This will be an open-label, single-arm, multicenter study (Figure 1). The primary endpoint of this study will be serious adverse events (safety profile); the secondary endpoint will include time to progression, overall survival, completion rate, and adverse events (efficacy profile). This study will be conducted in accordance with the Declaration of Helsinki, Clinical Trials Act, Ethical Guidelines for Medical and Health Research Involving Human Subjects, and all other applicable laws and guidelines in Japan. The protocol of this study was approved by the Institutional Review Board at Kanazawa University Hospital and is registered at the Japan Registry of Clinical Trials (jRCTs041180155).

We will first recruit 14 patients at Toyama City Hospital and Fukui-ken Saiseikai Hospital, subsequently adding those at the Kanazawa University Hospital, from October 2017 to December 2019. All patients will meet the inclusion criteria and no exclusion criteria will be applied (Textbox 1). Patients will be provided with comprehensive information about AFP-derived peptides and will provide written informed consent to participate in this study.

The treatment drug will be AFP-derived peptides (AFP 357 and AFP 403), which contain human leukocyte antigen (HLA)-A24–restricted cytotoxic T lymphocyte epitopes derived from tumor antigens expressed in HCC that are recognized by lymphocytes in patients with HCC at a high rate [11,13]. The AFP-derived peptides used in this study will be produced by Neo MPS Inc (San Diego, California) at Good Manufacturing Practice grade. One course of treatment will consist of the subcutaneous administration of 3 mg each of AFP 357 and AFP 403 on day 1 followed by 13 days of rest. Treatment will be repeated for up to six courses unless the following discontinuation criteria are applicable: obvious tumor progression based on radiological or physical findings, any adverse event that causes the discontinuation of protocol therapy, patient refusal, or death during protocol therapy. Before the treatment course starts, we will confirm that no grade 2 or higher adverse events exist. The administration of anticancer drugs other than AFP 357 and AFP 403 and procedures such as TACE, radiotherapy, hormonal therapy, or immunotherapy will not be permitted during the study. However, corticosteroids needed for fatigue; anorexia and emaciation; diuretics; amino acid preparations for ascites; hepatic edema; treatment of complications such as hypertension or diabetes mellitus; and antiemetics for nausea or vomiting will be permitted.

Table 1 shows the overall follow-up schedule. In this study, six courses of AFP-derived peptide administration are defined as the protocol treatment, and the entire study period is classified into the following three periods: pretreatment period, from consent to the start of treatment; treatment period, from the start of treatment to 30 days after the last administration (to assess adverse events related to protocol treatment); and follow-up period, from the end of the treatment period to death or to the final follow-up of this study. Before registration, hepatitis virus markers including hepatitis B virus antigen (HBsAg) and hepatitis C antibody will be assessed. Hepatitis B virus-DNA will be checked in positive HBsAg results. Hepatitis B surface antibody and Hepatitis B core antibody will be checked if HBsAg results are found to be negative. Chest x-ray, electrocardiogram, and tumor markers including AFP and des-carboxy-prothrombin will be assessed prior to TACE treatment. Patient’s general condition will also be evaluated using Eastern Cooperative Oncology Group (ECOG) performance status, body weight, encephalopathy, blood tests (complete blood count, blood chemistry, and coagulation), objective findings, contrast-enhanced computed tomography, and tumor markers within 14 days prior to registration.

During the treatment period, patients’ general condition including ECOG performance status, encephalopathy, and adverse events will be evaluated at every visit for the safety profile. Blood tests, chest X-ray, or electrocardiogram will also be performed if needed. Contrast-enhanced computer topography (CT) and tumor markers will be evaluated for the efficacy endpoint every 12 weeks after enrollment. Contrast-enhanced CT and tumor markers will also be evaluated until tumor progression is confirmed during the follow-up period.

The primary endpoint of this study will be the occurrence of serious adverse events (safety profile) during the treatment protocol. Serious adverse events are defined as any adverse events causing death, life-threatening condition, hospitalization (initial or prolonged), disability or permanent damage, or congenital anomaly/birth defect outcomes. Adverse events will be evaluated according to the Common Terminology Criteria for Adverse Events v 4.0 JCOG Version (Japanese translation of the NCI-Common Terminology Criteria for Adverse Events v 4.0) (CTCAE v 4.0 - JCOG). Each adverse event will be graded based on the definitions of grades 0-4. If a specific procedure is described based on grade, it will be graded for its clinical need. For example, if a patient has increased pleural effusions and oxygen or chest drainage is indicated, the patient may refuse. In such cases, the grading will be based on the medical judgment of what should have been performed rather than whether treatment was actually administered.

The secondary endpoints will be the efficacy profile including time to progression, overall survival, and completion rate as well as adverse events. The efficacy profile will be evaluated according to the Revised RECIST guidelines for the evaluation of the therapeutic efficacy of solid tumors Version 1.1 every 12±2 weeks after registration. The efficacy will be evaluated based on the same examination conditions as the baseline evaluation, such as slice width if imaging results are available. However, plain radiography and contrast studies with different modalities will be acceptable if a contrast allergy is detected prematurely and testing with the same modality cannot be continued.

Selected monitoring staff will conduct centralized data monitoring. The trial database will be monitored and reviewed annually by the selected monitoring staff, and data queries will be raised if necessary.

We first calculated the sample size for this study based on our in-house data on patients with HCC. Kanazawa University Hospital conducted TACE for 350 patients with HCC from January 2005 to December 2011. In this group of Japanese patients with HCC, approximately 60% of the individuals were HLA–A24-positive, and approximately 30% of the candidates were expected to drop out based on eligibility criteria and exclusion criteria. Considering that this is a safety confirmation study, the rate of obtaining consent for this study was suggested to be 30%, and as such, approximately 8 patients were expected to be recruited annually. On January 2019, the safety profiles of the 6 cases included in this study were reviewed by authorities of the Ministry of Health, Labour and Welfare, and the safety of the treatment protocol was confirmed. Based the advice of the Efficacy and Safety Committee, the 6 cases were designed as the main analysis target group for the safety confirmation part. Registration of additional cases continued at Fukui-ken Saiseikai Hospital, Toyama City Hospital, and Kanazawa University Hospital until December 31, 2019. The target number of patients in this extended cohort was set at 8 based on the expected number of patients at the cooperative research institution. In total, we included 14 patients from three hospitals.

Safety profiles will be summarized using appropriate descriptive statistical methods. If necessary, we will obtain an accurate 95% confidence interval based on the binomial distribution. Time to progression is defined as the period from the date of enrollment to the date of evaluation of tumor progression. Tumor progression includes both “progressive disease” based on imaging examination, evaluated according to RECIST version 1.1 (radiological progression), and tumor progression that cannot be confirmed by imaging examination (clinical progression). The date of the tumor progression evaluation will be determined by when imaging examination is performed for radiological progression and clinical judgement is made for clinical progression. Overall survival is defined as the period from the date of enrollment to the date of death from any cause. Cumulative survival will be estimated using the Kaplan-Meier method. Completion rate will be calculated as the number of patients receiving six courses of AFP-derived peptide divided by the number of patients enrolled.