Iwao Yabuuchi

Relation of Interferon Therapy and Hepatocellular Carcinoma in Patients with Chronic Hepatitis C

Hepatocellular carcinoma, a major cause of death in patients with cirrhosis, is one of the most prevalent malignant tumors worldwide, and its incidence is increasing [1-5]. After isolation of hepatitis C virus (HCV), most patients with chronic hepatitis and those with cirrhosis of unknown origin were found to be positive for anti-HCV [6-8]. Evidence suggests that HCV-related chronic liver disease plays a role in the development of hepatocellular carcinoma [9-13]. A high proportion of patients with hepatocellular carcinoma have anti-HCV, although the prevalence varies geographically. The highest rate of anti-HCV is in southern Europe and Japan, where about 70% of patients with hepatocellular carcinoma are positive for anti-HCV [5]. Interferon has been widely used to treat chronic HCV infection. A series of clinical trials showed that some patients who received interferon had sustained normalization of serum aminotransferase levels and elimination of serum HCV RNA [14-17]. Histologic improvement was also seen in patients who received interferon [14, 18-20]. It is important to determine whether interferon treatment also lowers the incidence of hepatocellular carcinoma in patients with chronic hepatitis C, but the recognized benefits of interferon make a randomized, controlled trial to address this question unethical. We did a retrospective study to compare the incidence of hepatocellular carcinoma in interferon-treated patients with HCV infection and histologically proven chronic hepatitis or cirrhosis with that in historical controls who did not receive interferon. We also examined the relation between response to interferon therapy and incidence of hepatocellular carcinoma. Methods Patients The interferon group comprised 419 consecutive patients with chronic hepatitis C who had undergone liver biopsy 1 to 2 weeks before interferon therapy and had started treatment between January 1992 and December 1993. The control group consisted of 144 consecutive patients with chronic hepatitis or cirrhosis who had undergone liver biopsy between January 1986 and December 1989. All patients had histologically proven chronic hepatitis or cirrhosis (Child-Pugh class A) and were positive for anti-HCV. Interferon Treatment In the interferon group, 176 patients received human lymphoblastoid interferon, 149 received recombinant interferon- 2a, and 94 received recombinant interferon- 2b for 6 months. The median total interferon dose was 480 mU (range, 282 to 800 mU). No patient had received interferon therapy before study entry. Contraindications to interferon treatment included pregnancy, presence of hepatitis B surface antigen, other types of liver disease, autoimmune disease, and any other serious illness. Efficacy of interferon therapy was categorized as follows. Patients with persistent normalization of alanine aminotransferase (ALT) levels during interferon therapy and follow-up were considered to have sustained response. Patients whose serum ALT level was normal at the end of the treatment but increased to an abnormal level after cessation of treatment were considered to have relapse. All other patients were classified as nonresponders. Follow-up Abdominal ultrasonography or computed tomography was performed every 4 to 8 months, and serum -fetoprotein was measured every 2 to 6 months. The diagnosis of hepatocellular carcinoma was confirmed by needle biopsy, by surgically resected tumor specimens, or by typical radiologic findings on hepatic angiography. The starting date of follow-up for patients in the interferon and control groups was defined as the date of liver biopsy. For both groups, the end of follow-up was the development of hepatocellular carcinoma or December 1991 in the control group and the time of the latest abdominal imaging in the interferon group. To detect hepatocellular carcinoma, follow-up examinations were done in 85.4% of controls and 90.7% of patients in the interferon group. The Osaka Cancer Registry was used [21, 22] to determine whether hepatocellular carcinoma had occurred in patients lost to follow-up. This population-based cancer registry has been operating since December 1962 with the cooperation of the Osaka Medical Association, the Department of Health of Osaka Prefecture, and Osaka Medical Center for Cancer and Cardiovascular Diseases. It covers all of Osaka Prefecture, which had a population of 8.6 million in 1995, and registers cases of cancer by using reports from hospitals and clinics and death certificates collected from health centers. One patient in each group who had been lost to follow-up was listed as having hepatocellular carcinoma in the Osaka Cancer Registry. Determination of the Presence of Hepatitis C Virus Antibody and Hepatitis C Virus RNA Hepatitis C virus antibody was measured by first-, second-, or third-generation enzyme-linked immunosorbent assays (Ortho Diagnostics, Tokyo, Japan). Serum HCV RNA was measured by reverse transcription polymerase chain reaction or complementary DNA assay, as reported elsewhere [23, 24]. Assessment of Liver Histologic Findings The histologic findings in liver biopsy specimens were scored by three of the authors in a blinded manner by using two scoring methods. For assessment of histologic staging, fibrosis score (F1 to F3 for chronic hepatitis and F4 for cirrhosis) was used; F1 indicated portal fibrous expansion, F2 indicated portal-portal septa without architectural distortion, F3 indicated portocentral septa with architectural distortion, and F4 indicated cirrhosis [25]. For assessment of histologic grading, a total score of histologic activity (components 1 to 3) of the Knodell histologic activity index was used [26]. Statistical Analysis Patients who did not complete the treatment protocol were included for analysis on an intention-to-treat basis. The chi-square test was used to compare the baseline characteristics of both groups. The Wilcoxon rank-sum test was used to assess a significant difference between tumor sizes in the two groups. The Kaplan-Meier method was used to calculate the cumulative incidence of hepatocellular carcinoma, and the log-rank test was used to compare the cumulative incidence of hepatocellular carcinoma between the groups. To estimate independent risk factors for the development of hepatocellular carcinoma, Cox proportional-hazards regression analysis was used. For analysis, interferon therapy, age, sex, serum ALT level, serum -fetoprotein level, platelet count, histologic staging, and activity scores were used as variables. A P value less than 0.05 was considered statistically significant. Data are expressed as medians and ranges and as risk ratios and 95% CIs. Results Table 1 shows the baseline characteristics of the interferon and control groups. The groups did not differ for age, sex, serum ALT level, or platelet count. In the interferon group, 387 patients (92%) had chronic hepatitis (128 had F1 disease, 138 had F2 disease, and 121 had F3 disease) and 32 (8%) had cirrhosis. In the control group, 124 patients (86%) had chronic hepatitis (30 had F1 disease, 38 had F2 disease, and 56 had F3 disease) and 20 (14%) had cirrhosis (P = 0.005). The proportion of patients with serum -fetoprotein levels greater than 20 ng/mL was higher in the control group (24%) than in the interferon group (15%) (P = 0.011). Table 1. Baseline Characteristics of Interferon-Treated Patients and Historical Controls with Chronic Hepatitis C In the interferon group, 151 patients (36%) had sustained response, 120 (29%) had relapse, and 148 (35%) were nonresponders. In the 143 patients with sustained response, serum HCV RNA was measured during follow-up. Sustained absence of serum HCV RNA was noted in 120 (84%) of these patients. Twenty-one patients could not complete the 6-month treatment protocol because of depression (5 patients), severe general fatigue (4 patients), skin eruptions (2 patients), severe reduction of serum platelet count (1 patient), pulmonary tuberculosis (1 patient), interstitial pneumonia (1 patient), severe nausea (1 patient), ischemic colitis (1 patient), cardiomyopathy (1 patient), hyperthyroidism (1 patient), and hypermenorrhea (1 patient). One patient stopped treatment because of his business, and one patient discontinued treatment after 3 months because hepatocellular carcinoma was diagnosed. Only 1 of the 21 patients who did not complete treatment showed sustained response; all others were nonresponders. Median follow-up was 47.6 months (range, 3.3 to 65.2 months) in the interferon group and 46.8 months (range, 6.9 to 71.6 months) in the control group. During follow-up, hepatocellular carcinoma was found in 19 controls (4 with F2 disease, 8 with F3 disease, and 7 with F4 disease). In the interferon group, 28 patients developed hepatocellular carcinoma during follow-up (2 patients with F1 disease, 5 with F2 disease, 13 with F3 disease, and 8 with F4 disease). A final diagnosis of hepatocellular carcinoma was made histologically in 17 patients in the interferon group (61%) and 11 controls (58%). In 11 patients (39%) in the interferon group and 8 controls (42%), a final diagnosis was made on the basis of typical angiographic findings. The maximum tumor sizes of hepatocellular carcinoma in the interferon and control groups at the time of discovery on ultrasonography or computed tomography were 20 mm (range, 10 to 52 mm) and 24 mm (range, 10 to 50 mm), respectively (P > 0.2). Figure 1 shows the cumulative incidence of hepatocellular carcinoma in the interferon and control groups, estimated by using the Kaplan-Meier method. The 4-year rate of hepatocellular carcinoma incidence was 6.6% in the interferon group and 12.2% in the control group (log-rank test, P = 0.040). Figure 1. Cumulative incidence of hepatocellular carcinoma (HCC) in interferon-treated patients (dotted line) and historical controls (solid line) with chronic hepatitis C. P Cox proportional-hazards regression analysis was performed to identify factors co

Declining Incidence of Hepatocellular Carcinoma in Osaka, Japan, from 1990 to 2003

Context Hepatitis C virus (HCV) infection in Japan began to spread during the 1920s, increased after World War II with an explosion in parenteral amphetamine use and paid blood donation, and decreased in the 1950s to 1960s with voluntary blood donation and penalties against amphetamine use. Evidence linking the trends in HCV infection to hepatocellular carcinoma rates in Japan is limited. Contribution Data from the Osaka Cancer Registry and 10 Osaka hospitals suggest that hepatocellular carcinoma rates began to decrease in 2000, mainly because of a decrease in HCV-associated cancer. Implication Control of HCV transmission within a population seems to be followed by a decrease in hepatocellular carcinoma. The Editors Primary liver cancer was the fifth most common cancer worldwide by 2000, with approximately 551000 new cases recorded (1). In most countries, hepatocellular carcinoma (HCC) comprises 85% to 90% of primary liver cancer cases. With some exceptions, developed countries, including the United States, have been experiencing an increase in the incidence of primary liver cancer, considered to be due at least in part to increased prevalence of chronic hepatitis C virus (HCV) infection (2). Japan has had one of the highest incidence rates of primary liver cancer among developed countries (age-standardized incidence rate in 1995, 25.5 per 100000 men and 7.7 per 100000 women) (3). Approximately 90% of liver cancer cases are HCC, which, in Japan, is mainly caused by chronic HCV infection rather than chronic hepatitis B virus infection (4). A recent report on the age-standardized incidence of primary liver cancer among Japanese men, which was calculated from 6 population-based cancer registries, showed a sharp increase that started in the mid-1970s but leveled off in the mid-1990s (5). These distinctive trends were thought to be due to the spread of HCV infection, which began in the 1920s and increased after World War II (68). Thus, HCV penetrated Japan earlier than Spain, Egypt, the United States, the former Soviet Union, South Africa, and Hong Kong, as evidenced by molecular clock analysis of the sequences of HCV isolates (8). However, recent temporal trends regarding incidence rates of HCC and the contribution of HCV infection have not been clearly documented in the Japanese population. We analyzed temporal trends for HCC incidence rates between 1981 and 2003 in Osaka Prefecture (population in 2005, 8.8 million) and interpreted these in the context of HCV infection rates. Methods Data Collection on Incident HCC Cases We obtained data on incident HCC cases from the Osaka Cancer Registry, which was established by the Osaka Prefectural Government in 1962. The registry collects reports on patients with newly diagnosed cancer, including demographic and cancer-related information, from all medical institutions in Osaka Prefecture (9). These have been routinely supplemented by death certificates gathered by the Osaka Prefectural Government (9). For patients with cancer who were enrolled in the registry on the basis of their death certificate, we contacted the issuing hospital to obtain information on diagnosis and treatment and to establish the date of HCC incidence, which we determined to be the time of diagnosis at that hospital. We site-coded the data according to the International Classification of Diseases for Oncology, Third Edition (10). We included patients with HCC (codes 8170 through 8180). The protocol was approved by the ethics committee of the Osaka Medical Center for Cancer and Cardiovascular Diseases. From 1981 to 2003, 48166 men and 15696 women with HCC were documented in the Osaka Cancer Registry. We calculated the annual age-standardized incidence rates of HCC (world population as a standard population) by sex between 1981 and 2003. To characterize temporal trends for HCC, we assessed 10-year, age-specific incidence rates of HCC between 1981 and 2003 in individuals age 50 to 79 years. We studied these particular age-specific rates because most HCV-related HCC cases in the Japanese population occur between the ages of 50 and 79 years (4). We used the annual population estimates from 1981 to 2003, which were based on the average population in each sex and age category for the Osaka Prefecture during the particular period, as denominators for calculating incidence rates. The annual population estimates were based on data from the 1980, 1985, 1990, 1995, 2000, and 2005 Japanese population censuses, with linear interpolation for the years in between. Statistical Analysis To identify years when a statistically significant change in the slope of the temporal trend in the incidence occurred, we applied the joinpoint regression model by using the Joinpoint Regression Program, version 3.0 (U.S. National Cancer Institute, Bethesda, Maryland). We assumed constant variance and uncorrelated errors (11) because we could not detect heteroskedasticity by the White test or autocorrelation by the Durbin-Watson test in men or women in any age group. We computed the estimated slopes describing the average annual change of incidence rate per 100000 persons and the corresponding 95% CIs for each trend by fitting a piecewise regression line to the rates, using calendar year as a regression variable. We used the permutation test method to identify years when a statistically significant change had occurred (P< 0.05) and set the number of randomly permuted data sets at 4499. We set the number of joinpoints to a minimum of 0 and a maximum of 3 in the Joinpoint Regression Program. Data Collection on Prevalence of HCV Infection among Patients with HCC The Osaka Cancer Registry does not collect serologic data on HCV infection in the registered patients. Therefore, we used data on HCV seropositivity from patients with HCC that was diagnosed at 10 hospitals in Osaka Prefecture (1 university hospital, 2 cancer centers, and 7 general hospitals) to estimate the prevalence of HCV infection in patients with HCC. We considered the HCC diagnosis confirmed when the patient had positive histologic or positive radiologic results by enhanced computed tomography or hepatic angiography. We collected data on the patients sex, date of birth, date of diagnosis between 1990 and 2003, first Chinese letter of the family name, and presence of hepatitis B surface antigen and antibody to hepatitis C (anti-HCV) as assessed by any commercially available kit. We did not collect the full first and family name for reasons of confidentiality. Because anti-HCV testing first became available in Japan in 1990, we collected data on patients whose HCC diagnosis was between 1990 and 2003. One investigator checked for duplication of the data set, because some patients might have been registered multiple times among the participating hospitals as a result of referrals and recurrence of HCC. We defined HCV-related HCC as occurring in patients who were HCV-seropositive at the time of diagnosis. We calculated the sex-specific, age-specific (50 to 59, 60 to 69, or 70 to 79 years), and period-specific (1990 to 1992, 1993 to 1995, 1996 to 1998, 1999 to 2001, or 2002 to 2003) prevalences of HCV seropositivity for patients with HCC. We then multiplied prevalence rates by the corresponding strata of the HCC incidence rate obtained from the Osaka Cancer Registry data. Thus, we derived the denominators from the general population in Osaka through the denominators of the HCC incidence rate and obtained the numerators by multiplying the prevalence rates by the HCC incidence rate. We calculated the incidence rate of nonHCV-related HCC by subtracting HCV-related HCC from total HCC. Thus, we describe trends for the estimated incidence rates of HCV-related and nonHCV-related HCC between 1990 and 2003 in Osaka Prefecture. We calculated the CI of the estimated rates by multiplying the lower and upper limits of the CI of the prevalence based on SE by the corresponding HCC incidence rate. Role of the Funding Source This study was supported by the Osaka Prefectural Government between 1990 and 2000 and Grants-in-Aid for Hepatitis Research of the Japanese Ministry of Health, Labor, and Welfare. There is no conflict of interest in the study. The funding sources had no role in the collection, management, or analysis of data. Results The age-standardized incidence rate of HCC in men increased between 1981 and 1987 from 29.2 to 41.9 cases per 100000 persons, then fluctuated until 1995. After that, it steadily decreased to 24.0 cases per 100000 persons in 2003 (Figure 1). Among women, the age-standardized incidence rate of HCC increased between 1981 and 1996 from 6.6 to 10.8 cases per 100000 persons, then gradually decreased to 7.3 cases per 100000 persons in 2003 (Figure 1). Figure 1. Trends in age-standardized (world population) incidence of hepatocellular carcinoma in Osaka, Japan, 19812003. Figure 2 shows the trends in the incidence of HCC among men and women age 50 to 59 years, 60 to 69 years, and 70 to 79 years in Osaka between 1981 and 2003. The HCC incidence rate increased from 1981 to 1986 among men age 50 to 59 years, from 1981 to 1995 among men age 60 to 69 years, and from 1981 to 2000 among men age 70 to 79 years (average annual change of the incidence rate [per 100000 persons], 10.0, 10.7, and 6.2, respectively) (Table 1). A striking downward trend occurred after the year of peak incidence in the 3 age groups (7.9 until 1996, 22.3 until 2003, and 12.4 until 2003, respectively). Among men age 50 to 59 years, there was a second joinpoint (a change from rapid to moderate decrease) in 1996, resulting in a slope of 3.1 until 2003. Among women age 50 to 59 years, 60 to 69 years, and 70 to 79 years, the incidence rates of HCC peaked in 1991, 1997, and 2000, respectively (Table 1). The rates in women seemed to increase slightly from 1981 until the year of the joinpoint, with slopes of 0.43, 2.07, and 3.10, respectively. Thereafter, HCC incidence rates in women decreased through 2003 at a

Frequent impairment of the spindle assembly checkpoint in hepatocellular carcinoma

Chromosomal instability (CI) leading to aneuploidy is one form of genetic instability, a characteristic feature of various types of cancers. Recent work has suggested that CI can be induced by a spindle assembly checkpoint defect. The aim of the current study was to determine the frequency of a defect of the checkpoint in hepatocellular carcinoma (HCC) and to establish whether alterations of genes encoding the checkpoint were associated with CI in HCC.

Lack of frameshift mutations at coding mononucleotide repeats in hepatocellular carcinoma in Japanese patients.

Microsatellite instability occurs frequently in hereditary nonpolyposis colorectal carcinoma, in sporadic gastrointestinal carcinoma, and in other tumors. In these tumors, slippage‐related frameshift mutations have been detected at coding mononucleotide repeats in genes such as those for transforming growth factor‐β receptor type II (TGFβRII), mannose 6‐phosphate/insulinlike growth factor II receptor (M6P/IGFIIR), hMSH3, hMSH6, and Bcl‐2–associated X protein (BAX). Because these genes regulate cell growth or repair DNA mismatches, loss of their function is thought to promote tumor development. The authors screened for these frameshift mutations and investigated the incidence of microsatellite instability (MI) in hepatocellular carcinoma (HCC) in Japan.

Classification of ultrasonographic images of small hepatocellular carcinoma using galactose-based contrast agent: relation between image patterns and histologic features

PurposeThe aim of this study was to establish the relation between observed ultrasonographic (US) images produced with a galactose-based contrast agent and histologic characteristics of small hepatocellular carcinomas (HCCs).Materials and methodsA total of 64 nodules in 64 patients, 22 well differentiated and 42 moderately differentiated with a histologically proven HCC, smaller than 3.0 cm in diameter and who had undergone hepatectomy were consecutively examined by contrast-enhanced US using a galactose-based contrast agent. Perfusion images were acquired by intermittent high-intensity, harmonic power Doppler sonography using a high pulse-repetition frequency and high-pass filter setting. Perfusion images of the arterial and late phases were classified into several patterns and compared with the histologic findings obtained from resected specimens.ResultsMost of the well- and moderately differentiated resected HCCs showed hyperechoic change during the arterial phase. However, 13 (59%) of the well-differentiated HCCs showed isoechoic change and 27 (64%) of the moderately differentiated HCCs showed hypoechoic change during the late phase. The difference is statistically significant (P < 0.0001). In a comparison of microscopic portal invasion (vp) of HCCs using enhanced US patterns, both vp(−) and vp(+) groups showed a high incidence of the hypervascular pattern during the arterial phase; in contrast, during the late phase 11 (73%) of 15 vp(+) nodules showed hypoechoic change with spotty signals. This difference is statistically significant (P < 0.0001) when compared with a high incidence (52%) of signal defect in the vp(−) group. The existence of well-differentiated components associated with the periphery of moderately differentiated HCCs also correlated closely with patterns during the late phase (P < 0.01).ConclusionsLate-phase contrast-enhanced US images of small HCCs with a galactose-based contrast agent are useful for predicting specific histologic characteristics.

Aromatase activity in human hepatocellular carcinoma. Relationship with the degree of histologic differentiation.

Human hepatocellular carcinomas (HCC) were examined aromatase activity, an enzyme that converts androgen into estrogen. Such activity was detected in all 13 specimens of HCC (mean activity, 120 fmol/30 min/mg microsomal protein). The activity tended to be lower in the HCC tissue than in the surrounding liver tissue (mean activity, 230 fmol/30 min/mg microsomal protein), although it was higher in the HCC tissue from three of eight patients with Edmondsons Grade 2 disease. This relationship was not found in the five with Grade 3 disease. On the whole, aromatase activity was significantly higher in specimens from patients with Edmondsons Grade 2 tumors than in the less differentiated Grade 3 type (P < 0.05). These observations suggested that aromatase activity was present in human HCC and was related to the degree of histologic differentiation. Cancer 1993; 71:56‐61.

Changes in serum hepatic fibrosis markers in biochemical responders to interferon therapy for chronic hepatitis C.

Serum hepatic fibrosis markers (7s domain of type IV collagen, N-terminal peptide of type III procollagen, and hyaluronate) were determined during and after a 6-month interferon treatment of patients with chronic hepatitis C. Changes in these markers were compared among the patients who showed a sustained normalization of serum alanine transaminase (ALT) levels with and without eradication of serum hepatitis C virus RNA (complete responders and biochemical responders) and nonresponders. In the case of complete responders, the serum 7s domain of type IV collagen and the N-terminal peptide of type III procollagen levels decreased at the end and 24 weeks after the end of the treatment. Hyaluronate levels were significantly decreased 24 weeks after the end of the treatment, as compared with those prior to the treatment. During and after interferon treatment, changes in these markers in the case of biochemical responders were nearly the same as those in the complete responders. These results suggest that serum hepatic fibrosis markers decrease in patients with chronic hepatitis C who show a sustained normalization of ALT after interferon treatment, even if serum hepatitis C virus RNA fails to be eradicated.

Ultra-deep sequencing analysis of resistance-associated variants during retreatment with simeprevir-based triple therapy after failure of telaprevir-based triple therapy in patients with genotype 1 hepatitis C virus infection

Simeprevir (SMV)‐based triple therapy is an effective retreatment option following failure of telaprevir (TVR)‐based triple therapy. However, it is unclear whether the persistence of resistance‐associated variants (RAVs) induced by TVR‐based therapy may reduce the treatment effect of SMV‐based therapy.