in patients with chronic hepatitis and in those with liver cirrhosis , detection of increasing incidences of hepatocellular carcinoma ( hcc ) requires careful monitoring every 36  months using ultrasound sonography ( us ) , computed tomography ( ct ) , magnetic resonance imaging ( mri ) , and frequent blood tests . once hcc is detected , patients experience repeated recurrence , despite the fact that a variety of hcc treatment methods have been developed , such as anti - viral agents , surgical treatment , percutaneous ethanol injection ( peit ) , radiofrequency ablation ( rfa ) , trans - catheter arterial chemoembolization ( tace ) , and molecular - target drugs . in the last 10  years , tumor vaccines generated from patients ' own formalin - fixed hcc tissues the autologous formalin - fixed tumor vaccine ( aftv ) has been reported to reduce the recurrence risk of primary resected hepatitis b virus - background hcc by 81% 1 . in one case , recurrence of multiple - recurrent hepatitis c virus ( hcv)-back ground hcc was clearly suppressed long - term without any additional treatment 2 . here , we report a case in which aftv injection resulted in suppression of multiple - recurrent hcv - background hcc recurrence . in this case , we detected a significant density of glypican-3 ( gpc3)-specific cytotoxic t lymphocytes ( ctl ) in the blood at 12  months after the last intradermal injection of aftv . a 55-year - old female patient was diagnosed with hcv - related hepatitis in 2002 . liver biopsy showed active chronic hepatitis accompanied by bridging fibrosis and necrotic inflammatory reactions . to reduce the activity of chronic hepatitis , the patient was treated with consensus interferon for 24  weeks . results showed complete remission of the hepatitis as liver transaminases normalized , as well as conversion of seropositive hcv to negative up to the present . when she was 57  years old , two 20-mm hccs were observed in left lobes s2 and s3 , and a left hepatic lobectomy was performed in june 2004 . in addition , two 10-mm hccs in s5 and s7 were treated with radiofrequency ablation ( rfa ) in september 2008 . the 10-mm hcc in s5 was detected again in january 2011 , and was treated with trans - catheter arterial chemoembolization ( tace ) and rfa . in january 2012 , a new 10-mm hcc was found in s6 , and a resection was done after treatment with rfa . pathologies obtained after the two hepatectomies showed poorly differentiated hcc in the 2004 specimen and moderately differentiated hcc in the 2012 specimen ( fig.1 ) . the liver function has been kept good and the bilirubin levels were < 2  mg / dl . the patient did not hope liver transplantation but desired aftv therapy because of the rapid decline in the recurrence interval , that is , 51- , 28- , 12- , and 4-months . pathology and glypican-3 ( gpc3 ) expression in hepatocellular carcinomas . ( a ) h&e staining and ( b ) gpc3 immunohistochemistry of representative tissue from a 2004-resected hepatocellular carcinoma ( hcc ) specimen of the present case . ( c ) h&e staining and ( d ) gpc3 immunohistochemistry from a 2012-resected specimen . gpc3 expression ( b , d ) was observed in areas with hcc ( arrows ) , but not in cirrhotic areas ( white stars ) . we prepared aftv as reported previously 3 using a total of 1.56  g of autologous hcc tissue recovered from formalin - fixed and paraffin - embedded tumors from the two operations . according to the therapeutic schedule employed 1 , a single course of aftv therapy applied from september to october 2012 consisted of the following : ( 1 ) a delayed - type hypersensitivity ( dth ) test with immunoadjuvant - free fixed hcc fragments applied to a forearm was initiated 48  h before the first aftv injection to check for allergic reaction . ( 2 ) up to three rounds of aftv were injected intradermally into the upper arm at 2  week intervals . ( 3 ) a second dth test , performed 2  weeks after the last aftv injection to the other forearm , showed a positive dth response . ( 4 ) ultrasound sonography and blood tests for tumor markers which were conducted every 3  months , and computerized tomography every 6  months , revealed no evidence of recurrence or generation of new hcc lesions for more than 27  months . immunohistochemistry to detect gpc3 4 in the hcc specimen resected in 2004 revealed strong expression of gpc in the hcc lesion ( fig.1b ) . we also detected gpc3 expression in the 2012 specimen resected after treatment with rfa ( fig.1d ) . however , we did not observe any gpc3 expression within cirrhotic lesions in either specimen ( fig.1b and d ) . to evaluate aftv - induced gpc3-reactive ctl , we carried out interferon--specific elispot assays as described previously 5 , using a target human hcc cell line expressing gpc3 ( sk - hep-1/gpc3 ) . the cell line was developed from sk - hep-1/hla - a0201/2402 cells by transfecting with human gpc3-gene . a control cell line ( sk - hep-1/vec ) although hla subtypes of the patient and the target cell line differ ( hla - a0206/3303 and hla - a0201/2402 , respectively ) , hla - a2 ( a0201)-restricted gpc3 epitopes are cross - reactive with hla - a0206 , allowing detection of gpc3-specific ctl that are reactive to the target cell line 5 . glypican-3-specific ctls were clearly observed in peripheral blood drawn 12  months after the first injection of aftv ( average 21.5 spots against sk - hep-1/hgpc3 cells vs. nine spots against sk - hep-1/vec control cells ) ( fig.2 ) . lymphocytes were prepared from the patient 's blood drawn at 12  months after autologous formalin - fixed tumor vaccine ( aftv ) injection . we detected hla - a2-restricted glypican-3 ( gpc3 ) peptide - specific cytotoxic t lymphocytes in the 12-month blood . sk - hep-1/hgpc3 , human gpc3 gene - transfected sk - hep-1 cells ( sk - hep-1 with hla - a a0201/2402 ) , a human hepatocellular carcinoma cell line unable to express gpc3 ; sk - hep-1/vec , control vector gene - transfected sk - hep-1 cell line . effector cells ( cd8 t cells ) , 1    10  cells / well ; target cells ( sk - hep-1 cells ) , 5    10  cells / well . gpc3 , a heparan - sulfate proteoglycans linked to the outer surface of cell membrane via a glycosylphosphatidylinositol anchor 6 , is frequently overexpressed in hcc 7,8 and in precancerous lesions in the liver 9 . for this reason , a recombinant humanized monoclonal antibody against gpc3 10 and a gpc3-peptide vaccine 11 have been developed for treatment of hcc . treatments with these new agents were well tolerated , and noted measurable immune responses and antitumor efficacy . however , clinical outcome were not necessarily prominent in these phase i trials compared to the previous expectations derived from preclinical studies . autologous formalin - fixed tumor vaccine differs from these specified molecule - targeted agents in that the autologous tumor , containing a wide array of unidentified antigens , is the source of antigens . thus , ctl induced with aftv made from tumor tissue must be polyclonal by nature ; for example , polyclonal ctl induced in vitro with a formalin - fixed carcinoembryonic antigen protein 12 . at present , no one provided any evidence that the impure tumor antigenic formalin - fixed autologous hcc tissue is truly active to induce a specific cellular immune response against a specified tumor antigenic molecule . the present case is the first one we could identify gpc3-specific cellular immune response based on the autologous formalin - fixed hcc . while we detected gpc3-specific ctl in the patient 's blood 12  months after aftv treatment , few gpc3-specific ctl were detected 16  months after the treatment ( data not shown ) . these results may suggest that , although the conspicuous life span of gpc3-specific ctl in blood is < 16  months , the immunoprotective effect of aftv continues beyond gpc3-specific ctl lifespan in blood , since this patient presently continued to show no evidence of hcc recurrence 32  months after the second resection of hcc . we consider two hypotheses to explain this continued protection : ( 1 ) the gpc3-specific ctl may have differentiated into memory t cells , which migrate to lymphatic tissues and are therefore not conspicuously detectable in peripheral blood . ( 2 ) the gpc3-specific ctl apoptosed after killing all target hcc cells in vivo , thereby eliminating all tumor antigens and preventing replicative stimulation to active ctl . this is the first case we could identify gpc3-specific cellular immune response based on impure tumor antigenic formalin - fixed autologous hcc tissue . present results suggest that aftv should be taken in consideration as one of treatment modalities in cases involving potential hcc derived from chronic hepatitis and liver cirrhosis .
key clinical messageautologous formalin - fixed tumor vaccine ( aftv ) suppressed re - recurrence for more than 32  months of multiple - recurrent hepatocellular carcinoma based on hepatitis c virus - induced liver cirrhosis in a case with previous recurrence interval , 51- , 28- , 12- , and 4-months . 
 we detected glypican-3-specific cytotoxic t lymphocytes in the peripheral blood at 12  months after aftv .