liver cancer is a highly common form of tumour accounting for 7% of all cancers . in addition , it is associated with substantial mortality being the third cause of cancer - related death . among liver cancers , hepatocellular carcinoma ( hcc ) represents a major global health problem most cases of hcc are secondary to viral hepatitis infection ( hepatitis b or c ) , alcoholism and cirrhosis . treatment and prognosis of hcc is based on the barcelona - clinic liver cancer ( bclc ) classification , established on validated prognostic variables , such as those related to tumour status ( size , number , vascular invasion , n1 , m1 ) , liver function ( child - pugh s ) and health status . hcc is included among the most chemo - resistant tumour types , and until 2007 no systemic drug was recommended for patients with advanced tumours , an unparalleled situation in oncology . sorafenib , an oral tyrosine kinase inhibitor that suppresses tumour proliferation and angiogenesis , emerged as the first effective systemic treatment for hcc after 30 years of research , and is currently the standard of care for patients with advanced disease . sorafenib has been the first drug , and remains the only one , to increase survival in patients with advanced hcc , receiving marketing authorization from european medicines agency ( emea ) and food and drug administration ( fda ) in 2007 . sorafenib is metabolited by cytochrome p450 ( cyp450 ) , particularly from the cyp3a4 isoform , producing two main metabolites : the n - oxide and the n - hydroxymethyl metabolite . this pharmacokinetic profile has been shown to determine significant drug - drug interactions with other concomitant therapies , including those with medicinal herbs , or with chemotherapeutic agent such as rifampicin . importantly , recent evidence showed that sorafenib metabolism might be impaired in hcc tumour cells . this latter finding might depend on the cancer - related alteration of the physiological pattern of cyp450 expression in the liver . the expression of cyp450 isoenzymes in the human body is substrate and tissue specific . of note , the largest proportion of cyp450 isoenzymes for drug metabolism are expressed in the liver . of the several cyp450 isoform , the cyp3a4 metabolizes about 30% of clinically used drugs , while the cyp3a7 is mostly expressed in the foetal liver although it converts to express cyp3a7 . in a previous study , we showed the overlapping expression between cyp3a4 and cyp3a7 in the foetal human liver during development . there is paucity of data available on cyp450 expression in the adult liver both in physiological as well as in pathological conditions , including hcc . here , we aimed to present novel findings on the expression patterns of cyp3a4 and cyp3a7 in hcc . we hypothesize that the presence of these cyp450 isoforms might have implications for personalized treatment of hcc . the positivity to immunostaining for cyp3a4 and cyp3a7 was evaluated in liver samples from 11 hcc carriers , 6 males and 5 females , aged 44 - 80 years . all procedures performed were approved by the ethics human studies committee of the university medical centre of cagliari ( according to the instructions of the declaration of helsinki ) . tissue section were then dewaxed , rehydrated through graded alcohols and pre - treated for 10 min with the heat - induced epitope retrieval in buffer ph 6.00 ( envision flex target retrieval solution low ph- dako denmark a / s , glostrup , denmark , code : k8004 ) for immunohistochemical analysis of cyp3a7 . slides were then incubated for 20 min at room temperature with anti - cyp450 3a4 rabbit polyclonal antibody at 1.1000 dilution and with anti - cyp450 3a7 mouse monoclonal antibody clone f19 p2 h2 at 1:50 dilution ( all produced by abcam , cambrige , uk , catalogue numbers : ab3572 , ab108624 and ab55840 , respectively ) . staining procedures were performed by envision flex+ ( dako , code : k8002 ) detection system and autostainerlink 48 instrument following dealer s instructions . negative control samples were incubated without mouse anti - cyp3a4 and anti - cyp3a7 polyclonal antibody . the histological examination was performed by three observers ( df , fl and ra ) , who independently assigned two different scores . the first score regarded the distribution of cyp3a4 and cyp3a7 in hcc and in the surrounding parenchyma : score 1 for focal distribution , score 2 for zonal distribution and score 3 for diffuse distribution . the second score concerned the intensity of immunostaining , which was defined as weak ( score 1 ) , medium ( score 2 ) and strong ( score 3 ) . in both score systems , the sum between the distribution score and the intensity score gave rise to the following final grading system : grade 0 for negative stain , grade 1 for a final score from 1 to 3 , grade 2 from 4 to 5 and grade 3 for a final score > 5 . all cases were then discussed at multisession microscope until a consensus between all three observers was obtained . in light of the evidence that sorafenib is metabolized through cyp3a4 and udp glucuronosyltransferase 1 family , polypeptide a9 ( ugt1a9 ) , we used publicly available bioinformatics resources , such as supercyp and transformer databases , to investigate its pharmacokinetic profile . furthermore , we tested whether other tyrosine kinases analogues of sorafenib underwent the same pharmacokinetic pathway . we also analysed whether the degree of immunostaining of cyp3a4 and cyp3a7 correlated significantly in the hcc samples using polychoric correlation coefficient . in light of the evidence that sorafenib is metabolized through cyp3a4 and udp glucuronosyltransferase 1 family , polypeptide a9 ( ugt1a9 ) , we used publicly available bioinformatics resources , such as supercyp and transformer databases , to investigate its pharmacokinetic profile . furthermore , we tested whether other tyrosine kinases analogues of sorafenib underwent the same pharmacokinetic pathway . we also analysed whether the degree of immunostaining of cyp3a4 and cyp3a7 correlated significantly in the hcc samples using polychoric correlation coefficient . immunoreactivity for cyp3a4 and cyp3a7 was detected in all cases of hcc immunostained , except one ( case number 11 ) for cyp3a7 . most cases showed a diffuse and strong positivity for cyp3a4 ( resulting in a final grading score equal to 3 ) . immunostaining for cyp3a7 was observed in a much lower number of cells than for cyp3a4 , with the majority of cases reporting the final grading score equal to 1 . cyp3a4 and cyp3a7 positivity in the surrounding liver was detected in all but 3 cases for cyp3a4 and all but 4 cases for cyp3a7 . cases number 1 , 3 and 4 showed a diffuse distribution of cyp3a4 ( score 3 ) in tumour cells ( figure 1 ) . cases number 2 , 5 , 6 , 10 and 11 presented a score of 2 for cyp3a4 in hcc . a score of 1 for cyp3a4 was found in cases number 7 , 8 and 9 ( table 1 ) . positivity for cyp3a4 was present also in the surrounding liver of 11 samples ( table 1 ) : samples number 1 and 10 showed a diffuse distribution of cyp3a4 ( score 3 ) ; number 2 , 3 , 6 , 7 and 9 showed a medium distribution of cyp3a4 in cases number 4 , 5 and 8 not classified ( nc ) . the surrounding parenchyma was positive for cyp3a4 in 8 out of 11 cases in which it could be evaluated ( table 1 ) . in cases number 1 ( figure 2 ) , 2 , 9 and 11 the surrounding area was affected by cirrhosis ( c ) ; cases number 3 ( figure 3 ) , 6 and 7 showed chronic hepatitis ( h ) ; the remaining 3 livers were nc . ten out of 11 hcc samples showed a medium or weak distribution for cyp3a7 ( score 2 and 1 ) , while 1 was negative ( table 2 ) . cases number 4 , 5 and 10 were characterized by a zonal distribution of cyp3a7 ( score 2 ) ( figure 4 ) , while the remaining cases showed a weak distribution for cyp3a7 ( score 1 ) . the surrounding parenchyma was positive for cyp3a7 in 8 out of 11 cases where it could be evaluated ( table 2 ) . cases number 6 , 7 and 9 showed moderate positivity of cyp3a7 ( score 2 ) , cases number 1 , 2 , 3 and 11 showed a weak distribution of the cyp3a7 ( score 1 ) , while the remaining were nc . the search in the supercyp and transformer databases did not show any interaction between sorafenib and cyp isoform cyp3a4 and cyp3a7 . interestingly , the bioinformatics analysis showed that other three tyrosine kinase inhibitors -imatinib , gefitinib and pazopanibare substrates of cyp3a4 ( with imatinib and pazopanib acting as inhibitors ) , while imatinib acts also as a substrate of cyp3a7 . the polychoric correlation coefficient showed a moderate concordance in the expression patterns of cyp3a4 and cyp3a7 in hcc samples ( rho= 0.26 , standard error= 0.2 ; goodness of fit test : pearson g2 = 4.64 ) . cases number 1 , 3 and 4 showed a diffuse distribution of cyp3a4 ( score 3 ) in tumour cells ( figure 1 ) . cases number 2 , 5 , 6 , 10 and 11 presented a score of 2 for cyp3a4 in hcc . a score of 1 for cyp3a4 was found in cases number 7 , 8 and 9 ( table 1 ) . positivity for cyp3a4 was present also in the surrounding liver of 11 samples ( table 1 ) : samples number 1 and 10 showed a diffuse distribution of cyp3a4 ( score 3 ) ; number 2 , 3 , 6 , 7 and 9 showed a medium distribution of cyp3a4 in cases number 4 , 5 and 8 not classified ( nc ) . the surrounding parenchyma was positive for cyp3a4 in 8 out of 11 cases in which it could be evaluated ( table 1 ) . in cases number 1 ( figure 2 ) , 2 , 9 and 11 the surrounding area was affected by cirrhosis ( c ) ; cases number 3 ( figure 3 ) , 6 and 7 showed chronic hepatitis ( h ) ; the remaining 3 livers were nc . ten out of 11 hcc samples showed a medium or weak distribution for cyp3a7 ( score 2 and 1 ) , while 1 was negative ( table 2 ) . cases number 4 , 5 and 10 were characterized by a zonal distribution of cyp3a7 ( score 2 ) ( figure 4 ) , while the remaining cases showed a weak distribution for cyp3a7 ( score 1 ) . the surrounding parenchyma was positive for cyp3a7 in 8 out of 11 cases where it could be evaluated ( table 2 ) . cases number 6 , 7 and 9 showed moderate positivity of cyp3a7 ( score 2 ) , cases number 1 , 2 , 3 and 11 showed a weak distribution of the cyp3a7 ( score 1 ) , while the remaining were nc . the search in the supercyp and transformer databases did not show any interaction between sorafenib and cyp isoform cyp3a4 and cyp3a7 . however , transformer databases confirmed that sorafenib undergoes glucuronidation through ugt1a9 . interestingly , the bioinformatics analysis showed that other three tyrosine kinase inhibitors -imatinib , gefitinib and pazopanibare substrates of cyp3a4 ( with imatinib and pazopanib acting as inhibitors ) , while imatinib acts also as a substrate of cyp3a7 . the polychoric correlation coefficient showed a moderate concordance in the expression patterns of cyp3a4 and cyp3a7 in hcc samples ( rho= 0.26 , standard error= 0.2 ; goodness of fit test : pearson g2 = 4.64 ) . the cyp450 superfamily in humans is formed by genes and pseudogenes encoding for functional mono - oxygenase . the most important function of cyp450 super - family are related to : i ) the detoxification of exogenous xenobiotics , such as carcinogens and foreign chemicals ; ii ) the decomposition of most of the currently used drugs ; iii ) the metabolism of endogenous growth factors , such as hormones , fatty acids , prostaglandins , cholesterol , vitamin d and bile acids . genetic polymorphisms of cyps have been studied as risk markers for hcc development in cell lines , in mouse and human patients . in previous studies the expression of cyp3a7 in foetal liver has been reported to start at the ninth week of gestation and to be abolished within the first postnatal week . cyp3a7 has been reported to be the dominant enzyme in the foetal liver , given that a subsequent age - related shift between cyp3a7 and cyp3a4 occurs after birth . however , cyp3a4 as well as cyp3a7 immunohistochemical positivity has been recently detected in foetal livers . it is conceivable that the reported changes in the expression of cyp3a7 ( up - regulation ) and cyp3a4 ( down - regulation ) occurring after birth , as well as the restriction of cyp3a7 expression just in the prenatal life , might not be as solid as previously established . in fact , the immunohistochemical patterns of cyp3a4 and cyp3a7 during development showed that the relationships between cyp3a4 and cyp3a7 are more complex then previously thought . the analysis of cyp genes in end - stage livers compared with normal donor liver showed that the expression of cyp3a4 , cyp3a5 and cyp3a7 was decreased in hcc . immunohistochemical cyp3a4 positivity was previously reported in hcc , hepatocellular adenomas ( hca ) and focal nodular hyperplasia ( fnh ) , suggesting that the expression of cyp3a4 may change in hca and hcc and indicating specific metabolic characterization in the subgroup of hca and hcc . our findings showed that both cyp3a4 and cyp3a7 were present in the vast majority of the hcc samples analysed , being detected in 10 out of 11 hccs . cyp3a7 was negative in just one hcc sample . regarding the comparison of the patterns of positivity of the two isoforms , we found that immunoreactivity for cyp3a4 was more diffuse and stronger than cyp3a7 in hcc cells . immunoreactivity for cyp3a4 was observed in the surrounding hepatocytes in 8 out of 11 cases , whereas immunostaining for cyp3a7 was found in the normal liver cells in 7 out of 11 cases . taken together , these findings suggest the existence of a marked inter - individual variability regarding cyp3a isoforms both in physiological tissue , as shown by differences among the surrounding livers , and in pathological one , as evidenced by differences observed among tumour cells . this finding confirms previous data on the expression of cyp3a4 and cyp3a7 in foetal human livers and suggests the need for a tailored individualized medicine in the therapeutic approach to hcc . of interest , the polychoric correlation confirmed to some extent this qualitative analysis by showing a low to moderate degree of concordance in the patterns of cyp3a4 and cyp3a7 immunostaining in hcc . conversely , our bioinformatic search did not highlight any interaction of sorafenib with cyp3a4 as well with cyp3a7 . the latter finding , however , appears to be consistent with the literature , with no reports of sorafenib cyp3a7-mediated metabolism . of interest is the evidence that other tyrosine kinase inhibitors analogues of sorafenib do undergo metabolism through cyp3a7 . the pharmacokinetic profile of sorafenib allows prioritizing the expression patterns identified in hcc in our sample . indeed , the absence of interaction between sorafenib and cyp3a7 , at least according to the current knowledge , renders less probable that specific alterations of cyp3a7 expression might be pharmacokinetically , and thus clinically , relevant . on the contrary an overexpression of cyp3a4 could lead to increased degradation of the drug and thus to clinical ineffectiveness . this could be clinically relevant given that sorafenib response rate remains still quite low at around 2% . the finding of a moderate concordance in the expression patterns of the two cyp isoform deserves a final comment . this finding is based on a small sample size and as such should be interpreted with caution . however , if the pattern of correlated positivity to immunostaining will be confirmed in larger cohort of patients , it will be possible to use immunostaining of either cyp isoforms in liver biopsies to study the pharmacokinetic profile on an individual basis . despite recent advances in diagnosis and management of hcc , the prognosis and the median survival of patients is still poor . therefore , the identification of new putative markers to improve the hcc prognosis became indispensable and opened a new research field . our group previously evaluated the expression of thymosin beta 4 ( t4 ) and thymosin beta 10 ( t10 ) in hcc tissues . showed an increase of selm expression and gpx4 and gpx7 over - expression in human hcc tissues by immunohistochemistry . our data suggest that immunohistochemistry for cyp3a4 and cyp3a7 should be introduced in clinical practice to further elucidate its relation with the clinical response to chemotherapy with sorafenib . immunohistochemistry for cyp isoenzymes might represent a prognostic marker for current management of hcc and may play a role along the way towards a personalized therapy for hcc .
hepatocellular carcinoma ( hcc ) represents a major global health problem , since more than 90% of primary liver cancers worldwide are hcc . 
 most cases of hcc are secondary to viral hepatitis infection ( hepatitis b or c ) , alcoholism and cirrhosis . 
 sorafenib , an oral tyrosine kinase inhibitor that suppresses tumor proliferation and angiogenesis , emerged as the first effective systemic treatment for hcc after 30 years of research , and is currently the standard - of - care for patients with advanced hcc . 
 sorafenib is metabolized by cytochrome p450 ( cyp450 ) , particularly from the 3a4 isoform , producing two main metabolites : the n - oxide and the n - hydroxymethyl metabolite . 
 we studied 11 hcc sample showing the presence of cyp3a4 and cyp3a7 in most of the samples analysed . 
 specifically , the immunoreactivity of cyp3a4 was stronger and more widespread than that of cyp3a7 . the cyp3a4 immunoreactivity was observed in surrounding hepatocytes in 8 out of 11 cases ; while the cyp3a7 immunostaining was found in normal liver cells , in 7 out of 11 cases . 
 these results suggest the existence of a marked inter - individual variability regarding the presence of the isoforms of cyp3a . 
 in addition , since sorafenib is metabolized by cyp3a4 , but not by cyp3a7 , an overexpression of cyp3a4 may lead to an increase in the degradation of the drug and then to clinical ineffectiveness 
 . these results might implicate the necessity of an individualized approach in the treatment of hcc as positivity to cyp3a4 in hcc liver samples might predict a scarce response to sorafenib .