A Phase I Study of Pegylated Arginine Deiminase (Pegargiminase), Cisplatin, and Pemetrexed in Argininosuccinate Synthetase 1-Deficient Recurrent High-grade Glioma

High-grade gliomas (HGG) are usually treated with a combination of surgery and chemoradiotherapy in the first-line setting, but, despite these measures, recurrence is nearly universal (1). At this point, treatment options are limited with no defined standard-of-care (2). Alkylating chemotherapy is most commonly used, but efficacy is poor with a progression-free survival (PFS) of around 1.5–3 months and an overall survival (OS) of 6-9 months (2). New treatment options are urgently required.

Arginine is a conditionally essential amino acid involved in the regulation of numerous cellular processes (3). These include cell signaling, proliferation (by modulating polyamine and nucleotide synthesis), vasodilatation (via nitric oxide), and hormone synthesis (3). Arginine also plays a crucial role in regulating the immune system in health and disease (4).

Auxotrophy is the inability of an organism to synthesize an organic compound necessary for its growth (5). Normal cells express the urea cycle enzymes argininosuccinate synthetase 1 (ASS1) and argininosuccinate lyase (ASL) that convert citrulline to arginine thereby providing an endogenous supply. However, ASS1 is downregulated in many tumors, commonly through hypermethylation of the ASS1 promoter (6, 7). This leads to increased pyrimidine synthesis and consequent tumor proliferation, but renders the tumor dependent on exogenous arginine (6, 8, 9). This can be exploited therapeutically using pegylated arginine deiminase (ADI-PEG20/pegargiminase), which catalyzes the conversion of l-arginine to citrulline and an ammonium ion (10). Importantly, low ASS1 expression is associated with a more aggressive clinical phenotype and a worse clinical outcome in several different cancer types (11–14).

Initial clinical trials showed that ADI-PEG20 is well tolerated with injection site tenderness being the most commonly reported adverse event. Allergic reactions have occurred, but anaphylaxis is rare (<1%). The most common laboratory toxicities were mild (grade 1 and 2) asymptomatic elevations in various serum chemistry values, while neutropenia has been described infrequently (13, 15, 16).

ADI-PEG20 has biological efficacy as a single agent in a range of tumors including melanoma, hepatocellular carcinoma, and mesothelioma (13, 15, 16). Furthermore, patients deficient in ASS1 gained the most benefit from monotherapy (17). In addition, preclinical studies of ADI-PEG20 showed that arginine deprivation suppressed the folate-dependent enzymes thymidylate synthase (TS) and dihydrofolate reductase (DHFR; ref. 12). This leads to the potentiation of pemetrexed, an inhibitor of TS and DHFR, when combined with ADI-PEG20 (12). Also, ADI-PEG20 potentiates the activity of cisplatin in melanoma cells by inhibiting synthesis of DNA repair enzymes that attempt to abrogate the effects of cisplatin (18). Therefore, ADI-PEG20 enhances the activity of both folate inhibitors and platinum agents. Furthermore, a phase I trial combining ADI-PEG20, pemetrexed, and cisplatin (ADIPEMCIS) has shown efficacy in patients with treatment-refractory ASS1-deficient mesothelioma or non–small-cell lung cancer, thus validating this clinical approach (19).

Preclinical findings have shown that 70% of HGG patient tissue samples were deficient in ASS1 (20). Arginine deprivation, either by exposure to arginine deiminase or arginase, inhibited the proliferation of patient-derived ASS1-deficient HGG cell lines and xenografts (20, 21). These preclinical studies established that targeting arginine auxotrophy is a potential therapeutic strategy in HGG that would have the benefit of partially overcoming the limitations imposed by the blood–brain barrier by acting in the peripheral blood rather than the tumor (22). This phase I expansion cohort assessed the safety and preliminary evidence for clinical activity using ADIPEMCIS in patients with recurrent or refractory HGG.

Eligible patients were 18 years of age or older with histologically confirmed HGG in which >50% of tumor cells did not express ASS1 as determined by IHC; 0 or 1+ by ASS1 IHC 1:500; from Polaris Pharmaceuticals, Inc.; refs. 7, 13, 19). Archived tissue was used for ASS1 assessment where the initial diagnosis was HGG. Patients who were initially diagnosed with a low-grade glioma were required to undergo a biopsy to confirm transformation to a HGG, which was subsequently used for ASS1 assessment. Patients must have failed first-line treatment with radiotherapy ± temozolomide (progressive disease and/or side-effects) and have evaluable disease by RECIST 1.1 (23). Any steroid dose must have been stable for at least 5 days prior to the patient receiving the first ADI-PEG20 dose. Patients had an Eastern Cooperative Oncology Group performance status of 0–1 as well as adequate hematologic, renal, and hepatic function. Exclusion criteria included radiotherapy or surgery within 4 weeks of first study treatment, ongoing toxic manifestations of previous treatments, and significant concomitant or uncontrolled intercurrent illness. Prior treatment with ADI-PEG20 was not allowed.

The study was performed in accordance with good clinical practice and the European Union Clinical Trials Directive, with approval from Leeds East (Yorkshire and The Humber) Ethical Review Board. All patients provided written informed consent.

This was an open-label, single-arm, expansion cohort of a phase I study conducted across two centers in the United Kingdom once the recommended phase II dose (RP2D) of ADIPEMCIS had been determined by the dose escalation cohort (19). The planned number of patients to be enrolled was ten. The RP2D was weekly ADI-PEG20 36 mg/m² intramuscularly plus pemetrexed 500 mg/m² and cisplatin 75 mg/m² intravenously once every 3 weeks (19). The initial dose of ADI-PEG20 was given at least 48 hours in advance of the first dose of cytotoxic drugs. To reduce pemetrexed toxicity, daily oral folic acid 400 μg and hydroxycobalamin 1,000 μg intramuscularly every 9 weeks were administered, starting at least 7 days prior to first dose. Oral dexamethasone 4 mg twice daily was given for 3 days with each cycle, starting the day before pemetrexed was due, if the patients were not already taking steroids or were on a lower dose. Treatment continued for a maximum of 6 cycles (18 weeks) after which patients who had stable disease or better were eligible to continue on single-agent ADI-PEG20 until disease progression.

A data cutoff of 15 November 2018 was used. The primary endpoints were safety, tolerability, and preliminary estimates of efficacy by best overall response rate as assessed by RECIST 1.1 (23). Secondary endpoints included PFS, OS, pharmacodynamics, and immunogenicity. Exploratory endpoints included analysis of ASS1/ASL epimutations.

The safety and tolerability of ADIPEMCIS was assessed using NCI Common Terminology Criteria for Adverse Events version 4.03.

Baseline imaging by diffusion-weighted MRI was undertaken within 21 days prior to the patient receiving the first ADI-PEG20 dose. MRI scans were performed every 6 weeks during ADIPEMCIS dosing and after every eighth weekly dose of ADI-PEG20 during the maintenance phase.

During combination treatment with ADIPEMCIS, blood samples were taken weekly for cycle 1 and then on days 1 and 15 for cycles 2–6 to analyze arginine and citrulline levels and for immunogenicity analyses, as described previously (13).

The methylation status of ASS1 and ASL were determined by methylation-specific PCR as described previously (20).

Between January 2016 and June 2017, 10 patients with HGG were enrolled from two centers in the United Kingdom (Table 1). The age range was 43–63 years with a median age at enrollment of 51 years. Ninety percent of patients were male and 80% were Caucasian. Twenty patients were screened with 18 (90%) being ASS1-deficient (Supplementary Fig. S1). It was not possible to determine the ASS1 score for 2 patients who were therefore excluded. The reasons for screen-failure in the other 8 patients were clinical deterioration (6, 60%) and withdrawal of consent (2, 20%).

For the 10 treated patients, 6 had glioblastoma, one of which had transformed from a grade II astrocytoma (Table 2). Three patients had anaplastic astrocytoma, one of which had transformed from a grade II astrocytoma. One patient had an anaplastic oligodendroglioma (1p/19q codeleted), which had transformed from a grade II oligodendroglioma. All patients with glioblastoma were isocitrate dehydrogenase (IDH) wild-type as assessed by R132H IHC as was 1 patient with anaplastic astrocytoma. Six patients had unmethylated O⁶-methylguanine-DNA methyltransferase (MGMT) promoters, 3 had methylated ones, and it was not determined for 1 patient. Ninety percent of patients had received two or more lines of systemic therapy prior to enrollment, including temozolomide, lomustine, and procarbazine/lomustine/vincristine (PCV).

The majority (74%) of adverse events (AE) were grade 1 or 2 (Table 3). The commonest grade 1–2 AEs seen in more than one patient related to at least one treatment agent were thrombocytopenia (5 patients, 50%), fatigue (3 patients, 30%), raised alanine aminotransferase (3 patients, 30%), nausea (2 patients, 20%), and dyspepsia (2 patients, 20%). Eight patients (80%) experienced at least one grade 3–4 AE with neutropenia (4 patients, 40%) and thrombocytopenia (3 patients, 30%) being the most frequent. There was 1 case of grade 3 neutropenic sepsis and one grade 3 rise in alanine aminotransferase level, both of which were treatment-related.

Two patients did not have any follow-up scans after the first cycle of treatment: patient E chose to withdraw from the study; and patient C suffered a combination of adverse events, clinical progression, and performance status deterioration.

Complete data were available for 5 patients over the 18-week period of blood sampling. Circulating plasma arginine levels fell rapidly upon commencing ADI-PEG20 and remained below 30% of baseline for the duration of the sampling period (Fig. 1A). A reciprocal rise in citrulline as expected was also maintained throughout the sampling period. Anti–ADI-PEG20 antibody titers rose during the first 4 weeks of ADI-PEG20 before plateauing at 10⁻² for the remainder of the study (Fig. 1B).

The best response by RECIST 1.1 was stable disease seen in 6 (80%) patients with 2 patients being nonevaluable due to lack of follow-up scans. No complete or partial responses were seen. The median PFS was 5.2 months [95% confidence interval (CI), 2.6–20.8) and the median OS was 6.3 months (95% CI, 1.8–9.7). PFS at six months (PFS6) was 20%. Individual patient responses are summarized in Fig. 2 with Kaplan–Meier plots shown in Supplementary Fig. S2. Notably, 1 patient with primary IDH wild-type, MGMT promoter unmethylated glioblastoma had a PFS of 20.8 months and remains alive currently (OS 24+ months). Moreover, re-resection of his glioblastoma at disease progression continued to demonstrate ASS1 deficiency by IHC (Supplementary Fig. S1). A retrospective analysis by Response Assessment in Neuro-Oncology (RANO) criteria was undertaken (24): 2 patients had a partial response as their best response to treatment, namely the 2 patients with the longest PFS of 20.8 and 9.3 months; 1 patient had stable disease; 2 patients had progressive disease; and 5 patients were nonevaluable owing to extensive necrosis. Median PFS by RANO was 2.5 months (95% CI, 0.0–9.2).

The promoter methylation status of ASS1 and ASL was determined for 8 of the patients on treatment using archived tissue, except where a HGG had transformed from a low-grade glioma when new biopsy tissue was used (insufficient material for 2 patients). There were no cases of ASL promoter methylation. Three patients had at least 1 tumor sample that demonstrated methylation of ASS1 promoter and there were 5 patients with unmethylated tumors (Table 2). Two samples from the same tumor were tested for patient G. Interestingly, 1 sample showed ASS1 methylation and the other did not, illustrating heterogeneity within the tumor.

This is the first reported use of arginine deprivation therapy in combination with cisplatin and pemetrexed (ADIPEMCIS) in recurrent HGGs, a biomarker-led approach. Patients enrolled in the trial had been heavily pretreated with 90% having received at least two prior lines of chemotherapy. Unsurprisingly, the commonest AEs were related to bone marrow suppression, including grade 4 neutropenia and thrombocytopenia, consistent with the chemotherapy. However, no bleeding events occurred and there was only 1 episode of neutropenic sepsis, in line with the expected rate. Overall, treatment was well tolerated.

Arginine suppression was observed for the duration of ADIPEMCIS treatment together with a concomitant rise in citrulline. The anti–ADI-PEG20 antibody titer rose to a peak of 10⁻² by week 4 and then plateaued at this level for the remainder of treatment. This is consistent with findings in patients with thoracic tumor treated with ADIPEMCIS (19).

Preliminary efficacy results showed PFS 5.2 months, OS 6.3 months, and PFS6 20% by RECIST 1.1. These are in line with historical findings for recurrent HGG, albeit toward the lower end of the range. This may be due to our population being more heavily pretreated as 90% had received at least two prior lines of chemotherapy, compared with only one line in most studies (25–28). In addition, ASS1 deficiency is associated with a worse prognosis in several different cancer types (11–14). However, given that our mixed population comprised grade III gliomas (better prognosis) and glioblastomas (worse prognosis), larger studies will be required to determine efficacy in these subtypes.

One patient with primary glioblastoma who had poor prognostic features (IDH-wild-type, unmethylated MGMT promoter) had a PFS of 20.8 months and remains alive currently (OS 24+ months). He had progressed after 3 cycles of adjuvant temozolomide and then again after 2 cycles (or 12 weeks) of PCV chemotherapy. He commenced ADIPEMCIS in November 2016 and completed the first 2 cycles at 100% dose, but required dose reductions in cisplatin and pemetrexed for cycles 3 and 4 before they were stopped from cycle 5 onwards owing to recurrent grade 3 and 4 thrombocytopenia. He continued on ADI-PEG20 monotherapy for a further 18 months. Radiologically, there had been a progressive increase in the enhancing component of the tumor from his first on-treatment MRI scan to 18% above baseline on the July 2017 scan (Fig. 3). However, arginine levels were suppressed in June 2018 after 16 months on ADI-PEG20 maintenance compared with baseline (data not shown) suggesting another mechanism causing disease progression. In July 2017, he was started on quinine sulfate 200 mg once daily by his physician for leg cramps. Subsequent scans showed that his tumor had stabilized before reducing in size, which was sustained for a further twelve months and was associated with behavioral improvements. Although this is only 1 patient with circumstantial evidence for an interaction, there is a preclinical basis for the effect. Autophagy is a known resistance mechanism of arginine deprivation therapy, and quinolone autophagy inhibitors have shown efficacy in preclinical tumor models treated with ADI-PEG20, including glioblastoma (20, 29). Indeed, recent studies confirm quinolone resensitization of glioma cell lines to targeted agents such as BRAF inhibitors, with clinical evidence of efficacy (30, 31). Thus, the potential synergy between ADI-PEG20 and autophagy inhibitors warrants further clinical investigation. At disease progression, he underwent debulking surgery where the histology reconfirmed glioblastoma and molecular analysis showed no mutations in IDH1/2, although interestingly MGMT promoter methylation was now present at a low level (>0-5%) and may reflect prior treatment with cisplatin (32). Intriguingly, his recurrent tumor on ADI-PEG20 monotherapy remained ASS1-deficient (Supplementary Fig. S1) implicating as yet unknown mechanisms of drug resistance to ADI-PEG20 that may include stromal provision of arginine or its precursors (9).

Platinum agents are an established treatment option in recurrent HGG (33, 34). However, to our knowledge, this is the first time pemetrexed has been trialed in this setting in more than one patient (clinical trial NCT00276783 recruited 1 patient only with HGG–J. Raizer; personal communication). This is despite preclinical evidence showing pemetrexed has activity in a xenograft model of glioblastoma and clinical studies showing that cisplatin/pemetrexed has efficacy in brain metastases (35–37). Furthermore, preclinical data has shown that ADI-PEG20 suppresses the folate-dependent enzymes TS and DHFR in primary glioblastoma cells (Supplementary Fig. S3A) and when combined with pemetrexed synergizes to decrease proliferation in an ASS1-negative glioma cell line, but not in an ASS1-positive cell line as has been seen previously in bladder cancer and malignant mesothelioma cell lines (Supplementary Fig. S3B; ref. 12).

This was an expansion cohort of the initial phase I TRAP trial, which was conducted in thoracic tumors (19). As such, RECIST 1.1 was used to evaluate PFS (23). This involves a unidirectional measurement of the target lesion, rather than a bidirectional one used by RANO (24). Despite these differences, the correlation between the two sets of criteria is usually good, although in our small sample set differences were seen (38). Patient A had a PFS that was around 2 months shorter by RANO than RECIST 1.1 criteria. In contrast, patient J had a sustained partial response by RANO but did have a progression event according to RECIST 1.1. Future studies will employ RANO criteria exclusively. Two patients had clinical progression, rather than radiological progression, which led to their data being censored by RECIST 1.1. Patient A appeared to have disease progression on the July 2017 MRI scan with new left frontal enhancement (Fig. 3A). However, this region was not measurable by RANO criteria owing to central necrosis and thin walls. These findings highlight the well-documented issues with measuring PFS in HGGs.

Ninety percent of patients screened were found to be deficient for ASS1 expression by IHC using a less stringent threshold of >50% ASS1 loss than used in earlier studies that mandated >95%–99% ASS1 loss (20). Therefore, it is proposed that ASS1 expression status will not be required at study enrollment to minimize time-to-treatment; however, subgroup analysis will be performed retrospectively. In our small patient cohort, ASS1 promoter methylation occurred in only 30% of patients and did not correlate with treatment response in contrast to in vitro findings (Table 2; ref. 20). Heterogeneity was seen in 1 patient indicating that this may be an underrepresentation of the true rate. Alternatively, methylation of ASS1 intron 1 may be a more important determinant than promoter methylation in clinical samples as shown in mesothelioma (11). Also, ASS1-deficiency appears to be less important for medical benefit when ADI-PEG20 is combined with chemotherapy, as has been shown with gemcitabine and nab-paclitaxel in pancreatic cancer and with folinic acid, fluorouracil, and oxaliplatin in hepatocellular carcinoma (39, 40).

In summary, the combination of ADI-PEG20, pemetrexed, and cisplatin was well tolerated in patients with recurrent aggressive gliomas. Preliminary estimates of efficacy were encouraging with the intriguing role of a quinolone-based autophagy inhibitor in combination with ADI-PEG20 needing further clinical investigation. Additional trials of ADI-PEG20 are planned in patients with HGGs.

P.E. Hall reports receiving speakers bureau honoraria from MSD. R. Shaffer is an employee of GenesisCare. A. Johnston has ownership interests (including patents) at Polaris Pharmaceuticals Inc. R. Jena is an employee of Microsoft. S. Jefferies is in private practice consultation for patients with CNS and thyroid tumors, and provides legal advice and does legal hearings for UK Health Ombudsman. B.-W. Wu has ownership interests (including patents) at Polaris Group. J.S. Bomalski has ownership interests (including patents) at Polaris Group. P.W. Szlosarek reports receiving commercial research grants and commercial research support from Polaris Pharma. No potential conflicts of interest were disclosed by the other authors.

The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health and Social Care.

Conception and design: N. Syed, A. Johnston, J.S. Bomalaski, T. Crook, K. O'Neill, P.W. Szlosarek

Development of methodology: J.S. Bomalaski, T. Crook, P.W. Szlosarek

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P.E. Hall, R. Lewis, N. Syed, R. Shaffer, M. Williams, F.P. Harris, R. Jena, S. Jefferies, K. Smith, T. Crook, K. O'Neill, D. Paraskevopoulos, R.S. Khadeir, M. Sheaff, S. Pacey, P.N. Plowman, P.W. Szlosarek

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P.E. Hall, X. Feng, A. Johnston, T. Matys, J.S. Bomalaski, K. O'Neill, R.S. Khadeir, M. Sheaff, P.W. Szlosarek

Writing, review, and/or revision of the manuscript: P.E. Hall, R. Lewis, N. Syed, R. Shaffer, M. Williams, X. Feng, A. Johnston, J.A. Thomson, F.P. Harris, S. Jefferies, K. Smith, J.S. Bomalaski, T. Crook, K. O'Neill, D. Paraskevopoulos, M. Sheaff, S. Pacey, P.W. Szlosarek

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): J. Evanson, S. Ellis, X. Feng, B.-W. Wu, J.S. Bomalaski, P.W. Szlosarek

Study supervision: R. Shaffer, P.W. Szlosarek

Others (chief investigator of the study): P.W. Szlosarek

We thank the patients and their families, the Experimental Cancer Medicine Centre staff at Queen Mary University of London, and University of Cambridge, and the neurosurgical teams at the referring hospitals and St. Bartholomew's Hospital for their support. We thank Polaris Pharmaceuticals Inc. for their trial sponsorship. This research was supported by the NIHR Cambridge Clinical Research Facility and Cambridge Experimental Cancer Medicine Centre. The Cambridge Human Research Tissue Bank is supported by the NIHR Cambridge Biomedical Research Centre. This work was supported by the Cancer Research UK Experimental Cancer Medicine Centre at Barts Cancer Institute, Queen Mary University of London (London, United Kingdom; C16420/A18066; C16420/A15583). P.E. Hall was supported by a National Institute for Health Research (NIHR) Academic Clinical Fellowship. N. Syed is supported by the Brain Tumour Research Campaign and Brain Tumour Research. M. Williams is supported by the NIHR/Imperial Biomedical Research Centre. S. Pacey is supported by the NIHR/Cambridge Biomedical Research Centre (The Human Research Tissue Bank). P.W. Szlosarek is supported by the Higher Education Funding Council for England. Polaris Pharmaceuticals, Inc. provided clinical trial support.

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