Sulfasalazine and temozolomide with radiation therapy for newly diagnosed glioblastoma
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.128280
Source of Support: None, Conflict of Interest: None
Background: A recent phase 1/2 clinical trial argued for caution for the use of sulfasalazine in progressive glioblastoma (GBM). However, the study enrolled patients with recurrent or progressive high-grade glioma indicating that patients recruited probably had severe disease. Thus, the study may not accurately reflect the effectiveness of sulfasalazine for GBM and we hypothesized that earlier sulfasalazine administration may lead to anticancer effects. Aim: The aim of this study was to investigate whether sulfasalazine can improve the outcomes of patients with newly diagnosed GBM. Subjects and Methods: A total of 12 patients were treated with temozolomide and sulfasalazine with radiation therapy after surgery. Twelve patients with primary GBM treated with temozolomide and radiation therapy formed the control group. Progression-free survival (PFS), overall survival (OS) and seizure-free survival (SFS) curves were obtained using the Kaplan-Meier method. The survival curves were compared using the log-rank test. Results: The median OS, PFS and SFS did not differ between the groups. Grade 3 or 4 adverse events occurred over the duration of the study in nine (75%) patients. The median SFS was 12 months in nine patients who received sulfasalazine administration for more than 21 days, which was strongly but not significantly longer than the 3 months observed in the control group (P = 0.078). Conclusions: Sulfasalazine treatment with temozolomide plus radiotherapy for newly diagnosed primary GBM is associated with a high rate of discontinuation due to hematologic toxic effects. This treatment may have no effect on OS or PFS, although it may improve seizure control if an adequate dose can be administered.
Keywords: Glioblastoma, seizure, side-effects, sulfasalazine, X-ray computed tomography
Gliomas are the most common primary tumors of central nervous system. These tumors orginate from glial cells, or glial progenitors and glioblastoma (GBM) is the most malignant type of glioma. , The prognosis of patients with GBM remains poor despite the administration of aggressive cytotoxic therapies.  The invasive nature of GBMs and the need to preserve the brain function almost always prevents the use of total surgical excision.  In spite of the wealth of preclinical and clinical research, treatment options for GBM remain scarce and the median survival of patients barely reaches 15 months regardless of the use of surgery, irradiation and chemotherapy (temozolomide).  Several molecular targets against GBM have been proposed, including nuclear factor κ-light chain-enhancer of activated B cells (NF-κB) and system xc− .
NF-κB is a transcription factor activated by the epidermal growth factor receptor signaling pathway. NF-κB is constitutively activated in GBM. ,, Activated NF-κB contributes to the growth and survival of tumor cells and NF-κB inhibition blocks tumor growth in both in vitro and in vivo models. , System xc− is a cystine-glutamate exchange transporter consisting of a light-chain subunit (X-ray computed tomography [xCT], SLC7A11) and heavy-chain subunit (CD98hc, SLC3A2).  The level of the xCT expression is correlated with the system xc− activity.  System xc− represents the primary viable pathway for glutamate release and cystine uptake in glioma cells.  xCT-mediated cystine uptake and glutamate release in cancer cells are highly associated with cell proliferation, chemoresistance, tumor invasion and tumor growth. ,, Furthermore, our previous study showed that the xCT expression is an independent predictive factor for both overall survival (OS) and progression-free survival (PFS) in GBM patients.  Therefore, we hypothesized that xCT inhibition may improve OS and PFS in patients with GBM.
The anti-inflammatory agent sulfasalazine is a known inhibitor of the system xc− and κB kinase, which is essential to the canonical pathway of NF-κB activation and is widely used to treat various diseases, including rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, ulcerative colitis and Crohn's disease.  Furthermore, sulfasalazine is considered to be safe.  Therefore, the effects of sulfasalazine on GBM have been encouraging. Nevertheless, the results of a recent phase 1/2 clinical trial urged caution in the use of sulfasalazine for GBM therapy.  In that study, the application of sulfasalazine may have been associated with grade 4 toxicities, as well as unfavorable magnetic resonance imaging changes; therefore, the trial was terminated after its interim analysis. However, that study enrolled patients with recurrent or progressive high-grade glioma,  indicating that patients may have been too severely ill or neurologically impaired. Therefore, that study may not accurately reflect the effectiveness of sulfasalazine for GBM and we hypothesized that earlier sulfasalazine administration may lead to anticancer effects.
Seizures are commonly associated with low-grade gliomas and are a well-known presenting sign in patients with malignant brain tumors,  although they can be refractory to conventional antiepileptic medications.  In fact, the incidence of seizures in patients with GBM ranges from 30% to 50% respectively; , therefore, the occurrence of seizures dramatically affects the quality of life of patients with GBM.  The etiology of tumor-associated seizures and their relationship with tumor growth are poorly understood. Recently, attention has focused on the neurotoxicity caused by glutamate. In fact, increased glutamate levels have been implicated in numerous seizure disorders and glutamate can reach neurotoxic levels immediately preceding and during spontaneous seizures.  Previous studies conducted in both humans and animal models with glioma have suggested that epileptiform activity originates within the peritumoral border, where invading tumor cells surround the neurons and the glutamate concentrations are quite high. ,,, Furthermore, sulfasalazine reduces epileptic activity in glioma-bearing mice, implicating the role of glutamate release through system xc−S in the generation of tumor-associated epileptic events, suggesting that sulfasalazine may be considered an adjuvant treatment for ameliorating peritumoral seizures associated with gliomas. 
The present study investigated whether sulfasalazine with temozolomide plus radiotherapy can improve the outcomes, including seizure outcomes, of patients with newly diagnosed GBM.
This study was conducted with the approval of the ethics committee of the National Defense Medical College. Only patients from whom written informed consent was obtained were included.
Patients and tissues
Nineteen consecutive patients were newly histologically diagnosed with primary GBM between 2008 and 2010. Tumor specimens were obtained through surgical resection in all cases. Formalin-fixed, paraffin-embedded sections were stained with hematoxylin and eosin and both histological and cytological diagnoses were established. The histological diagnoses and tumor grading were performed according to the grading system established by the World Health Organization.  xCT immunohistochemistry was performed as described in a later section. Seven patients from whom informed consent was not obtained or who had GBM with negative xCT immunoreactivity were excluded; therefore, 12 patients were treated with temozolomide and sulfasalazine with radiation therapy after surgery and were classified into the sulfasalazine group. Another 12 consecutive patients with histologically confirmed primary GBM with moderately/intensely positive xCT immunoreactivity treated with temozolomide with radiation therapy after surgery (without sulfasalazine) before 2008 were selected for the control group. The patient charts were retrospectively reviewed to record age, sex, Karnofsky performance status, mini-mental state examination score, type of surgery, the xCT expression, tumor location, post-operative antiepileptic agents, administration periods and side-effects of sulfasalazine, PFS, OS and seizure-free survival (SFS). PFS was defined as the time from surgery to the first sign of radiological progression according to McDonald's criteria.  OS was defined as the time from surgery to death from any cause. SFS was defined as the time from surgery to the first seizure or death from any cause.
Temozolomide with radiation therapy
After diagnosis, the patients received radiotherapy (fractionated focal irradiation in daily fractions of 2 Gy given 5 days/week for 6 weeks, for a total of 60 Gy) plus continuous daily temozolomide (75 mg/cm 2 /day, 7 days/week from the first to the last day of radiotherapy), followed by adjuvant temozolomide (150-200 mg/cm 2 for 5 days during each 28-day cycle)  until exhibiting drug intolerance. There was no limit to the number of adjuvant cycles.
The patients in the sulfasalazine group also received daily doses of 1, 2 or 4 g of oral sulfasalazine continuously after diagnosis until exhibiting complete remission, evidence of progression or drug intolerance. Blood samples were analyzed at least every 7 days following treatment initiation during the first 6 weeks and at least every 28 days during adjuvant therapy. Adverse events were evaluated according to the Common Terminology Criteria for Advance Events (CTCAE version 3.0) (Public Health Service, National Institutes of Health, National Cancer Institute, Bethesda, MD, USA).
Post-operative antiepileptic treatment
There was no defined standard for the use of antiepileptic agents. Three patients (one in the control group and two in the sulfasalazine group) who experienced seizures during the preoperative period received antiepileptic agents continuously starting in the preoperative period. Fifteen patients (10 in the control group and five in the sulfasalazine group) received antiepileptic prophylaxis based on the clinician's preference. The selection of specific antiepileptic agents was also based on the clinician's preference.
Analysis of xCT
Immunohistochemical staining was performed on paraffin sections with rabbit anti-human xCT polyclonal antibody clones (1:1,000) as the primary antibody. The analysis was performed as previously described.  Briefly, the entire invasive margin that was available in each specimen was analyzed in a high-power field (×400). The immunoreactivity was graded based on the estimated percentage of immunopositive cells (0-10%, negative; 11-50%, moderately positive; and 51-100%, intensely positive respectively) by two independent observers unaware of the clinical information. Weak (faint) diffuse staining was not scored as positive.
All statistical analyzes were performed using the SPSS version 11.0 software program (IBM Corporation, Armonk, NY, USA). The correlations between two groups were determined using the Mann-Whitney or Fisher's exact tests. PFS, OS and SFS curves were obtained using the Kaplan-Meier method. The survival curves were compared using the log-rank test. P < 0.05 were considered to be statistically significant.
A comparison of the baseline characteristics of the two groups is shown in [Table 1]. The patient characteristics did not differ between the two groups. Kaplan-Meier curves of OS, PFS and SFS are shown in [Figure 1]. The median OS, PFS and SFS were 11 months, 4 months and 7 months, respectively, in the sulfasalazine group, which did not differ from the 13 months, 4 months and 3 months, respectively, observed in the control group.
Grade 3 or 4 adverse events occurred over the duration of the study in nine of the 12 patients (75.0%). Eight patients (66.7%) developed Grade 3/4 leukopenia and seven patients (58.3%) developed Grade 3/4 neutropenia. Sulfasalazine was discontinued during the first 6 weeks in seven patients (58.3%) due to Grade 3/4 leukopenia or neutropenia in five patients, nausea in one patient and dysphagia in one patient. Five patients received daily sulfasalazine for 6 weeks without discontinuation; however, one patient decided to discontinue adjuvant sulfasalazine and another patient did not receive adjuvant sulfasalazine due to tumor progression. Therefore, adjuvant sulfasalazine treatment was completed in three patients (25.0%) who received 13 cycles, two cycles and one cycle, respectively. Adjuvant sulfasalazine was discontinued due to Grade 3/4 leukopenia or neutropenia in all three patients. The administration period was 21 days or less in all three patients. The median cumulative administration period during the first 6 weeks was 38 days (range, 3-42 days).
A further analysis was performed to investigate whether adequate sulfasalazine administration had any effect on OS, PFS or SFS. Adequate sulfasalazine administration was defined as an administration period of more than 21 days (half of 6 weeks) during the first 6 weeks of treatment. Therefore, nine patients who received sulfasalazine administration for more than 21 days were compared with the control group. The characteristics of the two groups are compared in [Table 2]. The patient characteristics did not differ between the two groups. Kaplan-Meier curves of OS, PFS and SFS are shown in [Figure 2]. The median OS and PFS were 14 months and 6 months, respectively, in the sulfasalazine group, which did not differ from the 13 months to 4 months, respectively, observed in the control group. The median SFS in the sulfasalazine group was 12 months, which was strongly but not significantly longer than the 3 months observed in the control group (P = 0.078).
The current study showed that it is difficult to continue sulfasalazine treatment with temozolomide plus radiotherapy for newly diagnosed primary GBM due to the high rate of side-effects (especially hematologic toxic effects). Sulfasalazine is considered to be relatively safe. Neutropenia and leukopenia are potentially dangerous, but rare reactions. Neutropenia and/or leukopenia are observed in only 2-4% of patients with rheumatoid arthritis treated with sulfasalazine.  The rates of neutropenia and leukopenia during temozolomide plus radiotherapy are less than 10%.  In our series, 75% of the patients who received sulfasalazine plus temozolomide with radiotherapy developed neutropenia and/or leukopenia. Therefore, sulfasalazine and temozolomide may express synergic hematologic toxic effects.
In addition, the current study showed that sulfasalazine treatment has no effect on OS or PFS. We suppose that the high rate of drug intolerance observed in this study may be the primary reason for this lack of effect. Previous studies have suggested that sulfasalazine can effectively penetrate the blood-brain barrier (BBB) in mice and humans, ,, although a more effective drug delivery system to cross the BBB is required to overcome drug intolerance and allow the drug to exert its anticancer effects.
Based on the current results, we conclude that our protocol of sulfasalazine and temozolomide with radiation therapy for newly diagnosed GBM should not be routinely applied. However, the current findings also imply that sulfasalazine is useful for achieving seizure control if an adequate dose can be administered. This result is consistent with the findings of a previous in vivo study that showed that sulfasalazine reduces the epileptic activity in glioma-bearing mice, implicating the role of glutamate release through system xc− in the generation of tumor-associated epileptic events.  We believe that inhibition of system xc− could more significantly improve seizure outcomes if the study samples were larger and the high rate of drug intolerance could be resolved.
There are several limitations associated with the current study. Retrospective analyzes are subject to observational and assessment biases. Furthermore, the number of patients in this series was small, which reduced the statistical power and increased the chance of type-2 errors. Although, there were no significant differences in antiepileptic agents between the two groups, the lack of a defined standard for the use of such agents in our series prevented an accurate assessment of the effects of sulfasalazine on seizure outcomes. Further investigations are needed to elucidate the optimal strategy for targeting system xc− and NF-κB in patients with GBM.
Sulfasalazine treatment with temozolomide plus radiotherapy for newly diagnosed primary GBM is associated with a high rate of discontinuation due to hematologic toxic effects. This treatment may have no effect on OS or PFS.
[Figure 1], [Figure 2]
[Table 1], [Table 2]