hepatocellular carcinoma ( hcc ) accounts for 85% to 90% of primary liver cancers   and the age - adjusted hcc mortality rate has increased in recent decades in japan .
similarly , a rising hcc trend has been reported in several developed countries in north america , europe , and asia .
hcc often develops in patients with liver cirrhosis caused by hepatitis b virus ( hbv ) , hepatitis c virus ( hcv ) , excessive alcohol consumption , or nonalcoholic fatty liver disease . of the hepatitis viruses that cause hcc ,
however , it has been reported that the absolute numbers and proportion of hbsag and hcvab negative hcc ( hcc - nonbc ) have both been steadily increasing in japan .
recently , radio - frequency ablation ( rfa ) and percutaneous ethanol injection ( pei ) have also been recognized as effective methods of achieving complete tumor necrosis for small hccs . however , the chance of curative treatment is often limited by several features of hcc itself .
the incidence of associated cirrhosis is also high , exceeding 80% in most series [ 1820 ] .
transcatheter intra - arterial chemoembolization ( tace ) , with which complete necrosis of hccs is thought to be difficult to achieve , is also impacted by the above factors . to increase opportunities for meaningful intervention and to improve survival , early detection of hcc by measuring alpha - fetoprotein ( afp ) and/or imaging screening
however , the poorer prognosis of patients with hcc - nonbc is reportedly attributable to its late detection in an advanced stage , owing to the lack of a surveillance system for early detection of hcc . in this retrospective cohort study ,
our aim was to characterize consecutive patients who had been diagnosed with hcc - nonbc during an 11-year period ( 19992010 ) at the centers comprising the nagasaki association study of liver disease ( nasld ) group .
we evaluated the clinical characteristics of patients with hcc - nonbc , their tumor stages , treatment , afp and dpc as potential biomarkers , and survival .
in total , 2638 patients with hcc diagnosed between 1999 and 2010 in the nasld were recruited for this study .
the diagnosis of hcc was based on afp and/or dcp levels , as well as the results of imaging techniques such as ultrasonography ( usg ) , computerized tomography ( ct ) , magnetic resonance imaging ( mri ) , and hepatic angiography ( hag ) , and/or liver biopsy . the diagnostic criteria included characteristic liver biopsy findings , elevated afp ( 20 ng / ml ) and/or dcp ( 40 ng / ml ) , and neovascularization on hag , ct , and/or mri .
the diagnosis of chronic hcv infection was positive for both anti - hcv , by a third - generation enzyme - linked immunosorbent assay ( elisa ) , and for hcv rna by polymerase chain reaction ( pcr ) .
the diagnosis of chronic hbv infection was based on the presence of hbsag ( enzyme - linked immunosorbent assay ; abbott laboratories ) ; serum afp was measured by radioimmunoassay ( abbott laboratories ) .
the history of alcohol intake was obtained from medical records ; habitual drinking was defined as an average daily consumption of an amount equivalent to 80 g of pure ethanol for a period of more than 10 years .
hcc etiologies were categorized into 4 groups : ( 1 ) hcc - b , hbsag positive , and hcvab negative ; ( 2 ) hcc - c , hcvab positive , and hbsag negative ; ( 3 ) hcc - bc , both hbsag and hcvab positive ; and ( 4 ) hcc - nonbc , both hbsag , and hcvab negative .
the significance of age , sex , body mass index ( bmi ) , alcohol intake , diabetes mellitus , underlying liver disease child - pugh score , platelet count , prothrombin time ( pt ) , albumin ( alb ) , total bilirubin ( bil ) , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , afp , dcp , and tumor - node - metastasis ( tnm ) stage were examined to identify possible relationships with hcc - nonbc using logistic regression analysis .
patients diagnosed with hcc were assessed for surgery based on the extent of lobar involvement and liver function status .
the extent of lobar involvement was evaluated based on a combination of usg , ct , mri , and hag findings .
patients were considered to be poor candidates for resection if they met any of the following criteria : ( 1 ) bilobar involvement , ( 2 ) evidence of tumor infiltration into the main portal vein or thrombosis of the vein , ( 3 ) evidence of extrahepatic metastases , ( 4 ) child s grade c cirrhosis , or ( 5 ) poor cardiac and/or respiratory status . if surgery was contraindicated or the patient refused to undergo an operation , rfa or pei therapy was the second treatment choice , offered to those with hccs less than 3 cm in diameter .
the remaining patients without main portal vein thrombosis or extrahepatic metastasis were advised to undergo tace , regardless of tumor size or number .
after initial treatment , the afp levels and liver functions of the patients were assessed every 1 to 3 months , and usg imaging was performed every 3 to 6 months during the follow - up period .
the assessment of treatment for recurrent hcc was based on lobar involvement and liver function status as described for the initial treatment .
rfa or liver transplantation to treat hcc was started at our institution in 2002 . furthermore
, none of the subjects in our study received either of these treatments for recurrent hcc during the follow - up period .
the survival duration was the time from the diagnosis of hcc until the time of death or the time of preparation of this manuscript .
the survival rate was analyzed using the kaplan - meier method , and differences between the survival probability curves were tested using the log - rank test .
descriptive summaries of study groups are reported as the median value ( sd : standard deviation ) and number ( % ) .
data were analyzed using the mann - whitney u test for continuous ordinal data , and the chi - square test with yates correction and fisher s exact test were used for intergroup comparisons to determine the association between 2 qualitative variables .
variables achieving statistical significance according to univariate analysis were subsequently included in the multivariate analysis using a logistic regression model and were described as hazard ratios ( hr ) with 95% confidence intervals ( ci ) .
in total , 2638 patients with hcc diagnosed between 1999 and 2010 in the nasld were recruited for this study .
the diagnosis of hcc was based on afp and/or dcp levels , as well as the results of imaging techniques such as ultrasonography ( usg ) , computerized tomography ( ct ) , magnetic resonance imaging ( mri ) , and hepatic angiography ( hag ) , and/or liver biopsy . the diagnostic criteria included characteristic liver biopsy findings , elevated afp ( 20 ng / ml ) and/or dcp ( 40 ng / ml ) , and neovascularization on hag , ct , and/or mri .
the diagnosis of chronic hcv infection was positive for both anti - hcv , by a third - generation enzyme - linked immunosorbent assay ( elisa ) , and for hcv rna by polymerase chain reaction ( pcr ) .
the diagnosis of chronic hbv infection was based on the presence of hbsag ( enzyme - linked immunosorbent assay ; abbott laboratories ) ; serum afp was measured by radioimmunoassay ( abbott laboratories ) .
the history of alcohol intake was obtained from medical records ; habitual drinking was defined as an average daily consumption of an amount equivalent to 80 g of pure ethanol for a period of more than 10 years .
hcc etiologies were categorized into 4 groups : ( 1 ) hcc - b , hbsag positive , and hcvab negative ; ( 2 ) hcc - c , hcvab positive , and hbsag negative ; ( 3 ) hcc - bc , both hbsag and hcvab positive ; and ( 4 ) hcc - nonbc , both hbsag , and hcvab negative .
the significance of age , sex , body mass index ( bmi ) , alcohol intake , diabetes mellitus , underlying liver disease child - pugh score , platelet count , prothrombin time ( pt ) , albumin ( alb ) , total bilirubin ( bil ) , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , afp , dcp , and tumor - node - metastasis ( tnm ) stage were examined to identify possible relationships with hcc - nonbc using logistic regression analysis .
patients diagnosed with hcc were assessed for surgery based on the extent of lobar involvement and liver function status .
the extent of lobar involvement was evaluated based on a combination of usg , ct , mri , and hag findings .
patients were considered to be poor candidates for resection if they met any of the following criteria : ( 1 ) bilobar involvement , ( 2 ) evidence of tumor infiltration into the main portal vein or thrombosis of the vein , ( 3 ) evidence of extrahepatic metastases , ( 4 ) child s grade c cirrhosis , or ( 5 ) poor cardiac and/or respiratory status . if surgery was contraindicated or the patient refused to undergo an operation , rfa or pei therapy was the second treatment choice , offered to those with hccs less than 3 cm in diameter .
the remaining patients without main portal vein thrombosis or extrahepatic metastasis were advised to undergo tace , regardless of tumor size or number .
after initial treatment , the afp levels and liver functions of the patients were assessed every 1 to 3 months , and usg imaging was performed every 3 to 6 months during the follow - up period .
the assessment of treatment for recurrent hcc was based on lobar involvement and liver function status as described for the initial treatment .
furthermore , none of the subjects in our study received either of these treatments for recurrent hcc during the follow - up period .
the survival duration was the time from the diagnosis of hcc until the time of death or the time of preparation of this manuscript .
the survival rate was analyzed using the kaplan - meier method , and differences between the survival probability curves were tested using the log - rank test .
descriptive summaries of study groups are reported as the median value ( sd : standard deviation ) and number ( % ) .
data were analyzed using the mann - whitney u test for continuous ordinal data , and the chi - square test with yates correction and fisher s exact test were used for intergroup comparisons to determine the association between 2 qualitative variables .
variables achieving statistical significance according to univariate analysis were subsequently included in the multivariate analysis using a logistic regression model and were described as hazard ratios ( hr ) with 95% confidence intervals ( ci ) .
the underlying causes of hcc were as follows : 474 ( 18% ) patients were positive for hbsag , 1533 ( 58% ) were positive for hcvab , 40 ( 2% ) were positive for both hbsag and hcvab , and 591 ( 22% ) were negative for hbsag and anti - hcv .
the cumulative 5-year survival rates of the patients with hcc - b , hcc - c , hcc - bc , and hcc - nonbc were 43% , 52% , 49% , and 47% , respectively ( figure 1 ) .
patients in the hcc - c group had a higher cumulative survival rate than those in the hcc - b and hcc - nonbc groups .
univariate and multivariate analyses were performed to identify factors independently related to hcc - nonbc . in the univariate analysis ,
the following 13 factors significantly influenced hcc - nonbc : age , sex , bmi , alcohol consumption , diabetes mellitus , underlying liver disease , platelet count , bil , ast , alt , afp , dcp , and tnm stage ( table 2 ) .
multivariate analysis identified age ( 70 years , hr 1.63 ) , sex ( female , hr 1.73 ) , bmi ( 25 , hr 2.12 ) , alcohol consumption ( not excessive , hr 3.41 ; excessive , hr 14.73 ) , diabetes mellitus ( hr 2.42 ) , underlying liver disease ( chronic hepatitis , hr 0.46 ; cirrhosis , hr 0.52 ) , platelet count ( < 116,000/l , hr 1.88 ) , ast ( < 56
ng / ml , hr 0.60 ; 200 ng / ml , hr 0.63 ) , dcp ( 20199 mau / ml , hr 1.64 ; 200 mau / ml , hr 2.08 ) , and tnm stage ( ii , hr1.67 ; iii , hr1.88 ; iv , hr 2.40 ) , as independent and significant factors associated with hcc - nonbc ( table 3 ) . the positive rate of afp ( 20 ng / ml ) in hcc - b , hcc - c , hcc - bc , and hcc - nonbc were 62% , 55% , 61% , and 44% , respectively ; whereas the positive rate of dcp ( 40 mau / ml ) were 67% , 55% , 75% and 77% , respectively ( figure 2 ) .
the positive rate of afp in hcc - nonbc was significantly lower than those in the hcc - b and hcc - c groups , whereas the positive rate of dcp was significantly higher than that in the hcc - b and hcc - c groups .
the median afp and dcp levels in hcc - b , hcc - c , hcc - bc , and hcc - nonbc according to tnm stage are presented in table 4 .
the median afp levels in hcc - b , hcc - c , hcc - bc , and hcc - nonbc were 60 ng / ml , 25 ng / ml , 29 ng / ml , and 13 ng / ml , respectively ; whereas the dcp levels were 4990 mau / ml , 418 mau / ml , 612 mau / ml , and 3077 mau / ml , respectively .
the median afp level in hcc - nonbc was significantly lower than those in the other groups ; whereas the median dcp level was significantly higher than that in the hcc - c group .
according to tnm stage , the median afp levels in hcc - nonbc with tnm stages i , ii , and iii were significantly lower than that in either hcc - b or hcc - c . in tnm stage iv ,
the median afp level in hcc - nonbc was significantly lower than that in either hcc - b or hcc - bc .
the median dcp levels in hcc - nonbc with tnm stages i and ii were significantly higher than that in either hcc - b or hcc - c . in tnm stage iii , the median dcp level in hcc - nonbc was significantly higher than that in hcc - c .
however , there were no significant differences in the median dcp level among tnm stage iv cases .
the survival rate of patients in the high dcp group ( 200 mau / ml ) was significantly lower than that of patients classified into the low dcp ( 40199 mau / ml ) and dcp negative ( < 40 mau / ml ) groups among those with hcc - b , hcc - c , and hcc - nonbc ( p0.001 ; log - rank test ) ( figure 3 ) .
the underlying causes of hcc were as follows : 474 ( 18% ) patients were positive for hbsag , 1533 ( 58% ) were positive for hcvab , 40 ( 2% ) were positive for both hbsag and hcvab , and 591 ( 22% ) were negative for hbsag and anti - hcv .
the cumulative 5-year survival rates of the patients with hcc - b , hcc - c , hcc - bc , and hcc - nonbc were 43% , 52% , 49% , and 47% , respectively ( figure 1 ) .
patients in the hcc - c group had a higher cumulative survival rate than those in the hcc - b and hcc - nonbc groups .
univariate and multivariate analyses were performed to identify factors independently related to hcc - nonbc . in the univariate analysis ,
the following 13 factors significantly influenced hcc - nonbc : age , sex , bmi , alcohol consumption , diabetes mellitus , underlying liver disease , platelet count , bil , ast , alt , afp , dcp , and tnm stage ( table 2 ) .
multivariate analysis identified age ( 70 years , hr 1.63 ) , sex ( female , hr 1.73 ) , bmi ( 25 , hr 2.12 ) , alcohol consumption ( not excessive , hr 3.41 ; excessive , hr 14.73 ) , diabetes mellitus ( hr 2.42 ) , underlying liver disease ( chronic hepatitis , hr 0.46 ; cirrhosis , hr 0.52 ) , platelet count ( < 116,000/l , hr 1.88 ) , ast ( < 56
iu / l , hr 2.48 ) , afp ( 20199 ng / ml , hr 0.60 ; 200 ng / ml , hr 0.63 ) , dcp ( 20199 mau / ml , hr 1.64 ; 200 mau / ml , hr 2.08 ) , and tnm stage ( ii , hr1.67 ; iii , hr1.88 ; iv , hr 2.40 ) , as independent and significant factors associated with hcc - nonbc ( table 3 ) .
the positive rate of afp ( 20 ng / ml ) in hcc - b , hcc - c , hcc - bc , and hcc - nonbc were 62% , 55% , 61% , and 44% , respectively ; whereas the positive rate of dcp ( 40 mau / ml ) were 67% , 55% , 75% and 77% , respectively ( figure 2 ) .
the positive rate of afp in hcc - nonbc was significantly lower than those in the hcc - b and hcc - c groups , whereas the positive rate of dcp was significantly higher than that in the hcc - b and hcc - c groups .
the median afp and dcp levels in hcc - b , hcc - c , hcc - bc , and hcc - nonbc according to tnm stage are presented in table 4 .
the median afp levels in hcc - b , hcc - c , hcc - bc , and hcc - nonbc were 60 ng / ml , 25 ng / ml , 29 ng / ml , and 13 ng / ml , respectively ; whereas the dcp levels were 4990 mau / ml , 418 mau / ml , 612 mau / ml , and 3077 mau / ml , respectively .
the median afp level in hcc - nonbc was significantly lower than those in the other groups ; whereas the median dcp level was significantly higher than that in the hcc - c group . according to tnm stage , the median afp levels in hcc - nonbc with tnm stages i , ii , and iii were significantly lower than that in either hcc - b or hcc - c . in tnm stage iv ,
the median afp level in hcc - nonbc was significantly lower than that in either hcc - b or hcc - bc .
the median dcp levels in hcc - nonbc with tnm stages i and ii were significantly higher than that in either hcc - b or hcc - c . in tnm stage iii , the median dcp level in hcc - nonbc was significantly higher than that in hcc - c .
however , there were no significant differences in the median dcp level among tnm stage iv cases .
the survival rate of patients in the high dcp group ( 200 mau / ml ) was significantly lower than that of patients classified into the low dcp ( 40199 mau / ml ) and dcp negative ( < 40 mau / ml ) groups among those with hcc - b , hcc - c , and hcc - nonbc ( p0.001 ; log - rank test ) ( figure 3 ) .
the age - adjusted mortality rate for hcc has increased over the past few decades in japan .
however , most patients are still diagnosed at an advanced stage and their survival time is therefore short .
patients with chronic hbv and/or hcv infection in addition to cirrhosis should be monitored with usg and/or ct and/or mri of the liver to detect tumors at an early stage .
there is a large population of individuals infected with hcv , hbv , or both in whom cancer is in the latency period . for those who harbor chronic hcv and/or hbv infections
, attention must be focused on the detection of hcc at an early stage . in this study ,
more than 75% of patients with hcc were positive for hbv and/or hcv . additionally , our data showed patients with hcc - nonbc to generally be diagnosed at an advanced stage .
. however , it will not be easy to select appropriate subjects for screening of hcc among those negative for both hbsag and hcvab .
persistently elevated afp is well known to be related to the presence of hcc and its determination can facilitate better identification of patients at risk .
however , in our present dataset , the median serum afp level in hcc - nonbc was not abnormal , whereas those in hcc - b and hcc - c cases were abnormal .
several reports have shown elevated afp to be a risk factor for hcc development in hcv and/or hbv patients [ 24,3036 ] .
however , our results suggest afp alone to be insufficient for hcc - nonbc surveillance . since liebman et al . demonstrated dcp to be a useful marker for hcc diagnosis ,
several investigations have made comparisons of the usefulness of dcp and afp for hcc diagnosis [ 3740 ] . however , whether afp is superior to dcp in all cases is still controversial .
one reason for these differences involves the use of different cut - off values in the various studies ( e.g. , 40 , 60 , and 100 mau / ml for dcp ; and 20 , 100 , and 200 ng / ml for afp ) .
other possible reasons include differences in the causes of the underlying liver diseases , and patients with cirrhosis tending to have higher afp levels than those with chronic hepatitis .
another possible reason for these differences might be etiological differences in liver diseases among the patients examined in prior studies . in this study ,
the median afp level in hcc - nonbc was significantly lower than that in either hcc - b or hcc - c , whereas the median dcp level was significantly higher .
our data suggest that dcp levels differ among liver diseases with different etiologies . the high value identified in our study
the biological function of afp is still not well identified . since afp is similar to albumin , it is possible that afp function as a carrier for several ligands such as bilirubin , fatty acids , steroids , heavy metals , flavonoids , phytoestrogens , dioxin , and various drugs .
the increase of afp levels to 500 ng / ml is correlated with the tumor size ; 80% of small hcc show no increase of afp concentration .
furthermore , sensitivity of afp decreases from 52% to 25% when tumor diameter is > 3 and <3 cm , respectively .
firstly , dcp is a more specific hcc marker than afp because other liver diseases do not cause an increase of dcp serum levels .
however , we often encounter patients with liver disease who have slightly elevated dcp levels , but undetectable hcc as assessed by imaging studies .
it has been reported that aberrant elevation of dcp is occasionally observed in patients with alcoholic cirrhosis , obstructive jaundice , or vitamin k deficiency .
recently , toyoda et al . measured a novel dcp ( nx - dcp ) in serum using a newly developed sandwich eclia with new anti - dcp monoclonal antibodies p11 and p16 , and reported preliminary data from only 20 hcc patients .
they showed that the dcp / nx - dcp ratio may be useful for the diagnosis of hcc among warfarin users .
neither dcp alone nor afp alone was optimal for the detection of hcc , but the combination of both markers enhanced sensitivity , indicating that these 2 markers are complementary .
several other studies have shown dcp and afp to be complementary , which is consistent with the production of dcp and afp in hcc occurring through different pathways , possibly explaining why sex , race , underlying liver disease , and hepatic disease etiologies had opposite effects on these 2 markers [ 4648 ] .
in conclusion , this retrospective cohort study demonstrated dcp to be more sensitive than afp for the diagnosis of early - stage cryptogenic hcc .
we advocate that dcp be used as the main serum test for detecting cryptogenic hcc .
 backgroundthe incidence of hepatocellular carcinoma ( hcc ) continues to increase in japan , but the clinical characteristics of japanese patients with hcc have not been well described . 
 the aim of this study was to determine the frequencies and utilities of elevated -fetoprotein ( afp ) and des - gamma - carboxy prothrombin ( dcp ) levels as biomarkers in cryptogenic hcc.material/methodsa total of 2638 patients with hcc diagnosed between 1999 and 2010 in the nagasaki association study of liver ( nasld ) were recruited for this study . 
 the cause of hcc was categorized into 4 groups ; hcc - b , hcc - c , hcc - bc , and hcc - nonbc . 
 the significance of factors was examined for hcc - nonbc using logistic regression analysis in all patients.resultsmultivariate analysis identified age , sex , bmi , alcohol consumption , platelet count , ast , alt , afp , dcp , and tnm stage as independent and significant risk factors for hcc - nonbc . according to tnm stage , the median afp levels in hcc - nonbc with tnm stages i , ii , and iii were significantly lower than in either hcc - b or hcc - c . in tnm stage iv , 
 the median afp level in hcc - nonbc was significantly lower than in either hcc - b or hcc - bc . 
 the median dcp levels in hcc - nonbc with tnm stages i and ii were significantly higher than those in either hcc - b or hcc - c . in tnm stage iii , 
 the median dcp level in hcc - nonbc was significantly higher than that in hcc-c.conclusionsdcp was more sensitive than afp for the diagnosis of early stage cryptogenic hcc . 
 dcp should be used as the main serum test for cryptogenic hcc detection . 
Background
Material and Methods
Patients
Treatment modalities
Statistical analysis
Results
Patient characteristics at enrollment
Univariate and multivariate analyses of the factors associated with HCC-nonBC
Comparison of biomarkers according to liver disease cause
Discussion
Conclusions