cancer vaccine targeting hepatocellular carcinoma ( hcc ) tumor antigens have been tested in clinical trials .
however , cancer vaccines using tumor - antigen - derived peptides have not demonstrated adequate antitumor efficacy in clinical trials for advanced hcc .
glypican-3 ( gpc3 ) , a carcinoembryonic antigen , is an ideal target for immunotherapy against hcc because it is overexpressed specifically in hcc ( 7281% ) and correlates with a poor prognosis .
gpc3 forms a complex with wnt molecules and promotes the growth of hcc by stimulating canonical wnt signaling .
we identified hla - a*24:02-restricted gpc3298306 ( eyilsleel ) and hla - a*02:01-restricted gpc3144 - 152 ( fvgefftdv ) peptides , both of which induce gpc3-reactive cytotoxic t - lymphocytes ( ctls ) without inducing autoimmunity .
we recently reported the safety , immunological , and clinical responses of a gpc3-derived peptide vaccine in a phase i clinical trial of patients with advanced hcc .
the results of that trial showed that gpc3 peptide - specific ctls increased in peripheral blood , and that many cd8-positive t cells infiltrated the tumors in some patients , demonstrating a correlation between the ctl response and overall survival following gpc3 peptide vaccination . based on these results
, we conducted a trial in patients with advanced hcc to assess the clinical outcome and whether tumor - infiltrating lymphocytes with an antitumor effect increased . in all cases , liver biopsies were performed before and after gpc3 peptide vaccination according to the protocol .
this trial was approved by the ethics committee of the national cancer center and registered with the university hospital medical information network clinical trials registry ( umin - ctr number 000005093 ) .
the patient described herein was the first case examined pathologically using autopsy specimens . here , we present the clinical course and pathological study , including an autopsy , of a patient with advanced hcc who revealed remarkable tumor lysis immediately after the second vaccination in an ongoing clinical trial of a gpc3 peptide vaccine .
a 62-year - old male had a history of asymptomatic chronic hepatitis c. in september 2009 , he was diagnosed with hcc .
abdominal ct ( ct ) scans showed four lesions in the liver , and the patient was treated four times with hepatic artery chemoembolization . in december 2010 , ct scans revealed a new lesion indicative of a tumor thrombus extending into the inferior vena cava .
. however , the sorafenib treatment was discontinued in january 2011 due to progressive multiple intrahepatic tumors .
as no established therapeutic regimens exist for this condition , he was offered participation in a clinical trial of a gpc3 peptide vaccine for advanced hcc .
the patient had a performance status of 0 , and child - pugh class b disease .
the patient did not have active hbv infection or rapidly progressive tumor thrombus before enrollment , met the eligibility criteria , and was enrolled after providing informed consent .
early - phase contrast - enhanced ct before treatment showed a maximum 68  51-mm tumor with multiple intrahepatic tumors and a 44  30-mm tumor invading the right atrium ( fig . 
pretreatment tumor markers were as follows :  fetoprotein ( afp ) , 852 ng / ml and des - gamma - carboxy prothrombin ( dcp ) , 1346 mau / ml . a liver biopsy was performed 1 week prior to gpc3 peptide vaccination according to the protocol . in april 2011 ,
3 mg of hla - a2-restricted gpc3144 - 152 peptide ( fvgefftdv ) ( american peptide co. ) emulsified with incomplete freund s adjuvant ( montanide isa-51vg ; seppic ) was injected intradermally as the vaccine following good manufacturing practice guidelines .
the patient had a low - grade fever on day 6 following the first vaccination , and inflammatory and hepatic parameters were elevated on day 12 ( fig . 
therefore , he received the second vaccination on day 26 after the first vaccination . on day 9 after the second vaccination
, the patient was admitted to our hospital with a high fever and general fatigue . on admission , the patient s c - reactive protein ( crp ) level ( 10.76 mg / dl ) and laboratory hepatic parameters were elevated .
one day after hospitalization , aspartate aminotransferase and alanine aminotransferase and levels were elevated to 1,580
as seen by early - phase contrast - enhanced ct scan , most tumors in the liver were not contrast enhanced .
in contrast , the size of the tumor thrombus in the right atrium increased to a maximum of 83  50 mm ( fig . 
levels of the tumor markers afp and dcp decreased temporarily to 634 ng / ml and 777 mau / ml , respectively . he was infused with a liver - supporting agent ( monoammonium glycyrrhizinate , glycine , and l - cysteine hydrochloride hydrate ) .
we did not perform a liver biopsy when the hepatic parameters were elevated because they improved promptly .
protocol treatment was discontinued due to progressive disease and he died 30 days after the second vaccination . based on the clinical course , we could not rule out the possibility that his condition had worsened as a result of the vaccine .
therefore , an autopsy was performed to determine the main cause of death and the elevated hepatic parameters , and to evaluate the anti - tumor effect of vaccination figure  1 .
( a ) contrast - enhanced ct scan before vaccination shows a 68  51-mm tumor with multiple intrahepatic tumors ( arrow ) and a 44  30-mm tumor invading the right atrium ( arrowhead ) .
( b ) contrast - enhanced ct after the second vaccination showing multiple low - density areas in the liver , indicating extensive tumor necrosis ( arrow ) .
by contrast , a tumor thrombus in the right atrium increased to a 83  50-mm tumor ( arrowhead ) .
approximately 1 week after the first vaccination , the patient began reporting general fatigue and showed intermittent fever .
inflammatory and hepatic parameters were elevated ( crp : pink line , ast : red line , t - bil : green line ) .
the abnormal laboratory parameters improved after observation . on day 9 after the second vaccination , the patient was admitted to our hospital as an emergency due to fever and general fatigue , which were similar to his previous symptoms .
inflammatory and hepatic parameters improved 1 week after hospitalization . however , his status gradually worsened , and he died on day 30 after the second vaccination .
ex vivo ifn- enzyme - linked immunospot ( elispot ) assays against gpc3 in 5  10 peripheral blood mononuclear cells ( pbmcs ) were performed before and after vaccination .
the  spot number indicates the number of gpc3 peptide - specific cytotoxic t - lymphocytes ( ctls ) .
the number of interferon ( ifn)- positive spots increased from 0 to 84 after the second vaccination .
a 62-year - old male had a history of asymptomatic chronic hepatitis c. in september 2009 , he was diagnosed with hcc .
abdominal ct ( ct ) scans showed four lesions in the liver , and the patient was treated four times with hepatic artery chemoembolization . in december 2010 , ct scans revealed a new lesion indicative of a tumor thrombus extending into the inferior vena cava .
. however , the sorafenib treatment was discontinued in january 2011 due to progressive multiple intrahepatic tumors .
as no established therapeutic regimens exist for this condition , he was offered participation in a clinical trial of a gpc3 peptide vaccine for advanced hcc .
the patient had a performance status of 0 , and child - pugh class b disease .
the patient did not have active hbv infection or rapidly progressive tumor thrombus before enrollment , met the eligibility criteria , and was enrolled after providing informed consent .
early - phase contrast - enhanced ct before treatment showed a maximum 68  51-mm tumor with multiple intrahepatic tumors and a 44  30-mm tumor invading the right atrium ( fig . 
pretreatment tumor markers were as follows :  fetoprotein ( afp ) , 852 ng / ml and des - gamma - carboxy prothrombin ( dcp ) , 1346 mau / ml . a liver biopsy was performed 1 week prior to gpc3 peptide vaccination according to the protocol . in april 2011 ,
3 mg of hla - a2-restricted gpc3144 - 152 peptide ( fvgefftdv ) ( american peptide co. ) emulsified with incomplete freund s adjuvant ( montanide isa-51vg ; seppic ) was injected intradermally as the vaccine following good manufacturing practice guidelines .
the patient had a low - grade fever on day 6 following the first vaccination , and inflammatory and hepatic parameters were elevated on day 12 ( fig . 
therefore , he received the second vaccination on day 26 after the first vaccination . on day 9 after the second vaccination
, the patient was admitted to our hospital with a high fever and general fatigue . on admission , the patient s c - reactive protein ( crp ) level ( 10.76 mg / dl ) and laboratory hepatic parameters were elevated .
one day after hospitalization , aspartate aminotransferase and alanine aminotransferase and levels were elevated to 1,580 iu / l and 1,112
as seen by early - phase contrast - enhanced ct scan , most tumors in the liver were not contrast enhanced .
in contrast , the size of the tumor thrombus in the right atrium increased to a maximum of 83  50 mm ( fig . 
levels of the tumor markers afp and dcp decreased temporarily to 634 ng / ml and 777 mau / ml , respectively . he was infused with a liver - supporting agent ( monoammonium glycyrrhizinate , glycine , and l - cysteine hydrochloride hydrate ) .
we did not perform a liver biopsy when the hepatic parameters were elevated because they improved promptly .
protocol treatment was discontinued due to progressive disease and he died 30 days after the second vaccination .
based on the clinical course , we could not rule out the possibility that his condition had worsened as a result of the vaccine .
therefore , an autopsy was performed to determine the main cause of death and the elevated hepatic parameters , and to evaluate the anti - tumor effect of vaccination figure  1 .
( a ) contrast - enhanced ct scan before vaccination shows a 68  51-mm tumor with multiple intrahepatic tumors ( arrow ) and a 44  30-mm tumor invading the right atrium ( arrowhead ) .
( b ) contrast - enhanced ct after the second vaccination showing multiple low - density areas in the liver , indicating extensive tumor necrosis ( arrow ) . by contrast
, a tumor thrombus in the right atrium increased to a 83  50-mm tumor ( arrowhead ) .
approximately 1 week after the first vaccination , the patient began reporting general fatigue and showed intermittent fever .
inflammatory and hepatic parameters were elevated ( crp : pink line , ast : red line , t - bil : green line ) .
the abnormal laboratory parameters improved after observation . on day 9 after the second vaccination , the patient was admitted to our hospital as an emergency due to fever and general fatigue , which were similar to his previous symptoms .
however , his status gradually worsened , and he died on day 30 after the second vaccination .
ex vivo ifn- enzyme - linked immunospot ( elispot ) assays against gpc3 in 5  10 peripheral blood mononuclear cells ( pbmcs ) were performed before and after vaccination .
the  spot number indicates the number of gpc3 peptide - specific cytotoxic t - lymphocytes ( ctls ) .
the number of interferon ( ifn)- positive spots increased from 0 to 84 after the second vaccination .
generally , ctls specific for tumor antigens can not be detected directly ex vivo ; they can be detected only after expansion by repeated in vitro stimulation with the antigenic peptide in conjunction with appropriate antigen - presenting cells .
this is attributed to the sensitivity of the assay and the low frequency of tumor - antigen - specific ctls .
gpc3 peptide - specific ctls in pbmcs , which can be detected directly ex vivo without in vitro stimulation , can provide strong immunological evidence .
the number of gpc3 peptide - specific ctls increased from 0 to 84 in 5  10 pbmcs after the second vaccination ( fig . 
this result led us to anticipate a good clinical response because the increased number of ctls and the specific ctl number correlated with the clinical response in a previous trial of the gpc3 peptide vaccine .
immunohistochemical staining showed expression of gpc3 and hla class i in the cytoplasm and membranes of the carcinoma cells and a few cd8-positive t cells in the carcinoma tissue before vaccination ( fig . 
( a ) pathological findings of liver biopsy specimens before vaccination . a microscopy image of a hematoxylin and eosin ( h&e)-stained section shows well - differentiated hepatocellular carcinoma ( hcc ) .
immunohistochemical staining for gpc3 and hla class i showed positivity in the cytoplasm and membranes of carcinoma cells , respectively .
( b ) macroscopic findings of the liver and heart before formalin fixation at the time of autopsy .
( a ) microscopic images of h&e - stained sections showing central necrosis of carcinoma tissue , whereas a cirrhotic nodule adjacent to the carcinoma tissue was not necrotic .
( b ) magnified image of the area enclosed within the white box in ( a ) showing that cancer cells exhibited a morphology ( left ) different from that of cirrhotic cells ( right ) .
( c ) cd8-positive t cells ( brown ) infiltrated the carcinoma cells accompanied by necrosis .
in contrast , no infiltration of cd8-positive t cells was detected within the cirrhotic nodule .
( d ) magnified image of the area enclosed within the red box in ( a ) showing necrosis and viable carcinoma cells .
( e ) positive immunohistochemical gpc3 staining was observed in only the cytoplasm of carcinoma cells .
( f ) cd8-positive t cells infiltrated the necrotic area and carcinoma tissue . a general autopsy ( with the exception of the brain ) was performed 2 h following death .
macroscopic findings of the liver revealed multiple macro - nodular lesions with central necrosis mainly in the right lobe ( fig . 
as the tumor occupied most of the right atrium , the main cause of death was circulatory failure due to progressive tumor thrombus ( fig .  2b , right ) .
a histological examination showed central necrosis in most of the tumor in the right lobe , and viable carcinoma cells remained around the necrotic tissue , whereas a cirrhotic nodule adjacent to the carcinoma tissue was not necrotic ( figs .  2c and 3a ) .
there was infiltration of cd8-positive t cells ( brown ) in the residual carcinoma , but not within the cirrhotic area ( figs .  2c and 3a ) .
we did not detect degeneration or necrosis of the hepatocytes in the non - tumor liver parenchyma of the left lobe .
these findings suggest that the elevated hepatic parameters in our patient were due to an antitumor effect .
we diagnosed that the cause of death was unlikely to be related to vaccine - induced liver injury .
we focused on the necrotic area around the cirrhotic nodules , in which cd68-positive macrophages ( brown ) aggregated ( fig . 
cd8-positive t cells also infiltrated the marginal zone between the necrotic area and noncancerous cirrhotic nodule , suggesting that carcinoma cells were attacked by cd8-positive t cells , which may have resulted in necrosis ( fig . 
the histology of the tumor thrombus in the right atrium was similar to that of the intrahepatic tumor . however , viable tumor cells remained in half of the tumor thrombus and little infiltration of cd8-positive t cells was detected ( data not shown ) .
cd8-positive t cells ( brown ) infiltrated only the carcinoma area , accompanied by necrosis .
( c ) cd68-positive macrophages ( brown ) aggregated in the necrotic area around the cirrhotic nodule .
( d ) cd8-positive t cells ( brown ) infiltrated the necrotic area but not the cirrhotic nodule .
based on the image in ( d ) , most of the lymphocytes were cd8-positive t cells .
generally , ctls specific for tumor antigens can not be detected directly ex vivo ; they can be detected only after expansion by repeated in vitro stimulation with the antigenic peptide in conjunction with appropriate antigen - presenting cells .
this is attributed to the sensitivity of the assay and the low frequency of tumor - antigen - specific ctls .
gpc3 peptide - specific ctls in pbmcs , which can be detected directly ex vivo without in vitro stimulation , can provide strong immunological evidence .
the number of gpc3 peptide - specific ctls increased from 0 to 84 in 5  10 pbmcs after the second vaccination ( fig . 
this result led us to anticipate a good clinical response because the increased number of ctls and the specific ctl number correlated with the clinical response in a previous trial of the gpc3 peptide vaccine .
immunohistochemical staining showed expression of gpc3 and hla class i in the cytoplasm and membranes of the carcinoma cells and a few cd8-positive t cells in the carcinoma tissue before vaccination ( fig . 
( a ) pathological findings of liver biopsy specimens before vaccination . a microscopy image of a hematoxylin and eosin ( h&e)-stained section shows well - differentiated hepatocellular carcinoma ( hcc ) .
immunohistochemical staining for gpc3 and hla class i showed positivity in the cytoplasm and membranes of carcinoma cells , respectively .
( b ) macroscopic findings of the liver and heart before formalin fixation at the time of autopsy .
( a ) microscopic images of h&e - stained sections showing central necrosis of carcinoma tissue , whereas a cirrhotic nodule adjacent to the carcinoma tissue was not necrotic .
( b ) magnified image of the area enclosed within the white box in ( a ) showing that cancer cells exhibited a morphology ( left ) different from that of cirrhotic cells ( right ) .
( c ) cd8-positive t cells ( brown ) infiltrated the carcinoma cells accompanied by necrosis .
in contrast , no infiltration of cd8-positive t cells was detected within the cirrhotic nodule .
( d ) magnified image of the area enclosed within the red box in ( a ) showing necrosis and viable carcinoma cells .
( e ) positive immunohistochemical gpc3 staining was observed in only the cytoplasm of carcinoma cells .
( f ) cd8-positive t cells infiltrated the necrotic area and carcinoma tissue . a general autopsy ( with the exception of the brain ) was performed 2 h following death .
macroscopic findings of the liver revealed multiple macro - nodular lesions with central necrosis mainly in the right lobe ( fig . 
as the tumor occupied most of the right atrium , the main cause of death was circulatory failure due to progressive tumor thrombus ( fig .  2b , right ) .
a histological examination showed central necrosis in most of the tumor in the right lobe , and viable carcinoma cells remained around the necrotic tissue , whereas a cirrhotic nodule adjacent to the carcinoma tissue was not necrotic ( figs .  2c and 3a ) .
there was infiltration of cd8-positive t cells ( brown ) in the residual carcinoma , but not within the cirrhotic area ( figs .  2c and 3a ) .
we did not detect degeneration or necrosis of the hepatocytes in the non - tumor liver parenchyma of the left lobe .
these findings suggest that the elevated hepatic parameters in our patient were due to an antitumor effect .
we diagnosed that the cause of death was unlikely to be related to vaccine - induced liver injury .
we focused on the necrotic area around the cirrhotic nodules , in which cd68-positive macrophages ( brown ) aggregated ( fig . 
cd8-positive t cells also infiltrated the marginal zone between the necrotic area and noncancerous cirrhotic nodule , suggesting that carcinoma cells were attacked by cd8-positive t cells , which may have resulted in necrosis ( fig . 
the histology of the tumor thrombus in the right atrium was similar to that of the intrahepatic tumor . however , viable tumor cells remained in half of the tumor thrombus and little infiltration of cd8-positive t cells was detected ( data not shown ) .
cd8-positive t cells ( brown ) infiltrated only the carcinoma area , accompanied by necrosis .
( c ) cd68-positive macrophages ( brown ) aggregated in the necrotic area around the cirrhotic nodule .
( d ) cd8-positive t cells ( brown ) infiltrated the necrotic area but not the cirrhotic nodule .
based on the image in ( d ) , most of the lymphocytes were cd8-positive t cells .
to date , the time to ctl induction and subsequent tumor response has been prolonged in cancer vaccine trials . by contrast , no discrepancy regarding the time between ctl induction and tumor response was observed in our phase i trial of a gpc3 peptide vaccine . in this case , central necrosis of each intra - hepatic tumor was observed at the time of a strong immunological response against the gpc3 peptide , immediately after the second vaccination .
a biopsy may be necessary to rule out vaccine - induced liver injury when the hepatic parameters are elevated .
however , the clinical course and autopsy results suggested that the elevated hepatic parameters in our patient were due to an antitumor effect .
therefore , a positive radiographic response following vaccination , suggesting tumor necrosis , could be evaluated as a treatment response .
necrosis was found in the center of each tumor ; therefore , the central necrosis caused by ischemia , in addition to cd8-positive t cells attacking tumor cells , may have led to tumor necrosis .
three findings support the hypothesis that tumor necrosis was caused by cd8-positive t cells , as follows : ( 1 ) the necrotic changes determined by ct after vaccination , accompanied by clinical laboratory data ; this was consistent with an immune response , although no tumor necrosis was evident on the ct before vaccination ; ( 2 ) no necrosis was evident in the left lobe ( no tumors ) of the autopsy liver specimen , but it was present in the right liver lobe ( tumors present ) ; and ( 3 ) cd8-positive t cells infiltrated residual viable tumor cells .
the analyses used in this study may contribute to identifying the pathological state after vaccination .
we detected infiltration of cd8-positive t cells into the hepatic tumors , but little infiltration of cd8-positive t cells into the tumor thrombus .
this discrepancy may have been caused by the heterogeneity associated with immune - escape mechanisms in tumor cells .
this case report of central necrosis in a patient with hcc might be regarded as spontaneous regression correlated with circulatory failure due to a massive tumor embolism .
it was not known whether the tumor necrosis was induced by ctls , ischemia , or other factors .
however , the infiltration of cd8-positive t cells into tumor cells supports immune - related necrosis .
the rate of spontaneous partial regression among patients with hcc is 0.406% compared with the control arm of a randomized clinical trial . in contrast , three of 33 patients who received gpc3 peptide vaccination in the phase i trial had suspicious tumor necrosis on ct scans . in one report ,
massive infiltration of cd8-positive t cells in the remaining liver tumor and tumor necrosis were identified by histological examination of a biopsy specimen after vaccination .
indeed , on - going clinical trials of the gpc3 peptide vaccine will provide additional information and further demonstrate the antitumor effect .
histological results at the estimated time of a strong gpc3-specific ctl response suggest that gpc3 peptide vaccination may be a promising approach to treat hcc .
an ex vivo ifn- enzyme - linked immunospot ( elispot ) assay was performed to evaluate the antigen - specific ctl response , as described previously .
briefly , peripheral blood ( 30 ml ) was obtained from the patient before the first vaccination and 2 weeks after each vaccination and centrifuged on a ficoll 
non - cultured peripheral blood mononuclear cells ( pbmcs ) ( 5  10/well ) were added to plates in the presence of 10 g / ml peptide antigens and incubated for 20 h. the gpc3 antigen used was the hla - a2-restricted gpc3144152(fvgefftdv ) peptide .
pbmcs with the hla - a2-restricted hiv1927 ( tlnawvkvv ) peptide ( proimmune ) were used as negative controls .
immunohistochemical staining with monoclonal antibodies against gpc3 ( clone , 1g12 ; biomosaics ) , hla class i ( clone , emr8/5 ; hokudo ) , cd8 ( clone , 1a5 ; novocastra ) , cd45 ( cloned 2b11 and pd7/26 ; ventana ) , and cd68 ( clone , kp-1 ; ventana ) was performed according to the manufacturer s protocol .
an ex vivo ifn- enzyme - linked immunospot ( elispot ) assay was performed to evaluate the antigen - specific ctl response , as described previously .
briefly , peripheral blood ( 30 ml ) was obtained from the patient before the first vaccination and 2 weeks after each vaccination and centrifuged on a ficoll 
non - cultured peripheral blood mononuclear cells ( pbmcs ) ( 5  10/well ) were added to plates in the presence of 10 g / ml peptide antigens and incubated for 20 h. the gpc3 antigen used was the hla - a2-restricted gpc3144152(fvgefftdv ) peptide .
pbmcs with the hla - a2-restricted hiv1927 ( tlnawvkvv ) peptide ( proimmune ) were used as negative controls .
immunohistochemical staining with monoclonal antibodies against gpc3 ( clone , 1g12 ; biomosaics ) , hla class i ( clone , emr8/5 ; hokudo ) , cd8 ( clone , 1a5 ; novocastra ) , cd45 ( cloned 2b11 and pd7/26 ; ventana ) , and cd68 ( clone , kp-1 ; ventana ) was performed according to the manufacturer s protocol .
 we recently reported the safety , immunological and clinical responses to a gpc3-derived peptide vaccine in a phase i clinical trial of patients with advanced hepatocellular carcinoma ( hcc ) . 
 we conducted a subsequent trial in advanced hcc to assess the histopathological findings before and after vaccination with the gpc3 peptide . here , we present the clinical course and the pathological study including the autopsy of a patient with advanced hcc in the ongoing clinical trial . 
 a 62-year old patient suffering from hcc refractory to sorafenib therapy received the gpc3 peptide vaccine . 
 the patient had fever and remarkably impaired liver function twice after vaccination . 
 contrast - enhanced ct after the second vaccination showed multiple low - density areas in the liver tumor , indicating tumor necrosis . 
 in contrast , the tumor thrombus in the right atrium increased . 
 the patient discontinued protocol treatment due to disease progression and died 30 days after the second vaccination . 
 an autopsy was performed to determine the main cause of death and to evaluate the antitumor effect of the vaccination . 
 a histological examination showed central necrosis in most of the intrahepatic tumor . 
 the main cause of death was circulatory failure due to tumor thrombus , which occupied most of the right atrium . 
 an immunohistochemical analysis revealed infiltration of cd8-positive t cells in the residual carcinoma , but not within the cirrhotic area . 
 ex vivo ifn- enzyme - linked immunospot analysis revealed vaccine - induced immune - reactivity against the gpc3 peptide . a histopathological examination at the estimated time of a strong immunological response demonstrated a gpc3 peptide vaccination - induced cytotoxic t - lymphocyte response with an anti - tumor effect . 
Introduction
Patient presentation
Results
Immunological analysis and autopsy
Discussion
Materials and Methods
Ex vivo interferon- (IFN-) enzyme-linked immunospot assay
Immunohistochemical analysis