2.1 Study design
This is a prospective, single-center, randomized, double-blind, clinical trial, which will be conducted at the Second Affiliated Hospital of Chongqing Medical University in China. The study was registered at the Chinese Clinical Trial Registry on 14th August 2021 (ChiCTR-2100049941). We aim to recruit 142 eligible subjects and allocate these to the Control group (standard TMZ chemoradiotherapy) or the Experimental group (TMZ plus LEV chemoradiotherapy) at a 1:1 ratio. All participants will undergo a treatment period of 34 weeks and a follow-up period of three years. The trial flow chart is shown in Figure 1. The study protocol rigorously follows the Standard Protocol Items: Recommendations for Interventional Trials 2013 (SPIRIT 2013) checklist (See Additional File 1).
2.2Recruitment and eligibility criteria
The enrollment is expected to be completed within three years (from the beginning of recruitment to the last patient). Enrolment is projected to start in November 2021. The recruitment advertisements for the study will be placed on the WeChat public website and the hospital website. Research staff (NH, YHT and GJZ) will be responsible for screening inpatients and outpatients according to the inclusion and exclusion criteria. Then, the investigator will introduce the protocol to the participants and explain the harms and benefits of this trial in detail.
The inclusion criteria are as follows: (1) aged 18–65 years, female or male; (2) newly diagnosed GBM (WHO grade IV) patients with maximal surgical resection; (3) patients within three months of surgery; (4) KPS ≥50; (5) adequate hematological, renal, and hepatic function. All patients should meet the following criteria: (a) absolute neutrophil count (ANC)≥1.5x10 9 /L, platelet count≥100x10 9 /L, (b) serum creatinine ≤ 1.5 x upper limit of the normal range for each institution (ULN), (c) total bilirubin level ≤ 1.5 x ULN (except patients with Gilbert Syndrome), (d) aspartate aminotransferase (AST) ≤ 3.0 x ULN; alanine aminotransferase (ALT) ≤ 3.0 x ULN, AST/ALT < 2.5 x ULN; and (6) the patient and his/her family members were informed and provided signed and informed consent.
The exclusion criteria are as follows: (1) prior chemotherapy within the last five years; (2) planned surgery for other diseases; (3) pregnant women or those that are breastfeeding; (4) patients with a known hypersensitivity to LEV and TMZ or any of the excipients of the products; (5) concurrent illness, including unstable heart disease despite appropriate treatment, a history of myocardial infarction within 6 months, and active hepatitis (Hepatitis B Virus (HBV) or Hepatitis C Virus (HCV)); and (6) patients with serious psychological disease.
The withdrawal criteria are as follows: (1) voluntary withdrawal during the intervention; (2) alcohol and/or drug abuse; (3) patients who take part in another trial concerning glioma; (4) any other reasons considered inappropriate by the investigators.
All patients will be allocated to the Experimental group or the Control group according to a blocked randomization sequence produced by SPSS 11 software for windows. Each group will be prescribed drugs for 34 weeks (including four weeks as an interval). The protocol interventions consist of chemotherapy and radiation therapy (RT). A chart showing the administration of interventions is shown in Table 1.
2.3.1 Co-interventions (RT)
Patients in both study arms will receive the same intensity-modulated radiation therapy (RT). The radiation dose will be 54 - 60Gy in 30 fractions (1.8 - 2.0 Gy once daily, 5 days per week, for six weeks). The particular volumes of radiation treatment will be defined by clinical target volume (CTV) using MRI data.
2.3.2 Experimental group
Concurrent chemoradiotherapy (CCRT): Patients will take TMZ one hour before meals in the per-oral form at a daily dose of 75 mg/m 2 continuously for 6 weeks during RT if the non-hematological toxicity ≤ grade 1 except for hair loss, nausea, and vomiting, according to the common toxicity criteria (CTC)(18). Patients in the experimental group will be given LEV tablets for the same duration. The initial treatment dose is 250 mg each time, twice a day. According to clinical effect and tolerance, the daily dose can be increased to 1500mg each time, twice a day. Dosage changes should be increased or decreased by 500 mg/time every 2–4 weeks, twice a day.
Adjuvant chemotherapy: Four weeks after the end of the CCRT, six cycles of adjuvant therapy with TMZ plus LEV will be carried out. The daily dose of TMZ in the first cycle will be 150mg/m 2 continuously for five days during each 28-day cycle. At the beginning of the second cycle, if the non-hematological toxicity of the first cycle CTC is ≤2 (except for alopecia, nausea, and vomiting), the ANC is ≥ 1.5×10 9/L, and platelet count is ≥ 100 ×10 9 /L, then the dose of TMZ can be increased to 200 mg/m2/day. The daily dose of LEV generally should not be less than 500mg, continuous for 6 cycles. Drugs dose adjustments will be conducted according to the liver and renal function. if the patient's creatinine clearance rate is less than 50 mL/min, the daily dose should be halved. For patients with mild and moderate liver impairment, there is no need to adjust the dosage.
2.3.3 Control group
Standard TMZ chemoradiotherapy will involve TMZ plus placebo concurrent chemoradiotherapy plus six cycles of adjuvant treatment. The dosage of TMZ will be the same as that of the experimental group; placebos for LEV (with the same dosage and appearance) will be given during treatment.
2.3.4 Concomitant care
Patients with glioma often suffer from epileptic seizures. In the clinic, antiepileptic drugs (AED) will be administered to certain patients, including levetiracetam (LEV), valproic acid (VPA), phenytoin, carbamazepine, and phenobarbital. To minimize the impact on the experiment, we will recommend these drugs except for LEV. In addition, metoclopramide tablets (10mg) will be permitted to address nausea and vomiting after taking TMZ. All of the medications used during the trial will be documented in a case report form (CRF).
2.4.1 Primary outcome:
The primary outcome is the PFS of patients, the time from randomization and group allocation to any recorded disease progression, and even death.
2.4.2 Secondary outcomes are as follows:
(1) overall survival (OS), the time from randomization and allocation to death for any reason,
(2) the Karnofsky Performance status scale (KPS). This scale evaluates the levels of patient activity and medical needs. On this scale, there are 11 categories; the score ranges from 100 (no evidence of disease) to 0 (dead).
(3) Objective response rate (ORR)=(CR+PR)/total number of cases × 100%. A complete response (CR) will be defined as the disappearance of all target lesions. A partial response (PR) will be defined as a minimum 30% reduction in the sum of diameters of the target lesions, taking baseline sum diameters at a reference. ORR will be assessed by MRI according to the response evaluation criteria in solid tumors (RECIST)(19).
(4) Incidence of adverse events (AEs). Adverse events (CTC-Toxicity ≥ grade3) will be recorded and analyzed based on the Common Terminology Criteria for Adverse Events (CTC-AE)(18).
2.4.3 Other measures:
Complete blood counts (CBC): ANC, platelet count; Liver and renal function: creatinine clearance rate, total bilirubin level, Aspartate Aminotransferase (AST), and Alanine Aminotransferase (ALT);
2.5 Baseline and a follow-up visit
Demographic assessment will be carried out before the interventions, including age, gender, past medical history, the molecular pathological indicators (1p/19q status, ABCG2 level, and MGMT promoter status), complete blood counts (CBC), KPS, head MRI, and hepatic and renal function. During treatment, CBC and hepatic-renal function will be monitored weekly. KPS score and head MRI will be acquired on the third and sixth cycle and then every three months over the three years. Adverse events will be monitored continuously during treatment, then every 3 months in the 3 years. A flow diagram of enrollment, interventions, and assessments for participants (participant timeline) is presented in Table 2.
2.6 Sample size
The determination of sample size was calculated by PASS 15.0 software. Based on a previous study and clinical assumptions (20), the median OS of standard care for GBM is 14.6 months while that of the experimental group (TMZ plus LEV ) is 24 months. Statistical parameters are set as follows: One-sided log-rank testing with 80% power; the probability of obtaining a false positive with a statistical test at 0.05; a three years follow-up time. Considering a potential dropout rate of 10% across both groups, it is estimated that 142 patients will need to be enrolled (71 participants per group).
2.7 Randomization and blinding
Eligible subjects will be randomly divided into two parallel pairs of groups in a 1:1 ratio in line with a software-generated random sequence stratified by operation situation (have or not) (produced via SPSS software). The allocation sequence will be concealed using sealed, opaque, and stapled envelopes that will not be opened for the participants and recruiters prior to study group assignment. The implementation of sequence generation and allocation concealment will be conducted by a researcher who will not be involved in the recruitment process. A specific investigator will be responsible for the processing of study-group assignments.
The participants and outcome assessors will be blinded to the group allocation until the end of the trial. The study data statistician will not be unaware of allocations and will not participate in the follow-up visit. Regular unblinding will be performed for the first time by the principal investigator and statistical experts to conduct statistical analysis according to the statistical plan. The second unblinding progress will be performed by researchers to determine which of the groups was the experimental group and which was the control group. To avoid unnecessary unblinding or harm, the emergency unblinding of allocation will be conducted if the patient meets the following criteria: (1) patients who severely violate the treatment plan; (2) patients have severe adverse events during intervention; (3) patients or their family members request to stop the trial, and (4) visitors lost in follow-up.
2.8 Data collection and management
A case report form (CRF) will be filled in for each participant before intervention. The collection of baseline data, along with primary and secondary outcomes, will be recorded completely in CRF. All information will be held independently as double copies in a computer and imputed into ResMan, an Internet-based Electronic Data Capture (EDC). The data files will be locked after a blind audit and confirmed to be reliable. The CRF will be owned by the principal investigator and shall not be provided to any third party in any form without the written approval of the principal who will oversee all of the final trial data. All patients will complete a registration form of follow-up to promote retention. For participants who lose from intervention, the reason for loss, survival condition, and recent outcome assessment will be collected by a researcher. Finally, all original paper files will be kept in the filing cabinet of sponsoring organization and clinical data will be saved for 5 years.
2.9 Quality control
A data monitoring committee (DMC), consisting of neurosurgery experts and statisticians, will be established; this will be independent of the sponsor and trial investigator. The DMC will periodically evaluate the progress of the clinical trials, safety data, and important efficacy data. Furthermore, the DMC will also perform an interim evaluation and advise if the trial should be modified or discontinued according to the results of the interim evaluation.
During the study, an inspector shall check the accuracy and completeness of the CRFs and compare them with the source document. The inspector must make certain that the subject’s dose changes, treatment changes, adverse events, combined medications, missed visits, and examination omissions are recorded in the CRFs. They must also verify that the withdrawal and missing visits of the selected subjects have been recorded and explained in the CRFs. In addition, the inspector will notify the researcher of the errors, omissions, or unclear handwriting in CRFs and ensure that correction, addition, or deletion is carried out by a researcher or authorized person. If necessary, the reasons for modification must be recorded in writing. After each supervision and inspection, the inspector will write to the principal in a prompt manner.
2.10 Statistical analysis
We will use SPSS 23.0 statistical software for Windows to perform statistical analysis. To ensure the reliability of our conclusions, we plan to use intention-to-treat (ITT) analysis as the main form of analysis.
2.10.1 Baseline description
Means ± standard deviation (SD) will be used to describe quantitative data that are normally distributed; medians and interquartile ranges (IQRs) will be used to describe non-normal distribution data. The constituent ratio or relative ratio will be used to describe the count data.
2.10.2 Comparison of baseline data
T-tests will be performed for quantitative data that are normally distributed. A non-parametric test will be used for quantitative data that is not normally distributed. The Chi-squared (χ 2) test will be used for counting data. A two-sided P value < 0.05 will be considered to be statically significant.
2.10.3 Comparison of therapeutic efficacy
Kaplan–Meier survival analysis with a Log-rank test will be used to compare survival data (PFS and OS) between the two arms. Confounding factors, including age, history of drug use prior to intervention, epileptic seizure, and molecular pathological indicators will be analyzed by Cox proportion hazards regression analysis. Results will be expressed as hazard ratios (HRs) and 95% confidence intervals (CIs). Furthermore, the KPS and the ORR (ranked data) of the two arms will be compared by Wilcoxon’s Rank-sum Test.
2.10.4 Safety evaluation
Incidence of adverse events (CTC-Toxicity ≥ grade3) will be expressed as the number of patients (percentage) and be analyzed by the Chi-square (χ 2) test.
2.11 Reports of adverse events
Adverse events (AEs) will be evaluated mainly by abnormal variations in laboratory data (including complete blood counts (CBC), liver function tests (total bilirubin level, AST, ALT), renal function tests (creatinine clearance, blood urea nitrogen), and clinical symptoms. The time of occurrence, severity level, management strategies, the causality related to experimental agents, and the endings of all adverse events (AEs) will be recorded and preserved during clinical trials and follow-up. All severe adverse events (CTC-Toxicity ≥ grade3) will be reported to the principal investigator and the research ethics committee (REC) in a timely manner. The researchers and clinical trial institutions will ensure that the subjects are treated appropriately and truthfully informed of the relevant information. When patients are informed of adverse reactions that are related to the trial by the provisions of China's Code of Quality Management for Drug Clinical Trials, the researchers will be responsible for the cost of dealing with the adverse reactions and the compensation that the patient may receive.