4Clin Pathol 1996;49:470-473

Expression of P-glycoprotein in hepatocellularcarcinoma: a potential marker of prognosis

Y Soini, N Virkajarvi, H Raunio, P PNakkb

AbstractAims-(1) To investigate the immunohis-tochemical expression of the multidrugresistance gene (MDR1) product P-glyco-protein in histological samples from 31hepatocellular carcinomas (HCCs); and(2) to correlate the results with cell prolif-eration, p53 expression, the disease-freeinterval, and cumulative patient survival.Methods-C219 (a monoclonal antibody),CM-1 (a polyclonal rabbit anti-humanantibody) and PC1O (a monoclonal mouseanti-human antibody) were used to detectexpression of P-glycoprotein, p53 andproliferating cell nuclear antigen (PCNA),respectively, by means ofthe avidin-biotinperoxidase method.Results-Membrane bound positivity forP-glycoprotein was observed in 20 (65%)of the 31 HCCs. Cytoplasmic globularpositivity was also seen in some cases.There were no significant associationsbetween expression of P-glycoprotein andcell proliferation (determined by PCNAinimunoexpression and the mitoticcount), or p53 expression. Patients withP-glycoprotein positive tumours had ashorter disease-free interval than thosewith P-glycoprotein negative tumours,and also had a shorter survival time.There was no difference in survival be-tween P-glycoprotein positive patientswho had or had not received chemo-therapy, suggesting that chemotherapy(mainly mitomycin-C ) did not affect sur-vival in these patients.Conclusions-Expression of P-glyco-protein in HCCs is associated with ashorter disease-free interval and shortersurvival time. As expression of P-glyco-protein was not associated with cell prolif-eration or expression of p53, its effect ondisease progression and survival seems tobe independent ofthese factors.(7 Clin Pathol 1996;49:470-473)

Keywords: MDR, liver, hepatocellular carcinoma, p53,PCNA.

P-glycoprotein is a 170 kilodalton, membranebound glycoprotein encoded by the multidrugresistance (MDR 1) gene' 2 andfunctions as anATP dependent pump propelling drugs andcytostatics out of the cell cytoplasm. Becauseof its involvement in drug resistance, theexpression of P-glycoprotein has been studiedin several different neoplasms and increasedP-glycoprotein expression has been found in

several epithelial tumours, such as those of thecolon and breast." In breast carcinomasexpression of P-glycoprotein has been associ-ated with p53 expression and cell proliferation,as evaluated by the Ki67 index.5 P-glyco-protein expression has also been associatedwith a shorter disease-free interval in carcino-mas of the breast, colon and kidney.4 78

Expression of P-glycoprotein has been stud-ied previously in liver cells and in hepatocellu-lar carcinomas (HCCs). In non-neoplastic liverP-glycoprotein is expressed in the biliarycanalicular pole of hepatocytes3 and in epithe-lial cells lining the biliary ducts; 60-70 % ofHCCs are P-glycoprotein positive.3 9 Activa-tion of the MDR1 gene in hepatic tissues hasbeen studied extensively in animal models;several rodent hepatocarcinogenesis modelsshow that enhanced MDR1 expression is asso-ciated with the later stages of carcinogenesis.'°

In this study we investigated P-glycoproteinexpression in 31 patients with HCC. The aimof the investigation was to compare P-glyco-protein expression with the disease-free inter-val, cumulative survival and other clinicalparameters.

MethodsSections from 31 HCCs (from 13 men and 18women) resected between 1983 and 1993 wereretrieved from the files of the Department ofPathology, Oulu University Hospital. All of thematerial had been fixed in 10% neutral forma-lin and embedded in paraffin wax. In all casesthe diagnosis had been based on conventionallight microscopy, according to the criteria oftheWorld Health Organisation." Table 1 showsthe histological type of the tumours, theirgrade and TNM status. The grade of thetumours was determined as suggested byEdmonson and Steiner"2 and the stage asrecommended by Spiessl et al." The casehistories of all the patients were reviewed andthe pertinent clinical data, including survival,stage,"3 age, and sex of each patient werecollated. Six of the patients were alive, five haddied of disease other than HCC or during sur-gery. Fourteen patients received chemotherapyfollowing surgery: all initially receivedmitomycin-C; two patients were subsequentlytreated with epirubicin and one with doxorubi-cin. One patient had a history of viral hepatitis,two patients had chronic aggressive hepatitis,one had primary biliary cirrhosis, and sevenhad cirrhosis. Mean (SD) age was 60.9 (15.5)years and the mean (SD) tumour size was 10.8(7.2) cm.

Department ofPathology,University of Oulu,Oulu UniversityHospital,Oulu, FinlandY SoiniN VirkajarviP Paakk6

Department ofPharmacology andToxicologyH Raunio

Correspondence to:DrYlermi Soini,Department of Pathology,University of Oulu,Kajaanintie 52 D,FIN-90220 Oulu,Finland.

Accepted for publication20 February 1996

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Table 1 Immunohistochemical expression ofP-glycoprotein in relation to histological type,tumour grade and stage, PCNA, immunohistochemical expression ofp53, and patientsurvival

Histological Tumour Survivaltype P-glycoprotein Grade PCNA (Oo) stage * p53 (months)

Compact + + III 10 T2NOMO - 0.2tTrabecular + III 30 T2NOMO - 18**Compact + III 40 T2NOMO - 12tCompact + + II 40 T3NOMO - 8tTrabecular + + II 40 T3NOMO - 25tTrabecular + + + II 55 T2NOMO - 54§Compact + III 70 T2NOMO - 36§Compact + III 90 T3NOMO + + 6tCompact + II 5 T3NOMO - 2tTrabecular + II 5 T3NOMO - I tTrabecular + + + II 5 T2NOMO - 36tCompact + III 70 T3NOMO - 5tTrabecular + + + II 30 T3NOMO + O.1tFibrolamellar + III 95 T2NOMO + 2tCompact + III 20 T2NOMO - 108**Compact + II 40 T3NOMO - 15tCompact + + II 70 T3NOMO - 3tCompact + + + II 5 T3NOMO - 6**Acinar + II 65 T2NOMO - 5tTrabecular + + + I 10 T3NOMO - 102tTrabecular - III 20 T2NOMO - 24**Compact - III 95 T2NOMO + 36§Compact - III 5 T2NOMO - 48§Acinar - II 20 TlNOMO - 55tTrabecular - III 30 T3NOMO - 36§Compact - III 70 T2NOMO + 4tCompact - II 70 T3NOMO + 2tCompact - II 5 T3NOMO - 34tCompact - III 95 T2NOMO + + + 108§Compact - III 10 T2NOMO - 60**Trabecular - I 30 T2NOMO - lot

*At the time of operation.Evaluation of P-glycoprotein and p53 immunoexpression: - negative; + = S 10% tumourcells positive; + + = S 30% tumour cells positive; + + + = > 30% tumour cells positive.tDied of disease.tDied during surgery or of other disease.§Still alive.**Lost to follow up at this stage.

IMMUNOHISTOCHEMISTRYSections, 5 gm thick, were cut from thespecimens and mounted on poly-l-lysinecoated glass slides (Sigma, St Louis, Missouri,USA), air dried overnight and stained within afew days. Slides were dewaxed in xylene andrehydrated through graded alcohol. Endog-enous peroxidase activity was blocked byimmersing the slides in 0.1 % hydrogen perox-ide in absolute methanol for 20 minutes. Non-specific binding was blocked by incubating the

Figure 1 P-glycoprotein immunostaining localised to the cell membrane(immunoperoxidase, x200).

slides in 20 % fetal calf serum in phosphatebuffered saline (PBS) for 20 minutes.

Sections were pretreated in a microwaveoven in 10 mM citric acid monohydrate, pH6.0, for three minutes and were then incubatedwith the primary monoclonal antibody (C219;Signet Laboratories, Dedham, Massachusetts,USA) (diluted 1 in 20) directed againstP-glycoprotein for three hours. The biotiny-lated secondary anti-mouse antibody (Dako,Glostrup, Denmark) (diluted 1 in 300) wasapplied, followed by the avidin-biotin peroxi-dase complex (Dako). Normal liver served asan internal positive control.To detect p53 expression, sections were first

incubated overnight at 4°C with a polyclonalrabbit anti-human p53 antibody (CM-i; No-vocastra Laboratories, Newcastle upon Tyne,UK) diluted 1 in 1000.14 A biotinylatedanti-rabbit immunoglobulin (diluted 1 in 100;Dako) and the avidin-biotin peroxidase com-plex were then added. Sections from a lungcarcinoma previously shown to be stronglypositive for p53 served as a positive control.14A mouse monoclonal IgG2a primary anti-

body (PC 10; Dako) was used to detectproliferating cell nuclear antigen (PCNA). Thesections were incubated with the primary anti-body (diluted 1 in 50) for one hour, followedby a secondary rabbit anti-mouse antibody(diluted 1 in 200) (Dako) and the avidin-biotinperoxidase complex. A lymph node withfollicular hyperplasia served as positive control.

In all cases sections were rinsed stringently,with several changes of PBS between eachstage. Diaminobenzidine was used as the chro-mogen. Sections were were lightly counter-stained with haematoxylin and mounted withEukitt (Kindler, Freiburg, Germany). Fornegative controls non-immune mouse or rabbitserum replaced the primary antibodies in allcases.

Expression of P-glycoprotein and p53 wasscored as follows: - = negative; + = < 10 %tumour cells positive; + + = s 30 % tumourcells positive; + + + = >30 % tumour cellspositive. In the case of PCNA, the percentageof positively stained cells was evaluated in eachsection. P-glycoprotein immunostaining wasmainly localised to the membrane, althoughcytoplasmic staining was seen in some sections.PNCA and p53 immunoreactivity was nuclear.Cell proliferation was also assessed by countingthe number of mitotic figures per 10 highpower fields.

STATISTICAL ANALYSISComparisons between groups were made usingthe two-tailed Student's t test. The significanceof associations was determined using Fisher'sexact probability test and correlation analysis.Survival data were analysed using the Kaplan-Meyer method. The difference between sur-vival in different groups was analysed using thelog rank test. Probability values of less than0.05 were considered significant.

ResultsP-glycoprotein was expressed in 20 (65 %) ofthe 31 HCCs. Immunoreactivity was mainlyconfined to the cell membrane, but in some

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Figure 2 Photomicrograph showing globular intracytoplasmic immunostaining(immunoperoxidase, x200).

cases globular intracytoplasmic staining N

also seen (figs 1 and 2). Adjaceht normal litissue was also stained positively. Some of

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Follow up (months)Figure 3 Kaplan-Meyer curve showing the difference in survival time in P-glycoproteipositive and negative tumours.

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proliferating bile ductules were P-glycoproteinpositive at their luminal surface.There was no difference in the immuno-

staining pattern observed in the different HCChistological types. Tumour grade or stage wasnot significantly associated with positiveimmunostaining, although tumours of lowergrade (p = 0.19) and higher stage (p = 0.13,Fisher's exact test) tended to be more heavilystained. P-glycoprotein expression was not sig-nificantly associated with cell proliferation, asmeasured by positive PCNA immunoexpres-sion (p = 0.92, two-tailed t test) and mitoticcounts (p = 0.96, two-tailed t test). Expressionof P-glycoprotein in p53 positive and negativetumours was not significantly different (p =0.18, Fisher's exact test). Patients withP-glycoprotein positive tumours had a shortersurvival time (mean (SD) 47.0 (12.0) months,95% confidence interval (CI) 23.4-70.6months) than those with P-glycoprotein nega-tive tumours (72.3 (14.3) months, 95% CI

wvas 44.3-100.3 months) (fig 3). The difference iniver 1 btthe survival time between these two groups was

not statistically significant (p = 0.18, log ranktest). The disease-free interval (no evidence ofrecurrence either clinically or on laboratoryinvestigation) was significantly shorter in pa-tients with P-glycoprotein positive (38.5 (10.9)months, 95% CI 17.1-60.0 months) than inthose with negative tumours (86.2 (13.3)months, 95% CI 60.1-112.2 months) (p <0.05, log rank test) (fig 4). The survival time ofpatients with P-glycoprotein positive tumourswho received chemotherapy (41.0 (15.3)months, 95% CI 11.1-70.9 months) did notdiffer significantly from that of all patientsstudied (60.0 (10.5) months, 95% CI 39.3-80.6 months) (p = 0.26, log rank test) or frompatients with P-glycoprotein positive HCCswho did not receive chemotherapy (28.3 (8.1)months, 95% CI 12.4-44.1 months) (p =

< 0.95).120 Seven patients also had liver cirrhosis, of

whom five had P-glycoprotein positive tu-in mours (p = 0.49, Fisher's exact test). There

was no statistically significant associationbetween P-glycoprotein expression and longterm medication for conditions other thanHCC-for example, for late onset diabetes,high blood pressure, heart failure, bronchialasthma, and others (data not shown).

DiscussionPrevious studies have reported thatP-glycoprotein positive breast, colon or renaltumours are associated with a shorter disease-free interval than their P-glycoprotein negativecounterparts.47 8 Our results suggest that this isalso true for patients with HCC (fig 3).Although, in the present study, the differencein survival time between patients with andwithout P-glycoprotein positive tumours didnot reach statistical significance, P-glyco-

I protein immunoexpression may be used to120 predict prognosis. A similar trend in survival in

HCC was reported by Itsubo et al.9 It is worthnoting, however, that most of the tissue studied

ours by Itsubo et al was obtained at necropsy,whereas in the present study all tissue was

0 20 40 60 80 100Follow up (months)

Figure 4 Kaplan-Meyer curve showing the difference in the disease-free interval inP-glycoprotein positive and negative tumours. Patients with P-glycoprotein positive tumohad a significantly shorter disease-free interval (p < 0. 05, log rank test).

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obtained at surgery from patients who had notundergone preoperative chemotherapy. Insome cases chemotherapy may induceP-glycoprotein expression,2 which may thenhave an adverse effect on prognosis.

Twelve patients underwent postoperativechemotherapy. All patients received mito-mycin-C; two patients subsequently receivedepirubicin and one doxorubicin. To testwhether chemotherapy influenced postopera-tive survival, we compared P-glycoproteinexpression in patients who did and did notundergo chemotherapy. No difference in thesurvival time was found, suggesting thatchemotherapy did not affect prognosis inpatients with P-glycoprotein positive tumours.The reason for this may be becausemitomycin-C was the main chemotherapeuticagent used in the present study and has beenfound to be insensitive to drug resistanceinduced by MDR1 in vitro."5

P-glycoprotein immunostaining was mainlyconfined to the cell membrane in HCCs, a pat-tern which was also seen in the adjacentnon-neoplastic liver cells. Intracellular globularimmunostaining was observed in some cases,and its perinuclear location suggested thatP-glycoprotein was present in the Golgiapparatus, confirming previous electron mi-croscopy studies.1Chin et al6 reported that mutant p53 can

activate the MDR1 promoter and Charpin etat found that expression of P-glycoprotein wasassociated with p53 expression and with a highproliferation rate in breast cancer. In contrast,however, De Angelis et al"' found no associa-tion between expression of p53 and P-glyco-protein in colorectal carcinomas. This was alsothe case in the present study, suggesting thatexpression of p53 and cell proliferation have noeffect on P-glycoprotein expression in HCC.

In conclusion, expression of P-glycoproteinin HCCs is associated with a shorter disease-free interval and shorter survival time. Asexpression of P-glycoprotein was not associ-ated with cell proliferation or expression ofp53, its effect on disease progression andsurvival seems to be independent of these fac-tors.

We are grateful to Ms Mirja Vahera and Ms Maria Tolppanenfor their technical assistance. This study was supported by theFinnish Cancer Societies and the Finnish Anti-TuberculosisAssociation.

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