A Systematic Review and Meta-Analysis on the Number of Adjuvant Temozolomide Cycles in Newly Diagnosed Glioblastoma

Background In newly diagnosed glioblastoma, radiation with concurrent and adjuvant (six cycles) temozolomide (TMZ) is the established standard of postsurgical care. However, the benefit of extending adjuvant TMZ therapy beyond six cycles has remained unknown. Methods We searched PubMed, Web of Science, Scopus, and Embase up to October 1, 2021. The search keywords were “glioblastoma,” “adjuvant chemotherapy,” and their synonyms. The data of randomized clinical trials were extracted and included in this meta-analysis if they had reported patients’ median overall survival (OS) or median progression-free survival (PFS). The standard and extended chemotherapy regimens were considered as adjuvant TMZ up to six cycles and beyond six cycles (up to a total of 12 cycles), respectively. The median OS and median PFS were pooled and compared. Results Four studies consisting of 882 patients (461 patients for the standard chemotherapy group and 421 patients for the extended chemotherapy group) were included in this meta-analysis. The extended TMZ regimen was associated with a nonsignificant improvement in PFS [12.0 months (95% CI 9.0 to 15.0) vs. 10.0 months (95% CI 7.0 to 12.0), P = 0.27] without corresponding improvement in OS [23.0 months (95% CI 19.0 to 27.0) and 24.0 months (95% CI 20.0 to 28.0), P = 0.73]. Conclusions In newly diagnosed glioblastoma, continuing adjuvant TMZ beyond six cycles did not shown an increase neither in PFS nor OS.


INTRODUCTION
Glioblastoma is the most common primary brain tumor of glial origin in adults. It is characterized by rapid progression, a high recurrence rate, and a dismal prognosis (1)(2)(3). Historically, the management of glioblastoma was maximal safe surgical resection (MSR) followed by radiotherapy. In the early 21 st century, a large randomized clinical trial (RCT) converted the standard of care to MSR, adjuvant chemoradiotherapy (CRT) [with concurrent temozolomide (TMZ), an oral alkylating agent], followed by TMZ for six cycles (4). Dismal prognosis of glioblastoma brought up the extended adjuvant TMZ (ext-TMZ) and changed the clinical practice to continue adjuvant TMZ up to 12 cycles or until tumor progression. Since then, numerous studies have attempted to compare the ext-TMZ and the standard Stupp protocol (std-TMZ). However, there is still no consensus on the duration of adjuvant TMZ (5).

Study Design and Types of Included Studies
This meta-analysis was conducted per the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guideline (29). It included RCTs comparing std-TMZ and ext-TMZ-as the first-line treatment in glioblastoma-in terms of median OS and median PFS.

Search Strategy
Two authors (S.A.J and F.A) independently searched the English literature for free-text and standard MeSH (Medical Subject Headings) terms in PubMed, Web of Science, Scopus, and Embase up to October 1, 2021. The search keywords were: high-grade glioma OR malignant glioma OR glioblastoma OR glioblastoma multiforme OR grade IV glioma OR grade IV astrocytoma OR GBM, adjuvant chemotherapy OR temozolomide OR temodar, AND extended OR long-term OR prolonged OR maintenance OR cycles OR months. Also, the two review authors handsearched the reference lists of the relevant articles to identify the possible missed RCTs. Thereafter, they downloaded all titles and abstracts retrieved by electronic searching to EndNote ™ V.20.0, removed duplicates, and excluded the studies that did not meet the eligibility criteria, clearly (mentioned below). Eventually, they debated on the disagreements to improve the search results.

Participants
Adult patients with glioblastoma who underwent surgery, radiotherapy (concurrent with TMZ), and adjuvant TMZ as the primary treatment.

Inclusion Criteria
RCTs comparing std-TMZ and ext-TMZ, in which the median OS and/or median PFS were reported.

Exclusion Criteria
Studies if (i) not following the standard treatment sequence of MSR, CRT (with TMZ), and adjuvant TMZ, (ii) submitted only as abstracts or proceedings from scientific meetings, (iii) lacking English full text or summaries, or (iv) including patients with recurrent glioblastoma were excluded. Eligible studies were assessed finally for quality of methodology (30, 31).

Data Extraction
The following data were extracted from the studies: (i) study information (the first author, year of publication, study country, sample size), (ii) patient baseline characteristics (age, sex ratio), (iii) intervention duration (std-TMZ, ext-TMZ), and (iv) treatment outcomes (median OS, median PFS). Only data from the first-line therapy of both groups were extracted.
The standard chemotherapy regimen (std-TMZ) was defined as ≤ 6 cycles of TMZ following MSR and adjuvant CRT. The extended chemotherapy regimen (ext-TMZ) was defined as > 6 cycles of TMZ (up to 12 cycles) following MSR and adjuvant CRT. The median OS and PFS were extracted directly from the text or the Kaplan-Meier survival curves.

Quality Assessment
Two investigators (S.A.J. and F.A) assessed the methodological quality and risk of bias of the included studies. All four included studies were assessed using Cochrane's Risk of bias tool (31). They resolved differences by discussion or appeal to a third review author (F.T) and presented results in a "Risk of bias" table. The risk of bias summary consists of 5 questions (also known as the Oxford quality scoring system), ranging from 0 to 5. Studies with a quality score less than 3 were regarded as poor quality and excluded from the study ( Table 1).

Statistical Analysis
The main objective of this meta-analysis was to compare the median PFS and OS for std-TMZ versus ext-TMZ as the first-line treatment of patients with glioblastoma. The individual patient data (IPD) is essential in the standard approach to pooled survival estimates (26). However, IPD was unavailable in this meta-analysis, and we used the median PFS and OS (weighted by the inverse of variance) to estimate the pooled median and 95% confidence interval (95% CI) of PFS and OS in each group of RCTs. The statistical heterogeneity between studies was evaluated using Cochran's Q test and quantified by I 2 statistics (high heterogeneity was defined as I² > 20% or P-value < 0.1). We applied Stata V.14.0 (Stata Corp, College Station, TX, USA) for the quantitative synthesis. The statistical significance level was set to 0.05.
A total of 882 glioblastoma patients were included in the four studies. Of these, 461 patients were treated by std-TMZ regimen, and 421 patients received ext-TMZ regimen. The median PFS of patients with glioblastoma who were treated by the standard or extended chemotherapy regimens is shown in Figure 2.   (19,20,23).The remaining study was a pooled analysis of four RCTs (26). The main characteristics of the eligible studies are shown in Table 2. In summary, the current meta-analysis did not demonstrate the survival benefit of prolonged adjuvant TMZ in newly diagnosed glioblastoma. This might be explained by adaptive resistance. To better understand this issue, we need to recognize the mechanism of action of TMZ. As an alkylating agent, TMZ acts as a prodrug and induces cell cycle arrest at G 2 /M through methylation of DNA or RNA. The methylated sites can remain mutated by DNA mismatch repair (MMR) proteins, dealkylated by the action of O 6 -methylguanine methyltransferase (MGMT), or be removed by the base excision repair (BER) enzymes [such as alkylpurine-DNA-N-glycosylase (APNG)]. Cells are TMZ sensitive when MMR is overexpressed and active. On the other hand, MGMT or BER proteins overexpression increases the resistance of glioblastoma cells to TMZ. In vitro studies have delineated several mechanisms of adaptive resistance to TMZ in glioblastoma cell lines. For example, increased MGMT protein expression (33), decreased Tumor Necrosis Factor-Alpha-Induced Protein 3 (TNFAIP3) expression (34), upregulation of Signal Transducer and Activator of Transcription 3 (STAT3) (35), loss of MSH6 MMR gene (36), or upregulation of NTL1 (a BER enzyme) (37). Therefore, prolonged adjuvant TMZ can promote the development of tumor-resistant clones with more aggressive features. This issue can contribute to a dismal prognosis in salvage therapy of tumor recurrence. A multicenter, phase II trial-evaluating the efficacy of continuous dose-intense TMZ for recurrent glioblastomaconcluded that patients who had received adjuvant std-TMZ got more benefit from therapy in comparison with ext-TMZ group (38).
Our study harbors several limitations. Lack of access to IPD and not reporting the hazard ratio in most of the studies are among the main ones. Besides, the current evidence on the role of MGMT methylation in the value of prolonged TMZ therapy beyond six months is either not addressed properly in the clinical trials (20, 23), or is assessed retrospectively (26). Therefore, prospective data on the predictive value of MGMT methylation is lacking, and our analysis cannot provide any comment in this regard. Among the included studies, only Balana et al. evaluated the role of MGMT on the survival of patients with glioblastoma receiving first-line adjuvant TMZ. By multivariate analysis, they showed that MGMT methylation was an independent factor for longer PFS and OS. However, this finding was not translated into the survival benefit of extended TMZ in patients with MGMT methylation (19).
In conclusion, prolonged adjuvant TMZ (beyond six cycles) did not provide OS and PFS benefits in patients with newly diagnosed glioblastoma. Considering this finding, along with the adverse effects of TMZ, the economic burden and psychosocial impacts of prolonged treatment can underscore the rationality of the current practice, which is 6 cycles of adjuvant TMZ. Further studies are needed to determine the predictive value of MGMT status on the long-term TMZ maintenance therapy. Moreover,

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
SJ, FT-H, and FA designed the meta-analysis, screened the studies and wrote the manuscript. FA analyzed data. AF, PP, and BP helped designed the study. All authors reviewed the manuscript finally. DF-P and AF wrote the initial draft. SJ, FA, PP, and BP approved the final submission of the manuscript.