Journal of Hepatology 1999; 30: 653459
Printed in Denmark . All rights reserved
Munksgaard Copenhagen

Copyright 8 European Association
for rhe Study of the Liver I999

Journalof Hepatology
ISSN 01684278

Interferon therapy lowers the rate of progression to hepatocellular
carcinoma in chronic hepatitis C but not significantly in an advanced stage:a retrospectivestudy in 1148 patients
Takeshi Okanoue’,
Masafumi

Yoshito Itoh2, Masahito Minami’, Shinichi Sakamoto’, Kohichiro
Sakamoto’, Kenichi NishioJi’ “l, Yoshiki Murakami’,
Kei Kashima’and The Viral Hepatitis Therapy Study Group*

Yasuii,‘Third Department of Internal Medicine, Kyoto Prefectural University of Medicine and =Department of Internal Medicine, Kyoto Prefectural
Yosanoumi Hospital, Kyoto. Japan

Background/Aim:
Hepatocellular
carcinoma frequently develops during the advanced stages ofchronic hepatitis C. We examined whether interferon
prevents the development of hepatocellular carcinoma
in chronic hepatitis C patients.
Methods: Japanese patients with chronic hepatitis C(n=1.148; 117 with portal fibrous expansion (Fl), 636
with bridging fibrosis (F2), 355 with bridging fibrosis
and architectural distortion (F3)) and 40 cirrhotic(F4) patients were treated with interferon. These patients were followed from 1 to 7 years after interferon
therapy. Blood tests and image analysis were seriallyperformed to assess response to interferon and to detect hepatocellular carcinoma. Fifty-five cirrhotic
type C patients (control F4) not receiving interferon
were enrolled in this study.
Results: Sustained (SR: 27.5%) and transient (TR:23.0%) responders totaled 50.5%, while 49.5% did
not respond to interferon. SR showed an improvementI

N 1996, AROUND30 000 patients died of hepatocellular carcinoma (HCC) in Japan, where this tumor is
the third leading cause of cancer deaths. Of these patients, SO-85% of patients were positive for anti-hepatitis C virus (HCV) antibody (1,2). Eighty to ninety

Received 1 July; revised 7 October; accepted 20 October 1998

Correspondence:
Takeshi Okanoue, Third Department ofInternal Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji Kamigyo-ku, Kyoto, 6020841 Japan. Tel: 81 75 251 5519. Fax: 81 75 2510710.
E-mail: okanoue@koto.kpu-m.ac.jp* Additional members of the Viral Hepatitis Therapy
Study Group are listed in the Appendix.

in disease stage reflected by increased platelet counts.
Fifty-two patients (9 F2, 36 F3, and 7 F4) developed
hepatocellular carcinoma in the follow-up period; 3
SR, 8 TR, and 41 non-responders (NR). The cumulative incidence of hepatocellular carcinoma in F2 wassignificantly lower (p=O.O19) in SR compared with
NR, but not in SR in F3 and F4 patients. However,the cumulative incidence of hepatocellular carcinoma
was significantly decreased in all SR (p=O.OOOl) andTR (p=O.O397) compared with all NR.
Conclusion: These results indicate that interferon
therapy in chronic hepatitis C patients lowered therate of progression of hepatocellular carcinoma insensitive cases but not in patients in an advanced
stage.
Key words: Hepatitis C virus infection; Hepatocellular carcinoma; Interferon therapy; Non-responder;
Sustained responder; Transient responder.

percent of these patients had cirrhosis (l-4). The remaining patients had advanced stages of chronic hepatitis. HCC develops in these patients after years of hepatic inflammation. Thus, it is reasonable to consider
that inhibiting the progression of chronic hepatitis may
decrease the development of HCC.
Interferon (INF) alpha and beta have been widelyused for treatment ‘of chronic hepatitis type C (CH-C)
since public health insurance began to cover this therapy in January 1992. Thirty to forty percent of patients
showed a sustained response after IFN (5,6). To assess
if IFN reduces the development of HCC, we compared
the annual incidence and cumulative rate of HCC insustained responders, transient responders, and nonresponders among CH-C patients who received IFN.
653

T. Okanoue et al.

Materials and Methods
Patients and treatment
Patients who were positive for hepatitis B surface antigen or had ahistory of heavy drinking (daily alcohol intake more than 60 g ofethanol for more than 5 years) were excluded from this study. One
thousandtwo hundred and ninety-nine
Japanese CH-C patients received IFN therapy between January 1989 and September 1995 at the
13 hospitals of the Viral Hepatitis Study Groups listed in the Appendix to this paper, the University Hospital, and Kyoto Prefectural Yosanoumi Hospital, of whom 1148 patients were followed up for morethan 1 year after IFN treatment. The other 151 patients did not visit
the 15 hospitals after IFN therapy, even when letters were sent tothem requiring them to attend for consultation
at one of the hospitals. Patients less than 18 years old and over 68 years old were excluded from this study.
All patients were positive for serum anti-HCV antibody by a firstor second-generation
enzyme-linked
immunosorbent
assay (ELISA;
Ortho Diagnostics,
Tokyo, Japan) and for serum HCV RNA. SerumHCV RNA was detected using reverse transcriptase-polymerase
chain
reaction (RTnested
PCR) as described previously (7). Patients were
negative for antibodies against human immunodeficiency
virus.
Among the cirrhotic type C patients visiting the Kyoto Prefectural
University Hospital from 1987 to 1995, 55 cirrhotic patients who had
not received IFN were enrolled as controls (control F4). The criteria
for the control cirrhotic patients were as follows: less than 68 years
old, not heavy drinkers, less than 2 mg/dl in total serum bilirubin, no
ascites, no HCC by ultrasonography(US) and computed tomography(CT) at entry, and followed up for more than 2 years.
Serum alanine aminotransferase(ALT), blood cell counts, andserum alpha fetoprotein(AFP) were measured before IFN therapy,
and in two-thirdsof the patients the amount of serum HCV RNA
and HCV genotype were determined
before IFN therapy. US was
performed
in all patients before therapy to document the absence of
space-occupying
lesions suggesting HCC. Serum ALT, platelet (PLT)
counts, and AFP were serially checked during the follow-up period.
All patients underwent liver biopsy prior to IFN treatment and all
cirrhotic patients underwent laparoscopy
and/or liver biopsy. Histologic criteria and staging of chronic hepatitis were based on the newclassification
by Desmet et al. (89) in which staging is defined asfollows: FO (no fibrosis), Fl (fibrous portal expansion),
F2 (bridging
fibrosis). F3 (bridging fibrosis with architectural
distortion).
and F4(cirrhosis). One hundred and seventeen patients (male: 70, female: 47,
age: 42.62 13.2 years) were in the Fl stage prior to IFN therapy, 636(male: 413, female: 223, age: 49.52 11.2 years) were in F2, 355 (male:
223, female: 132, age: 54.729.1 years) were in F3, and 40 (male: 18,
female: 22, age: 55.628.3 years) were in F4. In the control F4. average
age was 57.6i5.2
years: 28 were male and 27 were female, with average ages 58.624.5
years and 57.7e5.7
years, respectively. Of themales, 11 were <60 years and 17 were 260 years. Of the females, 19

TABLE

were <60 years, and 1 I were Z60 years. Serum ALT levels and PLT
counts in each stage are shown in Table 1. Serum ALT levels increased, and PLT counts decreased in proportion
to the progress ofthe stage of liver disease (Table 1I.
Of the 1148 patients, 582 received natural IFN alpha (nIFNo), 204received recombinant
IFN alpha 2a (rIFNa 2a), and 362 received
recombinant
IFN alpha 2b (rIFNa 2b). Patients received 3 MU to 10
MU of IFNn intramuscularly
daily or thrice a week for 16 to 24
weeks; the total amount of IFN was 252 to 780 mega units (MU). In
principle. the schedule of IFN therapy was daily injection of IFN for
the initial 2 weeks and then three times a week: however, administration of interferon was changed to three times a week if marked leukopenia (<2000/mm’)or thrombocytopenia(<30000mmi) was seen
during IFN therapy. All F4 patients received 3 or 6 MU of IFNn
daily for the initial 2 weeks and then three times a week for 22 to 24
weeks.
Written informed
consent to participate
in these trials was obtained from all patients, and all aspects of these studies were approvedby the Ethical Committees of the 15 participating
hospitals.
The patients were classified into three groups according to the response to IFN therapy. Sustained responders(SR) were defined as
patients with normal serum ALT levels (C-30 IU/l) for more than 6months after IFN treatment. Transient responders (TR) were definedas patients with normal ALT at the end of treatment
which thenincreased to 230 IU/l after IFN treatment. Non-responders(NR) hadelevated serum ALT levels despite IFN therapy. Patient demographics
are shown in Table 1.

The follow-up schedule was different for each stage. All patients had
serum chemistry
determinations
every month during the initial 6months, and serum AFP was checked every 3 months in F2, F3, andF4 patients. After IFN therapy, TR and NR patients had blood
chemistries performed
every 1 to 2 months, while SR patients werechecked every 1 to 3 months. Serum HCV RNA was examined at theend of treatment and then 6 and 24 months after IFN therapy. Of the1148 patients, 514 were mainly followed by their practitioners
but 271of the 514 patients had no consultation
at any of the 15 hospitals formore than 1 year during the follow-up period. We sent those patients
letters on July 1996 requiring them to attend for consultation
at oneof the 15 hospitals and to undergo image studying.
In principle, US was performed once a year for FI patients, twice
yearly for F?, and three times a year for F3. F4. and control F4
patients. The 271 patients did not come in for imaging studies during
the follow-up period for early detection of HCC. In principle, CT wasdone once a year for F2 and twice yearly for F3. F4, and control F4.
Dynamic CT and/or magnetic resonance imaging (MRI) were done
when hepatic space-occupying
lesions were found by US. To obtain adiagnosis of HCC, angiography
and/or US-guided tumor biopsy were1

Characteristics

of patientswith chronic

liver diseases

type C at baseline

Stage of liver disease (sex)Age’ (years)

ALT* (IU/l)

PLT count3( X 104!ml)

Follow-up(months)

Fl (n=117) (70 M, 47 F)
F2 (n=636) (413 M, 223 F)
F3 (n=355) (223 M, 132 F)
F4 (n=40) (18 M, 22 F)

42.6213.2
49.5511.2
54.729.1
55.6t8.3

92.3178.5
114.8297.5
129.2292.2
107.1+41.8

21.0+5.2
18.OZZ5.1
14.6~4.2
9.722.5

42.02 17.8
39.52 13.9
41.0+13.2
45.7-t 12.0

Control

57.625.2

118.2138.4

11.112.0

67.1228.3

F4 (n=55)(28 M, 27 F)

Data are expressed as mean?SD.
Tukey’s q-test was used for the statistical analysis of age, ALT, PLT count, and follow-up’ (Fl vs F2, F2 1/s F3; p=O.OOOOl, F3 rs F4, p=O.95160).
* (Fl vs F2; p=O.O7617, F2 rs F3; p=O.O9126, F3 vs F4; p=O.48092).
s (Fl rs F2; p=O.O0030, F2 YSF3; p=O.O0043, F3 vs F4; p=O.90215).
4 No significant differences were noted in their follow-up periods among the 4 groups.

6.54

period.

period+’Interferon, chronic hepatitis C and hepatocellular carcinoma
TABLE

2

Results

Effects of interferon

therapyon chronicof interferon

liver disease type C

Stage of liverdiseaseResponse

therapy

SR

TR

NR

Fl (n=l17)
F2 (n=636)
F3 (n=355)
F4 (n=40)

47 (40.2%)’
194 (30.5%)2
73 (20.6%)3
2 (5.0%)39 (33%)156 (24.5%)
62 (17.5%)
7 (17.5%)

31 (26.5%)
286 (45.0%)
220 (62.0%)
31 (77.5%)

SR: sustained responder, TR: transient responder, NR: non-responder.
’ p=O.O008 (Fl vs F2), * p=O.OOl (F2 vs F3), and 3 p=O.O269 (F3 vsF4) when Wilcoxon’s rank-sum test was used for statistical analysis.

performed
in all patients with suspected HCC by image analysis orwhen significant elevations of serum AFP occurred.
Mean follow-up periods were 42.0t17.8
months in Fl, 39.5213.9
months in F2, 41.0t13.2
months in F3, 45.7212.0
months in F4,
and 67.1228.3
months in control F4 (59.2230.9
months in males,
70.7224.6 months in females) (Table 1). No significant differences inthe duration of follow-up periods were noted among the Fl, F2, F3,
and F4 groups; however, the follow-up period of control F4 was significantly (p=O.O000046) longer than F4.
Statistical methods
SAS software (SAS Institute Inc.) was used for statistical analysis.
Tukey’s q-test was used to evaluate differences in age, serum ALT
level, and PLT counts among the four groups (Fl, F2, F3, and F4)prior to IFN therapy. Differences in the follow-up periods were compared by means of Student’s t-test. Wilcoxon’s rank-sum test was used
to evaluate differences in the efficacy of IFN therapy and the sex
ratios in the four groups. Wilcoxon’s rank-sum test was used to estimate differences in the changes in PLT counts before and 2 years afterIFN therapy in SR and NR. Cox proportional
hazard model wasused to evaluate whether sex, age, stage of liver disease, serum ALT
level, and PLT count are risk factors of hepatocarcinogenesis.
The
cumulative incidences of HCC among total SR, TR, and NR in thefour groups were evaluated using the log-rank test. The cumulative
incidences of HCC among SR, TR, and NR in the four groups andthe control F4 were also evaluated using the log-rank test.

TABLE

3

Changesof platelet

countafter interferon(IFN)

therapy

Response to IFN therapy
The distribution of response in each stage of liver disease is shown in Table 2. The rate of SR was well correlated with the stage of liver disease (Fl vs F2; p=0.0008, F2 vs F3; p=O.OOOl, F3 vs F4; p=O.O269). No
significant differences were noted in the response to
IFN therapy between males and females in any group.
Among the SR in each group of patients, two of the
40 SR in Fl, 12 of the 156 SR in F2, and six of the 61
SR in F3 were positive for serum HCV RNA 6 months
after the cessation of IFN therapy. Of these 20 SR with
hepatitis C viremia, four patients became symptomaticduring the follow-up period: 11, 14, 20, and 24 months
after IFN therapy, respectively.
Platelet counts
PLT counts were significantly (p<O.Ol) lower with theprogression of liver disease (Fl vs F2, F2 vs F3, andF3 vs F4). PLT counts were compared before and 24
months after IFN therapy in all groups (Table 3).
Platelet counts were significantly (p=O.OOOl) increasedin SR in F2 and F3 patients 2 years after IFN therapy.
In NR, PLT counts were significantly decreased in F2(p=O.O171), F3 @=0.0053), and F4 (p=O.O372) 2 years
after IFN therapy compared with their counts before
IFN therapy.
Serum AFP levels before IFN therapy
Of the 1148 patients two cases showed levels of morethan 100 ng/ml of serum AFP at the start of IFN therapy. However, these serum levels of AFP decreased lessthan 50 ng/ml during IFN treatment.

in chronic

liver disease type C

Stage of liverdiseaseEffect of IFN
therapyNo. of cases

Platelet

Before IFN therapy

2 years after IFN therapy

Fl (n=95)

SR
TR
NR

40
30
25

20.525.5
21.225.1
21.624.9

21.325.2
21.925.5
20.525.9

F2 (n=523)

SR
TR
NR

156
125
242

18.4Z5.1
18.624.7
17.425.2

19.8?5.0’
18.224.7
16.9rc_5.02

F3 (n=302)

SR
TR
NR

61
54
187

15.o-c4.3
14.624.5
14.4k4.1

17.754.6’
14.124.8
13.9?4.53

F4 (n=35)

SR
TR
NR

2
6
27

9.9k3.3
10.623.7
9.722.5

13.227.4
10.3*3.1
8.3”2.24

Wilcoxon’s signed-rank
test was used to evaluate
3 p=O.O053, 4 p=O.O372).

the differencein PLT counts

count

before(104/ml)and 2 years after IFN

therapy(’ p=O.OOOl,’ ~~0.017,

65.5

T. Okanoue et al.
TABLE

4

Hepatocellular

carcinomain chronic

hepatitisand liver cirrhosis

Hepatocellular

Male/female
Age (years old)’
No. of HCC in SR/TR/NR
Follow-up period’ (months)
Serum AFP level’ (ng/ml)
Tumor size’ (mm)’ At the time of detection

Incidence

cirrhosis

type C

F3 (n=36)

F4 (n=7)

Total (n=52)

Control

8/l
47-67
O/1/8
13-52
I-137
9-30

1 l/25
51--70 (61.225.6)
3/5/B
14-61 (32.3t2.9)
2-824 (95.8-t 196.5)
8-45 (18.619.2)

413
54-68
O/l/6
1448
13-66
8-43

37115
47-70 (61.2?6.0)
3/l/4213-61 (32.42 12.9)
im-824 (75.4kl66.8)
8-45 (19.429.6)

1311
55-74 (65.624.9)(59.0~8.1)(32.3213.4)(27.5241.9)(20.927.6)
carcinoma(62.414.5)(32.1% 13.0)(28.62 19.6)(19.1?12.7)

F4 (n=20)

26-120 (67.1228.3)
12-398 (122.3~~139.4)
12-37 (25.1 k 10.8)(HCC).

carcinoma

5

Annualand cumulative
incidencesof hepatocellular
carcinoma(HCC) in each stage of chronic liver disease type C after interferon(IFN) therapy
Stage ofliver disease

Effect of
IFN therapy

No. of
HCC

Annual incidenceof HCC (“A>)

Fl (n=ll7)

SR (n=47)
TR (n=39)
NR (n=31)

0
0

0

0

0

SR (n= 194)
TR (n=l56)
NR (n=286)

0

0

1
8

0.2
0.9

F3 (n=355)

SR (n=73)
TR (n=62)
NR (n=220)

3
6
27

1.2
2.8
3.6

0.106
0.726

F4 (n =40)

SR (n=2)
TR (n=7)
NR (n=31)

0
1
6

0
3.7
5.1

0.54790.803122

6.9

F2 (n=636)

and in control

in each stage

HCC was detected in 52 patients after IFN treatment(Table 4); 15 were detected within 24 months, and 37
were detected 24 to 61 months after IFN therapy. Of
the 52 cases, one (0.6%) was detected in the 156 TR ofF2, eight (2.8%) of 286 NR of F2, three (4.1%) of 73
SR of F3, five (8.1%) of 62 TR of F3, and 28 (12.7%)of 220 NR of F3. One (14.3%) of seven TR was F4
and six (19.4%) of 31 NR were F4. No HCC was noted
in Fl. The detection time of HCC in these 3 groups
after IFN treatment was as follows: 22.726.5 months
in SR, 22.Ok7.7 months in TR, and 34.4212.9 months
in NR, respectively. The annual incidence of HCC was
as follows: in F2: O”/;,in SR, 0.2% in TR, and 0.9”A in
NR; in F3: 1.2% in SR, 2.8% in TR, and 3.6% in NR;
and in F4: 0% in SR, 3.7% in TR, and 5.1% in NR(Table 5). The cumulative incidence of HCC was significantly (p=O.O190) lower for all SR patients in F2

TABLE

therapyF2 (n=9)of hepatocellularof hepatocellular

carcinoma

type C after interferoncompared with NR, but not for SR compared with NR
in F3 (Table 5). No significant differences were noted
for TR vs NR in F2 and F3 or SR 1’sNR in F4; however, the cumulative incidence of HCC was significantly
lower in all SR @=O.OOOl) and TR Cp=O.O397) compared with all NR (Fig. 1). In control F4, HCC wasnoted in 22 patients during their follow-up periods andthe annual incidence of HCC was 6.9 %; 6.5% in themale patients <60-year-old and 11.9% in Z60-year-old
patients, respectively. In females, it was 3.8% in <60year-old patients and 7.7% in Z60-year-old patients.
The annual incidence of HCC in patients over 60 years
old was double that in patients ~60 years old; however,no significant difference was noted between sexes. No
significant difference was noted either in the cumulative incidence of HCC between F4 and control F4.
The ages at which patients who received IFN developed HCC ranged from 47 to 68 years (mean?SD:
61.126.0 years), and their mean ages were 59.Ok8.1
years in F2, 61.255.6 years in F3. and 63.324.5 years
in F4. Thirty-eight were male and 14 were female.
Their mean ages were 61.125.9 years (males) andp-value’

0

0.019
0.168

Rank test
p=0.0001
p=o.o397

Follow-up (years)

Control(n=55)

F4

p-value’: statistical analysis of cumulative incidence of HCC between
SR and NR, and TR and NR in each stage of liver disease estimatedby log-rank test.

656

Fig. 1. Cumulative incidence of heputocellular carcinoma inchronic hepatitis and cirrhotic type C patients who received
interferon. HCC was found sign$cantlJ1 less often in SR(p=O.OOOl) and TR (p=O.O397) compared with NR.

Interferon, chronic hepatitis C and hepatocelhdar carcinoma
TABLE

6

Risk factors for the developmentof hepatocellular
carcinoma
after
interferon therapy analyzed by basic multivariate
model of the variables of sex, age, stage of liver disease, serum ALT, and PLT count
Variables

p-value

Conditionalrisk ratio and
95% confidence intervalSex (male vs female)Age (years)
Stage of liver diseaseF2 vs F3F2 vs F4
Serum ALT
PLT count

0.02570.00280.459 (0.231-0.910)
1.064 (1.022-1.109)

0.00670.03730.76550.0815

3.102
3.522
0.999
0.929

Statisticalanalysis

was performed(1.369-7.029)(1.077-11.523)(0.994-1.004)(0.855-1.009)

using the log-rank

test.

61 .1+6.6 years (females). The mean ages of the 3 response groups were as follows: 67.023.0 years in SR,
63.3k4.5 years in TR, and 60.3t6.1 years in NR.
There were no significant differences in the age at
which HCC developed between males and females andamong SR, TR, and NR. The incidence of HCC wassignificantly (p=O.O02) higher in males than females.
Cox proportional hazard analysis of risk factors for
HCC in all patients was performed with the 5 variables
mentioned above. Age and stage of liver disease wereextracted as independent risk factors in the development of HCC (Table 6).
The size of HCC ranged from 8 to 45 mm in diameter (mean&SD: 19.429.6 mm, in Table 4). In control
F4, ages at detection of HCC ranged from 55 to 74
years (mean&SD; 65.624.7 years in all patients,
65.824.3 years in male and 64.925.8 years in female
patients); the size of HCC was 25.1510.8 mm.
Serum AFP levels at detection of HCC ranged from
1 to 824 ng/ml in the 52 HCC patients and 12 to 398
ng/ml (meant SD: 122? 139 ng/ml)in control F4 (Table4). The average serum AFP level of the 52 HCC cases
was 75? 167 (mean?SD) ng/ml; however, only eightcases showed more than 100 ng/ml AFP at detectionof HCC.
During the follow-up period in 1148 patients, threepatients died of cancer (gastric cancer, breast cancer,
and pancreas cancer). One patient died of brain tumor,
one patient died of myocardial infarction and another
patient died as a result of a traffic accident.

Discussion
We analyzed the effects of IFN therapy for CH-C andcirrhosis on the incidence of subsequent HCC. Development of HCC was significantly reduced in both sustained (SR) and transient responders (TR). The present study also demonstrated that PLT counts, reflecting the stage of chronic hepatitis, were significantly
improved in SR after IFN therapy.

The annual incidence of HCC has been reported tobe 1.4% in moderate-staged chronic hepatitis, 3% in
advanced-staged chronic hepatitis, and 5 to 7% in cirrhotic type C patients in Japan (4,10,11) which isaround double that in southern Europe (12,13). In thepresent study, the annual incidence of HCC was 0.9%
in non-responders (NR) in F2, 3.6% in NR in F3, and5.1% in NR in F4. Thus, the stage of liver disease is
an important factor in the development of HCC inchronic liver disease type C. The present data also demonstrated that the age of the patients was an importantfactor in the development of HCC in CH-C patients.
However, sex was not an independent risk factor in thepresent study. This might be due to the larger numberof female than male F4 patients.
The causal mechanisms for development of HCC are
unknown in patients with hepatitis C virus (HCV) infection. Recent experimental study in transgenic mouseusing HCV core protein demonstrated that HCV itself
was directly involved in the development of HCC (14),but HCC is mainly detected in patients with advanced
stages of chronic hepatitis or cirrhosis and rarely seenin asymptomatic HCV carriers. Tarao et al. (15,16) reported that hepatocellular proliferation is a risk factor
for the development of HCC. Maintenance of serumtransaminases at low levels may protect against the development of HCC. Hepatocyte necrosis, cell damage,and increases in hepatocyte replication result in increased DNA damage, influencing hepatocarcinogenesis. Thus, development of HCC may be delayed or
prevented in SR and TR.
Another possible mechanism for prevention of HCC
is the direct or indirect effects of IFN. IFN has many
immunologic and biologic actions, such as inhibitionof cell division via transcription factor, interferon regulatory factor (IRF) (17-19), growth inhibitory actions
through change in signal transduction (17,20,21), andactivation of natural killer cells (22) and T-cell activity(23), resulting in tumor suppression (23-25). Lai et al.
(25) reported that IFN treatment of patients with advanced HCC improved survival rates. Thus, it is possible that direct effects of IFN are responsible for the
low incidence of HCC in TR and NR.
HCC was mainly detected in NR with advancedstage chronic hepatitis or cirrhosis. These present results agree with the report by Kasahara et al. (26), whodemonstrated that the risk factors for HCC and for
its incidence after IFN therapy in CH-C patients were
present in NR, older patients and males. They also reported that the degree of fibrosis was not a significant
risk factor for the development of HCC; however, in
our present study the stage of liver disease was an independent risk factor for the development of HCC.

657

T. Okanoue et al.

The cumulative incidence of HCC was significantly
lower in SR than in NR in F2, but was not significantly
lower for TR vs NR in F2 and SR vs NR in F3 patientsin the present study. PLT counts appear to reflect thestage of chronic hepatitis and the increase in PLT
counts in SR of F2 and F3, and suggests improvementin the stage of liver disease after IFN therapy.
The growth rate (doubling time) of HCC is variable(26-30), ranging from 1 to 22 months (average 4 to
6 months). Sheu et al. (27) reported that the median
detectable subclinical period for HCC was 3.2 years.
Nevertheless,well-differentiatedand hypovascular
HCC have been reported to have a significantly longer
doubling time than hypervascular HCC (30). Our
study demonstrated that the doubling time of HCC
which were 2 to 5 cm in diameter was around 3 months(unpublished data). It may take more than 6 years until
HCC of diameter around 1 cm are detected by US or
CT, when the doubling time of HCC is assumed to be
90 days. The volume of one HCC cell is described by
the following equation, when we assume that the volume of an HCC cell is equal to that of a normal hepatocyte. The average diameter of a normal hepatocyte
is considered to be 35 pm. Thus, the volume (Vl) of
one hepatocyte is estimated as follows:

The volume of HCC 1 cm in diameter is expressed asfollows:
314X3.14 (5000)“+2.9~10”pm’.

The next equation can be used when we assume thatone HCC cell might grow logarithmically to the volume of HCC which are 1 cm in diameter. The doublingtime (DT)=(t2-t,)In2/lnVz-lnV,
Vl= 1.26X lo4&m3), V2=2.94~ 10” @m3), DT=90 days.
Using these data in the above equation, we can thenget the following result:t2-t, =2208 days (around 6.0 years).
In the process of the development of HCC, many factors may affect the growth of HCC, including
apoptosis and other immunological effects, possibly resulting in slower growth of HCC. Therefore, it isreasonable to consider that most HCC cases detectedin the present follow-up periods had developed prior
to interferon therapy, and that the growth rate of HCC
was delayed by IFN in sensitive cases.
Nishiguchi et al. (31) reported that IFN reduced thedevelopment of HCC in HCV-infected cirrhotic patients, but, some questions were raised about this result(32,33). This study (3 1) included very small numbers ofsubjects and noted a marked reduction in HCC, even
6.58

though SR comprised only 16% of the subjects who
received IFN. This SR rate is similar to that in F3stages in our study. A European group (34) reported
that the response to interferon-alpha with biochemical
resolution for cirrhosis type C was 9”/;, and that IFN
therapy did not significantly diminish the rate of thedevelopment of HCC compared with cirrhotic patients
without IFN therapy, supporting our present data.
However, a recent retrospective cohort study demonstrated that JFN-alpha lowered the rate of progressionof cirrhosis to HCC (35).
In the present study, there were only 40 cirrhotic patients (F4), and two cases (5.0%) were SR. The annual
incidence of HCC was 5.1% NR in F4, which was influenced by the patients’ age at entry, as demonstrated
in this study. Furthermore, no significant differenceswere noted in the cumulative incidence of HCC among
SR, TR, and NR patients with advanced liver disease(F3 and F4). The discrepancy between our results andthose of the international study group (35) might bedue to the difference in patients’ backgrounds, because
the annual rate of development of HCC in cirrhotic
type C patients in Japan was double that in European
countries. Especially, in Japanese cirrhotic patients ahigh frequency of undetectable-sized HCC had alreadydeveloped before IFN treatment, resulting in the failure of inhibition of HCC by IFN therapy. Further
studies are needed to define the effects of IFN on thedevelopment of HCC in advanced-stage hepatitis C patients.
In conclusion, IFN therapy for chronic hepatitis C
patients resulted in 27.5% sustained responders, 23.0%
transient responders, and 49.5%) non-responders. Development of HCC was significantly reduced or delayed in both sustained and transient responders with
CH-C, but not significantly in advanced-stage patients.

Appendix
In addition to the study authors, the investigators inthe Viral Hepatitis Therapy Study Group included: M.
Miyoshi, Department of Internal Medicine. Kyoto
First Red Cross Hospital; S. Takamori, Second Department of Internal Medicine, Matsushita Memorial
Hospital; T. Ogasawara, and T. Nakajima, Center ofDigestive Disease, Ohtsu Municipal Hospital; K. Kagawa, Department of Internal Medicine, Fukuchiyama
City Hospital; Y. Katsuma, Department of Internal
Medicine, Kyoto Municipal Hospital; M. Takeda, Department of Internal Medicine, Rakuwakai-Marutamachi Hospital; Y. Nakagawa and H. Tada, Department
of Digestive Disease, Hoshigaoka-Kohseinenkin
Hospital; M. Ohta, Department of Internal Medicine, Saiseikai Kyotofu Hospital: Y. Sawa, Department ofInterferon,Internal Medicine, Aiseikai Yamashina Hospital; H.
Kanaoka and T Takeuchi, Department of Internal
Medicine, Notogawa City Hospital; M. Matsumoto,Internal Medicine. Ayabe Municipal Hospital; M. Mizuno, Internal Medicine, Yodogawa Christian Hospital; Takami, Department of Internal Medicine, National Sabae Hospital.

Acknowledgements
This study was supported in part by a grant no.
10470139 from the Ministry of Education, Japan. The
authors express their thanks Dr. Hiroaki Asano, Department of Computer Science, College of Medical
Technology, Kyoto Prefectural University of Medicine,for his advice on the statistical analysis of this study.

References
1. The Liver Cancer Study Group of Japan. Primary liver cancer inJapan: clinicopathologicalfeatures and results of surgical treatment. Ann Surg 1990; 211: 277-87.
2. Okuda K. Epidemiology
and clinical aspects of HCC. J Gastroenter01 Hepatol 1993; 8: S14.
3. Takano S, Yokosuka 0, Imazeki F, Tagawa M, Omata M. Incidence of hepatocellular
carcinomain chronic hepatitis B and C:
a prospective study of 251 patients. Hepatology
1995; 21: 650-5.
4. Ikeda K, Saitoh S, Koida I, Arase Y, Tsubota A, Chayama K,
et al. A multivariate
analysis of risk factors for hepatocellular
carcinogenesis:a prospective
observationof 795 patients withviral and alcoholic cirrhosis. Hepatology
1993; 18: 47-53.
5. Yoshioka K, Kakumu S, Wakita T, Ishikawa T, Itoh Y, Takayanagi M, et al. Detection of hepatitis C virus by polymerase
chain
reaction and response to interferon-therapy:
relationship to genotypes of hepatitis C virus. Hepatology
1992; 16: 293-9.
6. Tsubota A, Chayama K, Ikeda K, Arase Y, Koida I, Saitoh S, etal. Factors predictive of response to interferon-atherapy in hepatitis C virus infection. Hepatology
1994; 19: 1088-94.
7. Okanoue T, Yasui K, Sakamoto S, Minami M, Nagao Y, Itoh Y,
Kagawa K, et al. Circulating
HCV RNA, HCV genotype, andliver histology in asymptomatic
individuals reactive for anti-HCV
antibody and their follow-up study. Liver 1996; 16: 241-7.
8. Scheuer PJ. J. Classificationof chronic hepatitis: a need for reassessment. J Hepatol 1991; 13: 3724.
9. Desmet VJ, Gerber M, Hoofnagle
JH, Manns M, Scheuer PJ.
Classificationof chronic hepatitis: diagnosis,
grading and staging. H1994; 19: 1513-20.
10. Takano S, Yokosuka 0, Imazaki F, Tagawa M, Omata M. Incidence of hepatocellular
carcinoma in chronic hepatitis B and C:
a prospective study of 251 patients. Hepatology
1995; 21: 650-5.
11. Kato Y, Nakata
K, Omagari
K, Furukawa
R, Kusumoto
Y,
Mori I, et al. Risk of hepatocellular
carcinomain patients withcirrhosis in Japan. Cancer 1994; 74: 22348.
12. Colombo M, de Franchis R, Ninno ED, Sangiovanni
A, De Fasio C, Tommasini M, Donato AS, et al. Hepat&ellular
carcinomain patients with cirrhosis. N Engl J Med 1991; 325: 675-80.
13. Sheu JC, Sung JL, Chen DS, Lai MY, Wang TH, Yu JY, et al.
Early detection of hepatocellular
carcinoma
by real-time ultrasonography.
a prospective study. Cancer 1985; 56: 660-6.
14. Moriya K, Fujie H, Shintani Y, Yotsuyanagi H, Tsutsumi T, Ishibashi K, et al. The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat Med 1998; 4: 1065-7.
15. Tarao K, Ohkawa S, Shimizu A, Harada M, Nakamura
Y, ItohY, et al. Significance of hepatocellular
proliferationin the development of hepatocellular
carcinoma from anti-hepatitis
C viruspositive cirrhotic patients. Cancer 1994; 73: 1149-54.

chronic hepatitis C and hepatocellular

carcinoma

16. Tarao K, Ohkawa S, Tamai S, Miyakawa K. Sustained low serum
GPT level below 80 INU in HCV-associatedcirrhotic patients bymultiagent therapy prevents development of hepatocellular
carcinoma [abstract]. HEPACOM
1995; Suppl 3: S162.
17. Harada H, Kitagawa M, Tanaka N, Yanmamoto
H, Harada K,
Ishihara M. Anti-oncogenic
and oncogenic potentials
of interferon regulatory factors-l
and-2. Science 1993; 259: 9714.
18. Tanaka N, Ishihara M, Kitagawa M, Harada H, Kimura T, Matsuyama T, et al. Cellular commitment
to oncogene-induced
transformation
or apoptosis is dependent on the transcription
factor
IRF-1. Cell 1994; 77: 829-39.
19. Tanaka N, Ishihara M, Lamphier MS, Nozawa H, Matsuyama
T, Mak TW, et al. Cooperationof the tumor suppressors
IRF-1
and p53 in response to DNA damage. Nature 1996; 381: 8168.
20. Diaz MO, Rubin CM, Harden A, Ziemin S, Larson RA, Le Beau
MM, et al. Detection of interferon genes in acute lymphoblastic
leukemia. N Engl J Med 1990; 322: 77-82.
21. Hannigan GE, Williams BRG. Signal transduction
by interferonalpha through arachidonic
acid metabolism.
Science 1990; 251:2047.
22. SwaminathanN,
Lai CM, Beilharz MW, Klinken SP Biological activities of recombinantmurine interferon alpha 1 and 4: large difference in antiproliferative
effect. Antivir Res 1992; 19: 149-59.
23. Chen LK, Tourvielle B, Burns G, Bach FH, Mathieu-Mahul
D,
Seaportes M, et al. Interferon: a cytotoxic T lymphocyte differentiation signal. Eur J Immunol 1986; 16: 767-70.
24. Tabata Y, Uno K, Yamaoka T, Ikeda Y, Muramatsu
S. Effects
of recombinant- interferon-gelatin
conjugate on in viva murine
tumor cell growth. Cancer Res 1991; 51: 5532-8.
25. Lai C-L, Lau JYN, Wu P-C, Ngan H, Chung H-T, Mitchell J, etal. Recombinantinterferon-ain inoperable hepatocellular
carcinoma: a randomized
controlled trial. Hepatology
1994; 17: 38994.
26. Kasahara
A, Hayashi
N, Mochizuku
K, Takayanagi
M,Yoshioka K, Kakumu
S, et al. Risk factors for hepatocellularcercinoma and its incidence after interferon treatment in patientswith chronic hepatitis C. Hepatology
1998; 27: 1394402.
27. Sheu J-C, Sung J-L,, Chen D-C, Yang P-M, Lai M-Y, Lee C-S, etal. Growth rate of asymptomatic
hepatocellular
carcinomaandits clinical implication.
Gastroenterology
1985; 89: 259966.
28. Ebara M, Ohto M, Shinagawa T, Sugiura N, Kimura K, Matsutani S, et al. Natural history of minute hepatocellular
carcinoma
smaller than three centimeterscomplicating
cirrhosis.
Gastroenterology 1986; 90: 289-98.
29. Barbara L, Benzi G, Gaiani S, Fusconi F, Zironi G, Siring0 S, etal. Natural history of small untreated hepatocellular
carcinoma in
cirrhosis: a multivariate
analysis of prognostic
factors of tumor
growth rate and patient survival. Hepatology
1992; 16: 132-7.
30. Saitoh S, Ikeda K, Koida I, Tsubota A, Arase Y, Chayama K, etal. Serial hemodynamic
measurement
in well-differentiatedhepatocellular carcinomas.
Hepatology
1995; 21: 15304.
31. Nishiauchi
S, Kuroki T, Nakatani
S, Morimoto
H, Takeda T,
Nakaima
S, et al. Randomised
trial of effects of interferon-cc on
incidence of hepatocellular
carcinoma in chronic active hepatitis
with cirrhosis. Lancet 1995; 346: 1051-5.
32. Harper SE, Dienstag JL. Can interferon alpha treatment prevent
hepatocellular
carcinoma in patients with chronic hepatitis C infection and compensatedcirrhosis? Hepatology
1996; 23: 93&3.
car33. Grimm I, Shaheen N. Can interferon prevent hepatocellular
cinoma in hepatitis C virus-induced
cirrhosis? Gastroenterology
1996; 110: 2019-21.
34. Fattovichi G, Giustina G, Degos F, Tremolada F, Diodati G, Almasio P, et al. Morbidity and mortality in compensatedcirrhosis
type C: a retrospective
follow-up study of 384 patients. Gastroenterology 1997; 112: 463-72
Interferon-a
Hepatocellular
Carcinoma
Study
35. International
Group. Effect of interferon-aon progression of cirrhosis to hepatocellular carcinoma:a retrospective
cohort study. Lancet 1998;
351: 1535-9.

659