumours with absent or low expression of arginine regeneration enzymes, argininosuccinate synthase (ASS) and ornithine transcarbamylase (OTC), are reliant on exogenous source of arginine for growth and hence more sensitive to arginine deprivation [
3,
5]. In HCC cell lines and patients’ tumour tissues, most of the tumours have no or very low expression of the ASS or OTC on immunohistochemical (IHC) examination [
6,
7]. This phenomenon renders HCC a potential cancer type to be benefited from the approach of treatment with arginine deprivation.
PEG-BCT-100 is a pegylated recombinant human arginase 1 protein expressed in
E. coli which is purified and formulated for human dosing. In clinical trials, PEG-BCT-100 is efficacious in inducing arginine depletion safely and in a dose-dependent manner [
8,
9]. In clinical studies, a dose of 2.7 mg/kg/week was found to be the optimal dose of PEG-BCT-100 for sustained arginine depletion for human clinical studies [
8]. Regarding evaluation of predictive biomarker for PEG-BCT-100, patients were not mandated to have pre-treatment tumour tissues in previous clinical trials hence evaluations of ASS and OTC were not available to correlate with treatment outcomes [
8]. In the present study, we set to evaluate in a phase II setting the treatment efficacy of PEG-BCT-100 in post sorafenib HCC and to correlate treatment outcomes with tumour expression of ASS and OTC (NCT02089763). All patients must have freshly obtained tumour biopsies or archival tumour tissue obtained within 6 months prior to commencement of treatment for analyses of protein expression of ASS and OTC.
Patients and methods
Study design and endpoints
This study was an open-label, single-arm Phase II study to evaluate the safety and efficacy of PEG-BCT-100 in chemo naïve patients with prior treatment with sorafenib for HCC. The primary endpoint was time to progression (TTP), which was defined from the time of commencing study treatment (i.e., Cycle 1 Day 1) to the first documentation of objective tumour progression. The secondary endpoints were OS, PFS, radiological response (RR), disease control rate (DCR), type and number of adverse events (AEs), and quality of life (QoL). This clinical trial also explored predictive and prognostic functions of tissue biomarkers with protein expression of ASS and OTC in tumours, as determined by immunohistochemistry. Intended sample size was 30 patients. The study protocol was approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee and was conducted in accordance with the Declaration of Helsinki.
Eligibility
All patients were recruited at the Prince of Wales Hospital in Hong Kong. Key inclusion criteria included: clinical diagnosis of HCC according to AASLD criteria; age; prior first-line treatment with sorafenib lasting for at least 14 days; adequate haematological and renal function; bilirubin ≤2 x upper limit of normal (ULN) and alanine transaminase (ALT) ≤ 3 x ULN; Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. All patients must be fit and willing to undergo a needle biopsy of tumour unless there were archival tumours within 6 months of recruitment to the clinical trial. Key exclusion criteria included: Liver function of Child-Pugh class of B or C; presence of ascites not controlled by medications; prior malignancy and prior treatment with arginine depletion agent.
Study treatment
Patients were treated with PEG-BCT-100 at 2.7 mg/kg intravenous injection once every week on Day 1, 8 and 15 every 3-weekly cycle. There is no interruption between each cycle. This dosage is equivalent to the dose of 1600 IU/kg weekly administration as used in the previous phase clinical trial [
8,
10]. All patients received PEG-BCT-100 till progressive disease, intolerable toxicity, or patients’ consent withdrawal.
Study assessments
Routine blood tests were measured during screening period, baseline and Day 1 of each 3-weekly cycle. Serum alpha-fetoprotein (AFP) was assessed on day 1 of every cycle. Tumour response was assessed according to the Response Evaluation Criteria In Solid Tumours (RECIST) 1.1 criteria and modified RECIST 1.1 criteria with computed tomography every 6 weeks. Adverse events were evaluated with the Common Terminology Criteria for Adverse Events (CTCAE) 4.0. Quality of life was measured using the European Organization for Research and Treatment of Cancer quality of life questionnaire (EORTC QLQ C-30) and QLQ-HCC18 questionnaires.
Expression of ASS and OTC in the tumour were performed using standard immunohistochemical (IHC) staining protocol with 3, 3′-diaminobenzidine (DAB) as chromogen and polymer amplification. The expression was semi-quantitatively assessed by the Histoscore which was the product of the intensity of staining (graded as: 0, non-staining; 1, weak; 2, median; or 3, strong) and the percentage of positive cells.
Statistical analyses
Demographic data were summarized by descriptive statistics. Time-to-event data were estimated and plotted with the method of Kaplan-Meier analyses; median estimates and confidence limits will be given. RR and DCR were provided with corresponding exact 95% 2-sided confidence interval with standard methods based on the binomial distribution. Pearson Chi-square test were performed to explore the association between RR and the level of intensity of OTC and ASS. PFS between groups with different level of OTC and/or ASS would be compared by log-rank test, and Cox proportional hazards model were applied for PFS with the biomarkers as predictors. The hazard ratio (HR) and corresponding 95% confidence interval (CI) were estimated. SAS® Software version 9.2 (SAS Institute, Cary, NC, USA).
The sample size and stopping rule was determined using a single-stage design and the number of patients with progressive of disease observed at 2 months. With reference to the previous clinical trial on second-line treatment in HCC, the progression rate at 2-month was around 0.7 for second-line treatment of advanced HCC using placebo (11). Had the progression rate at 2 months been 0.7 or higher, the regimen would have been considered inactive. For a clinical trial with a power higher than 0.8 and a false positive rate of lower than 0.09, the sample size was calculated to be 30.
Discussions
In this Phase II clinical trial, we evaluated the safety, tolerability and efficacy of PEG-BCT-100 in chemo naïve post-sorafenib HCC. Pre-treatment tumour ASS and OTC IHC expression levels were mandated and studied to evaluate the association between these marker expressions and treatment outcomes such as TTP, PFS and OS. The protocol mandated no previous chemo to remove any confounding factors of previous lines of chemotherapy, which may negatively impact on treatment outcomes. As a post-hoc analysis we also studied the OS difference in those patients who had arginine depletion with PEG-BCT-100 over one cycle.
As with the previous published data on PEG-BCT-100, results of current study confirm the safety and tolerability of PEG-BCT-100 [
10]. Although the clinical trial failed its primary endpoint in extending TTP to beyond 2 months, there are nevertheless two clinically relevant findings that warrant discussion. First, ASS-negative tumour confers OS advantage over ASS-positive tumour: patients with ASS-negative tumour were found to have doubled the OS compared to the ASS+ ones (35 versus 15 weeks). On the contrary, the OTC status, whether positive or negative, were found not to associate with tumour response or OS. These data suggest that ASS but not the OTC expression is a more clinically relevant biomarker for PEG-BCT-100, and ASS negativity should be evaluated as a patient enrichment strategy in future clinical studies on PEG-BCT-100. Second, the duration of arginine depletion is found to be crucial for clinical benefits of arginine deprivation. In the current study, an OS advantage was demonstrated in patients with PEG-BCT-100 treatment
>3 weeks (25.29 weeks vs. 16.57 weeks). This OS advantage in prolonged arginine depletion with PEG-BCT-100 is consistent with the two studies of ADI-peg, which is a pegylated arginine deiminase. In the ADI-peg global HCC phase III study [
11], the authors reported OS advantage for those patients who had arginine depletion over 6 weeks. In another Asian ADI-peg HCC study [
12], it was similarly found that OS benefits were only observed in patients who had arginine depletion
>3 weeks as compared to those with <3 weeks. Above findings all indicate that 3 to 6 weeks is an optimal duration of clinically meaningful arginine deprivation, which is important for clinical trial design on arginine deprivation.
The survival advantage in ASS-negative HCC could have implication in the treatment of other ASS-negative tumours since a number of common tumours are arginine auxotrophic by virtual of the negative ASS status, including melanoma, prostate cancer, neuroblastoma, acute myeloid leukaemia, glioma and some sarcoma [
7]. In the US study of PET-BCT-100, it was shown that durable complete remission was observed in a patient with metastatic melanoma which was subsequently found to have negative ASS expression on immunohistochemistry [
9]. Similar observation was made with another arginine depletion treatment: amongst 9 patients with ASS-negative thoracic cancers were given chemotherapy and ADI-peg, 7 of them had partial responses [
13]. To further explore above concept in other cancer types, a number of clinical trials are ongoing, including the Phase II clinical trials on PEG-BCT-100 either singly in paediatrics cancers such as neuroblastoma and glioma (EUDRACT no:2017–002762-44) and another clinical trial on PEG-BCT-100 in combination with low dose cytarabine and in acute myeloid leukaemia (EUDRACT no:2011–000749).
About the potential clinical application, it is understood that tyrosine kinase inhibitors and check-point inhibitor immunotherapy are the current standard treatment for HCC. However, personalized treatment with biomarker for selection remains elusive for HCC. Our study provide preliminary evidence on applying ASS-negativity in tumour tissues as a biomarker for selection of patients for arginine deprivation therapy. There remains room for developing this strategy as monotherapy in later lines of systemic treatment for HCC. Further, the combination of arginine deprivation with other anti-cancer treatment could improve the efficacy in ASS-negative HCC [
14]. Hence it is clinically important to evaluate combinational strategy on PEG-BCT-100 with other existing treatments in HCC. Finally, the existent and emerging immune-oncology treatments are associated with high cost and the potential occurrence of hyperprogression in HCC [
15,
16]. For HCC patients who fail or could not afford targeted agents/immunotherapy, arginine depletion therapy with PEG-BCT-100 could still be an attractive and affordable treatment option.
In conclusion, the current phase II clinical trial shows that PEG-BCT-100 monotherapy is well tolerated in second-line post-sorafenib HCC, with moderate anti-tumour activity. In the first time, it is shown in human that ASS-negativity in tumour confers survival advantage over ASS positive patients, which support the potential role of ASS-negative as a predictive biomarker for patient selection. Further studies are now underway to further confirm ASS-negative as a predictive OS biomarker for treatment of arginine auxotrophic cancers with PEG-BCT-100.
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