Abstract B53: An autopsy case of a hepatocellular carcinoma patient with remarkable tumor necrosis immediately following glypican-3-derived peptide vaccination.
Authors: Sawada Yu , Yoshikawa Toshiaki , Fujii Satoshi , Takahashi Mari , Nakatsura Tetsuya 
Published: August 2014

Abstract
Introduction: We recently reported vaccine's safety, immunological and clinical responses in a phase I clinical trial of GPC3-derived peptide vaccine for advanced HCC patients (Clin Cancer Res 2012; 18: 3686-96). The phase I trial showed that GPC3 peptide-specific CTLs increased in peripheral blood, that many CD8 positive T cells infiltrated tumors in some patients, and the correlation between the CTL response and overall survival after the GPC3 peptide vaccination. On the basis of these results, we conducted the next trial for advanced HCC to assess histopathological findings after GPC3 peptide vaccination. We present here the clinical course and pathological study including autopsy of an advanced HCC patient, which had revealed remarkable tumor lysis immediately after the second vaccination in the ongoing clinical trial of GPC3 peptide vaccine.
Study design: The trial was designed to assess clinical response, and whether tumor-infiltrating lymphocytes with anti-tumor effect are increased indeed after GPC3 peptide vaccination for advanced HCC (UMIN-CTR number: 000005093). In all cases of the trial, liver biopsies are going to be performed before and after the vaccination according to the protocol. 3.0mg of HLA-A*02 -restricted GPC3144-152 peptide (FVGEFFTDV) was administered intradermally in a liquid form emulsified with incomplete Freund's adjuvant (Montanide ISA-51VG, SEPPIC). The vaccination was scheduled every two weeks.
Clinical course: A 62-year old patent, suffering from HCC refractory sorafenib therapy, was offered participation in the clinical trial of GPC3 peptide vaccine for advanced HCC. Contrast-enhanced CT before the vaccination showed multiple tumors in the liver and tumor invading into the right atrium. According to the protocol, liver biopsy was performed before the vaccination. Histopathological examination of the specimen revealed well-differentiated hepatocellular carcinoma. Immunohistochemical staining showed the expression of GPC3 and HLA class I on the membrane of carcinoma cells, and that there were little CD8-positive T cell in the specimen of liver biopsy before the vaccination. This patient had fever up, general fatigue, and remarkable impaired liver function twice subsequently after the vaccination. Contrast-enhanced CT after the second vaccination depicted multiple low density areas in the liver, indicating extended tumor necrosis. On the other hand, tumor embolism in the inferior vena cava and the right atrium was increased. Unfortunately, on day 30 after the second vaccination, he died. The autopsy was performed to determine the main causes leading to death and to evaluate the antitumor effect of vaccination.
Autopsy study and immunological analysis: The main cause of death was diagnosed with circulatory failure due to tumor embolism, which occupied most of the right atrium. Histopathological examination showed the central necrosis in most of the tumor and viable carcinoma cells remained slightly around the necrotic tissue. Immunohistochemical staining showed that the infiltration of CD8 positive T cells near the residual carcinoma cells was observed, while it was not observed in the cirrhotic nodule. CD68 positive macrophages aggregated in the necrotic area around cirrhotic nodules. CD8 positive T cells also infiltrated there, suggesting probable that carcinoma cells might had been attacked by CD8 positive T cells, which might had leaded to necrosis. Ex vivo IFN-γ ELISPOT analysis of peripheral blood lymphocytes revealed vaccine-induced immune-reactivity against GPC3 peptide.
Conclusion: Histopathological examination at the estimated time of the strong immunological responses can prove GPC3 peptide vaccination induce GPC3 peptide-specific CTL responses with the anti-tumor effect.
Citation Format: Yu Sawada, Toshiaki Yoshikawa, Satoshi Fujii, Mari Takahashi, Tetsuya Nakatsura. An autopsy case of a hepatocellular carcinoma patient with remarkable tumor necrosis immediately following glypican-3-derived peptide vaccination. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B53.