Correlation between CT features and liver function and p53 expression in hepatitis, cirrhosis and hepatocellular carcinoma

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1 ONCOLOGY LETTERS Correltion between CT fetures nd liver function nd p53 expression in heptitis, cirrhosis nd heptocellulr crcinom YAHUI HU, JING WU, SHA LI nd XIAOXIAO ZHAO Deprtment of Nucler Medicine, Zhengzhou Centrl Hospitl Affilited to Zhengzhou University, Zhengzhou, Henn , P.R. Chin Received December 27, 2017; Accepted June 27, 2018 DOI: /ol Abstrct. This study imed to investigte the correltion between CT fetures nd liver function nd p53 expression in heptitis, cirrhosis nd heptocellulr crcinom (HCC). Forty ptients with HCC, 30 ptients with cirrhosis nd 30 ptients with chronic heptitis were enrolled between December, 2015 nd December, At the sme time, norml liver tissues collected from 30 ptients with heptic hemngiom were used s the norml control group. All the ptients were scnned by CT. Averge body surfce re, left outer lobe nd cudte lobe volume, nd the proportions of left outer lobe nd cudte lobe to the whole liver were clculted. Biochemicl indexes of liver function were determined. The pthologicl tissues of ll the subjects were nlyzed. Compred with the control group, totl liver volume of the HCC group ws significntly reduced (P<0.05). Compred with the HCC group, the volume of the left outer lobe incresed in the heptitis group nd the cirrhosis group. Compred with control group, cudte lobe volume incresed significntly in the heptitis group (P<0.05). Compred with the control group, the volume of the left outer lobe nd the proportion of cudte lobe to the whole liver volume ws significntly incresed in ll three groups (P<0.05). Liver function relted indictors in the HCC nd cirrhosis groups were significntly different from those in the control group (P<0.05). The expression level of p53 in HCC ws significntly higher thn tht in the control group (P<0.05). The ccurcy of dignosis by using both p53 nd CT ws higher thn the use of p53 or CT lone. CT cn ccurtely mesure the volume of ech lobe of the liver, nd p53 hs importnt clinicl vlues in the dignosis of liver diseses. Thus, the resonble combintion of the two cn effectively improve the dignostic ccurcy. Correspondence to: Dr Yhui Hu, Deprtment of Nucler Medicine, Zhengzhou Centrl Hospitl Affilited to Zhengzhou University, 195 Tongbi Rod, Zhengzhou, Henn , P.R. Chin E-mil: h559d3@163.com Key words: heptitis, cirrhosis, heptocellulr crcinom, computed tomogrphy, tumor suppressor gene Introduction At present, heptocellulr crcinom (HCC) is the second most common cuse of cncer-relted deth in Chin, nd the incidence of HCC in recent severl yers hs shown n incresing trend (1,2). The mortlity rte of HCC is high. Heptitis, cirrhosis nd liver cncer belong to the trilogy of liver disese, nd most ptients with HCC often hve vrying degrees of cirrhosis (3,4). The key fctor for the high mortlity rte of HCC is the immture erly dignosis of HCC. HCC in erly stges usully shows no specific symptoms, nd most ptients re dignosed t dvnced stges, leding to the high mortlity rte (5,6). Therefore, erly dignosis nd tretment remin crucil in the tretment of HCC. Previous findings showed tht heptitis, cirrhosis nd HCC re ccompnied with different chnges in liver volume (7). In ddition, the occurrence of HCC is directly relted to the inctivtion of tumor suppressor genes nd bnorml cell prolifertion (8). p53 is mjor tumor suppressor gene ssocited with most mlignncies, including HCC (9,10). In the present study, the volume of whole liver, left lterl lobe nd cudte lobe ws mesured by 16-slice spirl CT nd the correltions between those fetures nd liver function were exmined. Expression of p53 in liver tissue of ptients with heptitis, cirrhosis nd HCC ws detected. The specificity nd sensitivity of dignosis of liver cncer were improved by combining the two different indexes. Mterils nd methods Inclusion nd exclusion criteri. Ptients with HCC, cirrhosis nd chronic heptitis t the Zhengzhou Centrl Hospitl Affilited to Zhengzhou University (Henn, Chin) were selected between December, 2015 nd December, The ptients were dignosed by pthologicl exmintions ccording to the dignostic criteri estblished by WHO. This study ws pproved by the Ethics Committee of Zhengzhou Centrl Hospitl Affilited to Zhengzhou University (Henn, Chin), nd ll ptients signed written informed consent. Inclusion criteri for the study were: ptients received surgicl tretment; ptients received no rdiotherpy or chemotherpy before surgery; ptients with primry HCC; ptients with complete clinicl record. Exclusion criteri for the study were: ptients without ccurte pthologicl dignosis; ptients

2 2 HU et l: CT FEATURES AND LIVER FUNCTION IN HEPATIC DISEASE Figure 1. Conventionl double-phse 16 spirl CT scnning. treted with rdiotherpy or chemotherpy before surgery; ptients with metsttic liver cncer; ptients with contrindictions for surgery. Clinicl dt. Clinicl dt of those ptients were collected. These ptients were selected using the Hospitl Informtion System (HIS), dignosed by Pthemtology nd conformed to the stndrd. There were 40 ptients with HCC, 30 ptients with cirrhosis nd 30 ptients with chronic heptitis. At the sme time, norml liver tissues collected from 30 ptients with heptic hemngiom were used s the norml control group. All norml liver tissues were observed under microscope (Olympus Corportion, Tokyo, Jpn), nd no inflmmtory cell infiltrtion, fibrosis nd regenertion nodules nd hemngiom lesions were observed. Methods Mesurement of liver volume. Siemens Senstion 16 spirl CT scnner ws used for conventionl double-phse scnning. The re from the top of the diphrgm to the lower edge of liver nd spleen ws scnned within breth-hold. During scnning, bolus injection of ml non-ionic contrst gent elixir ws performed t speed of ml/sec. Spirl collimtion ws 1.5 mm, moving speed ws 12 mm/sec, scnning lyer thickness ws 7.00 mm nd reconstruction intervl ws 5.00 mm. After scnning, the originl dt were subjected to 5 mm portl venous phse reconstruction, nd were imported into Volume softwre. Edge of the liver lobes ws identified from top of the septum to the lower edge of the liver nd gllbldder nd inferior ven cv were voided. The volume of the liver ws clculted with the threshold of HU (Fig. 1). Observtion index. Before CT exmintion, the ptient's height nd weight were recorded to clculte body surfce re. In order to void individul differences, volume of the whole liver, left outer lobe nd cudte lobe per unit body surfce ws clculted. Liver function test. Biochemicl indexes of liver function were tested using TBA-120FR utomtic biochemicl nlyzer (Toshib, Tokyo, Jpn). Immunohistochemicl detection of p53 expression. Tissues were fixed in 10% neutrl formlin, followed by prffin-embedding nd slicing. Tissue sections were trnsferred to glss slides nd bked t 70 C overnight. The slides were deprffinized 3 times using xylene, nd pssed grded concentrtions ethnol for hydrtion. Citric cid buffer ws used for ntigen retrievl. After wshing 3 times with phosphte-buffered sline (PBS), sections were incubted with 3% H 2 O 2 for 15 min t room temperture. The sections were incubted with primry rbbit nti-humn P53 monoclonl ntibody (1:700; ct. no. 2527; Cell Signling Technology, Inc., Dnvers, MA, USA) overnight t 4 C in wet box. After wshing 3 times with PBS, sections were incubted with secondry got nti-rbbit monoclonl ntibody (1:1,000; ct. no. 8114; Cell Signling Technology, Inc.) for 30 min t room temperture. After wshing 3 times with PBS, DAB color development ws performed in wet box, nd color development ws stopped by rinsing with tp wter. The sections were soked in hemtoxylin for 1-3 min, nd fter wshing, the sections were soked in hydrochloric cid lcohol for sec. The sections were rinsed, dehydrted, vitrificted nd seled with neutrl resin. Sttisticl nlysis. SPSS 20.0 sttisticl softwre (IBM, Armonk, NY, USA) ws used for the sttisticl nlysis. Mesurement dt were expressed s men ± stndrd devition (SD), nd subjected to one-wy ANOVA with LSD s the post hoc test. Intrgroup comprisons were performed using t-test. Countble dt were expressed s percentge (%), nd comprisons were performed using χ 2 test. P<0.05 ws considered to indicte sttisticlly significnt difference. Results Chnges in liver volume nd correltions with liver disese. Clculted body surfce re of ech group ws substituted into the formul for the clcultion of stndrd liver volume. No significnt differences in clculted liver volume were found between ptient groups nd control group (P>0.05). However, liver volume nd volume index mesured by CT in

3 ONCOLOGY LETTERS 3 Tble I. Chnges in liver volume nd correltions with liver disese. Groups Cses Formul volume (cm 3 ) CT volume (cm 3 ) Volume index Volume chnge rte (%) Heptitis group ± ± ± ±3.31 Cirrhosis group ± ± ± ±4.52 HCC group ± ± ± ±5.04 Control group ± ± ± ±2.25 t-test P-vlue Compred with control group, P<0.05. HCC, heptocellulr crcinom. Tble II. Liver volume of ech group. Totl liver Left outer Cudte lobe Totl liver volume/ Left outer lobe volume/ Cudte volume/body volume body surfce lobe body surfce lobe surfce Groups Cses (cm 3 ) volume (cm 3 ) volume volume (cm 3 ) volume (cm 3 ) volume (cm 3 ) Heptitis group ± ± ± ± ± ±13.11 Cirrhosis group ± ± ± ± ± ±10.57 HCC group ± ± ± ± ± ±5.98 Control group ± ± ± ± ± ±6.99 t-test P-vlue Compred with control group, P<0.05. HCC, heptocellulr crcinom. Tble III. Proportions of left outer lobe nd cudte lobe to the whole liver. Left outer lobe/ Cudte lobe/ Groups Cses whole liver (%) whole liver (%) Heptitis group ± ±1.45 Cirrhosis group ± ±1.45 HCC group ± ±1.42 Control group ± ±0.73 t-test P-vlue Compred with control group, P<0.05. HCC, heptocellulr crcinom. HCC groups were significntly lower thn those in other group. Liver volume chnge rte in HCC group ws ± 5.04%, which ws significntly higher thn tht in other three groups (P<0.05) (Tble I). Comprison of liver lobe between ptient groups nd control group. No significnt differences in totl liver volume nd unit surfce re were found between the heptitis, cirrhosis nd control groups (P>0.05), but significntly reduced vlues were found in the HCC group (P<0.05). Left outer lobe volume nd unit surfce re volume in the heptitis, HCC nd cirrhosis groups were significntly lrger thn those in the control group (P<0.05). Cudte lobe volume nd unit surfce re volume in the heptitis group were significntly lrger thn those in the remining groups, nd no significnt differences were found mong the cirrhosis, HCC nd control groups (P>0.05). No significnt differences in the proportions of left outer lobe nd cudte lobe to the whole liver were found mong the groups (P>0.05) (Tbles II nd III). Liver function-relted indictors in ech group. Liver function-relted indictors in the HCC nd cirrhosis groups were significntly different from those in the control group (P<0.05). The results showed tht the liver function of heptitis ptients ws norml. However, with the ggrvtion of liver dmge, liver dysfunction becme more nd more obvious (Tble IV). p53 expression in ech group. Positive rte of p53 expression ws 45.00% (18/22) in the HCC group nd 3.33% (1/29) in the cirrhosis group. The positive rte of p53 expression in cirrhosis nd heptitis ws not significntly different from tht in control group (P>0.05), but there were significnt differences between HCC group nd control group (P<0.05) (Tble V). Correltions between heptitis, cirrhosis nd HCC CT fetures nd liver function index ALB. Heptitis, cirrhosis nd HCC CT fetures showed high degree of consistency with

4 4 HU et l: CT FEATURES AND LIVER FUNCTION IN HEPATIC DISEASE Tble IV. Liver function-relted indictors in ech group. Groups Cses ALB (g/l) TP (g/l) ALB/GLB Pche (V/l) TBIL (µmol/l) TBA (µmol/l) Heptitis group ± ± ± ± ± ±0.11 Cirrhosis group ± ± ± ± ± ±0.22 HCC group ± ± ± ± ± ±0.24 Control group ± ± ± ± ± ±0.99 Compred with control group, P<0.05. HCC, heptocellulr crcinom. Tble V. p53 expression in ech group. p Positive Negtive Groups Cses n (%) n (%) Heptitis group 30 0 (0.00) 30 (100.00) Cirrhosis group 30 1 (3.33) 29 (96.67) HCC group (45.00) 22 (55.00) Control group 30 0 (0.00) 30 (100.00) Tble VII. Correltions between heptitis, cirrhosis nd HCC CT fetures nd p53 expression. Groups Heptitis group Cirrhosis group HCC group Control group HCC, heptocellulr crcinom. Correltion vlues r= -0.94, P<0.05 r= -0.96, P<0.05 r= -0.91, P<0.05 r= -0.93, P<0.05 Compred with the control group, P<0.05. HCC, heptocellulr crcinom. Discussion Tble VI. Correltions between heptitis, cirrhosis nd HCC CT fetures nd liver function index ALB. Groups Heptitis group Cirrhosis group HCC group Control group HCC, heptocellulr crcinom. Correltion vlues r=0.96, P<0.05 r=0.91, P<0.05 r=0.94, P<0.05 r=0.92, P<0.05 the expression level of liver function indictor ALB, nd they were positively correlted (Tble VI). Correltions between heptitis, cirrhosis nd HCC CT fetures nd p53 expression. Heptitis, cirrhosis nd HCC CT fetures showed high degree of consistency with the expression level of p53, nd they were negtively correlted (Tble VII). Dignostic ccurcies of liver diseses by using CT. Sensitivity, specificity, positive predictive rte nd negtive predictive rte of CT were 50.80, 98.90, nd 62.30%, respectively, while the vlues of p53 were 78.30, 77.80, nd 74.50%, respectively. Sensitivity, specificity, positive predictive rte nd negtive predictive rte of CT combined with p53 ws 33.60, 98.90, nd 54.92%, respectively. The dt suggest tht the ccurcy of dignosis of liver disese by both using p53 nd CT ws higher thn the use of p53 or CT lone (Tble VIII). In Chin, 110,000 ptients succumb to HCC, mking this disese the leding cuse of deth in ptients with chronic liver disese, especilly those with cirrhosis (11,12). The inhibition of heptitis or cirrhosis remins mjor issue for the clinicl tretment of HCC tht needs to be resolved (13). Erly dignosis is the most effective wy to improve prognosis nd increse the survivl rte of ptients with HCC (14,15). HCC is directly relted to cirrhosis; thus, it is crucil to detect chronic liver disese (16). Liver volume cn directly reflect the number of heptocytes, which cn be used to ssess the conditions of liver disesess. Liver volume now hs been widely used in the dignosis nd tretment of cute nd chronic liver diseses (17,18). In this study, in the stge of heptitis nd cirrhosis, the volume of whole liver ws not significntly reduced compred with tht in the control group, but significntly decresed in the HCC group. In ddition, the proportion of left outer lobe to whole liver ws incresed in ech ptient group compred with tht in control group, nd no significnt differences were found mong the ptient groups. The dt suggest tht enlrgement of left outer lobe cn be observed in both heptitis nd cirrhosis; thus, the proportion of left outer lobe to whole liver ws bsolutely incresed. The whole liver volume ws decresed in HCC group, thus, the proportion of left outer lobe to whole liver ws reltively incresed. Whole liver volume ws not decresed in the stges of heptitis or cirrhosis, but left outer lobe volume ws incresed to mintin norml compenstory function. With the development of disese, whole liver volume ws decresed nd compenstory function ws not needed. Thus, left outer lobe volume ws not be further incresed.

5 ONCOLOGY LETTERS 5 Tble VIII. The dignostic ccurcy of CT nd p53 on HCC/%. Positive Negtive Items Sensitivity Specificity predictive rte predictive rte LR + LR - Single method CT p Combined method Positive in t lest one method Positive in both methods HCC, heptocellulr crcinom. p53 protein dysfunction nd the development of humn tumors re closely correlted with ech other. The occurrence nd development of HCC re directly relted to the bnorml expression of p53 (19,20). In this study, it ws found tht p53 expression level in the control group nd chronic liver disese groups ws reltively low, but the expression level ws significntly incresed in the HCC group, indicting tht p53 my serve s dignostic mrker for HCC. p53 ws combined with CT to increse dignostic ccurcy. It ws found tht prllel combintion of p53 nd HSP70 incresed specificity nd reduced sensitivity, while seril combintion plyed n opposite role. Therefore, the resonble combintion of p53 nd CT cn be used to dignose HCC, which is more relible nd ccurte thn the use of p53 or CT lone. Therefore, the rtionl use of p53 nd HSP70 in series or in prllel cn increse both sensitivity nd specificity, which is conducive to the erly dignosis of HCC. Acknowledgements Not pplicble. Funding No funding ws received. Avilbility of dt nd mterils The dtsets used nd/or nlyzed during the current study re vilble from the corresponding uthor on resonble request. Authors' contributions YH recorded nd nlyzed the clinicl dt of ptients. JW ws involved in dt cquisition. SL ws responsible for indictor index. YH nd XZ nlyzed liver function. All uthors red nd pproved the finl mnuscript. Ethics pprovl nd consent to prticipte This study ws pproved by the Ethics Committee of Zhengzhou Centrl Hospitl Affilited to Zhengzhou University (Henn, Chin), nd ll ptients signed written informed consent. Ptient consent for publiction Not pplicble. Competing interests The uthors declre tht they hve no competing interests. References 1. Li H, Wng S, Wng G, Zhng Z, Wu X, Zhng T, Fu B nd Chen G: Yes-ssocited protein expression is predictive mrker for recurrence of heptocellulr crcinom fter liver trnsplnttion. Dig Surg 31: , Ji X, Zhng Q, Du Y, Liu W, Li Z, Hou X nd Co G: Somtic muttions, virl integrtion nd epigenetic modifiction in the evolution of heptitis B virus-induced heptocellulr crcinom. Curr Genomics 15: , Bhnssy AA, Zekri ARN, El-Bstwisy A, Fwzy A, Shett M, Hussein N, Omrn D, Ahmed AAS nd El-Lbbody SS: Circulting tumor nd cncer stem cells in heptitis C virus ssocited liver disese. World J Gstroenterol 20: , Chen X, Jing W, Yue C, Zhng W, Tong C, Di D, Cheng B, Hung C nd Lu L: Heprnse contributes to trns-endothelil migrtion of heptocellulr crcinom cells. J Cncer 8: , Gehrmnn M, Cervello M, Montlto G, Cppello F, Gulino A, Knpe C, Specht HM nd Multhoff G: Het shock protein 70 serum levels differ significntly in ptients with chronic heptitis, liver cirrhosis, nd heptocellulr crcinom. Front Immunol 5: 307, Kng GH, Lee BS, Lee ES, Kim SH, Lee HY nd Kng DY: Prognostic significnce of p53, mtor, c-met, IGF-1R, nd HSP70 overexpression fter the resection of heptocellulr crcinom. Gut Liver 8: 79-87, Tremosini S, Forner A, Boix L, Viln R, Binchi L, Reig M, Rimol J, Rodríguez-Lope C, Ayuso C, Solé M, et l: Prospective vlidtion of n immunohistochemicl pnel (glypicn 3, het shock protein 70 nd glutmine synthetse) in liver biopsies for dignosis of very erly heptocellulr crcinom. Gut 61: , Liu K, Zho X, Gu J, Wu J, Zhng H nd Li Y: Effects of 12C6 + hevy ion bem irrdition on the p53 signling pthwy in HepG2 liver cncer cells. Act Biochim Biophys Sin (Shnghi) 49: , Liu K, Lee J, Kim JY, Wng L, Tin Y, Chn ST, Cho C, Mchid K, Chen D nd Ou JJ: Mitophgy controls the ctivities of tumor suppressor p53 to regulte heptic cncer stem cells. Mol Cell 68: e5, Chi Y, Xioyu L nd Hiyn W: Correltion between expression levels of PTEN nd p53 genes nd the clinicl fetures of HBsAg positive liver cncer. J BUON 22: , EI-Emshty HM, Sd EA, Toson EA, Abdel Mlk CA nd Gdelhk NA: Apoptosis nd cell prolifertion: Correltion with BCL-2 nd p53 oncoprotein expression in humn heptocellulr crcinom. Heptogstroenterology 61: , 2014.

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