Impairment of IFN-α production capacity in patients with hepatitis C virus and the risk of the development of hepatocellular carcinoma

Abstract

AIM: To determine the utility of interferon (IFN) -α production capacity in patients with hepatitis C virus (HCV) infection for the measurement of immuno-surveillance potential and for the early detection of hepatocellular carcinoma (HCC) by investigating the Sendai virus (HVJ) stimulated IFN-α production capacity of patients with HCV infection.

METHODS: HVJ stimulated IFN-α production was determined in a large number of patients with HCV infection and the development of HCC was monitored for 3 years in patients with liver cirrhosis (LC).

RESULTS: IFN-α production capacity decreases gradually with the progression of liver disease from chronic hepatitis (CH) to HCC. A significant correlation between the duration of HCV infection and impaired IFN-α production capacity was observed. IFN-α production in patients who developed HCC within 3 years was significantly lower than that of patients who remained in LC without developing HCC.

CONCLUSION: Measurement of IFN-α production in LC patients may be useful for the early detection of HCC.

Key Words: IFN-α production capacity; Hepatitis C virus; Hepatocellular carcinoma

INTRODUCTION

The natural history of chronic hepatitis C virus (HCV) infection entails a gradual progression from chronic hepatitis (CH) to liver cirrhosis (LC) over 10-20 years leading to hepatocellular carcinoma (HCC) within 30 years[1-3]. Some common factors influencing the development of HCC include the degree of liver fibrosis, old age, sex (male), high alanine aminotransferase (ALT) level and a history of diabetes and alcohol consumption[4,5]. In addition, the development of HCC is affected by viral factors[6] and host immune factors[7]. Although recent progress in hepatitis research has illustrated the role of viral factors, analysis of the role if any played by immune factors has been insufficient. Several investigators have discussed the impairment of NK cell function, cytotoxic T lymphocyte (CTL) function and dendritic cell (DC) function in hepatitis C patients[8-11].

In previous papers, we have reported about the impairment of HVJ induced IFN-α production in whole blood cultures of patients with lung cancer, mylodysplastic syndrome, pulmonary tuberculosis, HIV, diabetes mellitus and IgA nephritis[12,13]. Additionally, we mentioned that IFN-α production in patients with lung cancer gradually decreases with the progress of cancer[12]. In the present study, we examined the IFN-α production in a large number of patients with HCV infection and LC who were monitored for more than 3 years for the development of HCC. IFN-α production in patients who developed HCC within 3 years was significantly lower than that of patients who remained in LC without developing HCC. These results suggest that IFN-α production could be a parameter with promising anti-tumor immuno-surveillance potential, i.e., measurement of IFN-α production in patients with HCV infection may be useful for the early detection of HCC.

MATERIALS AND METHODS

Subjects

IFN-α production in whole blood cultures was measured in 155 healthy individuals and 82 patients with chronic HCV infection including CH, LC and HCC. Blood was drawn after receiving informed consent.

The patients with HCV infection were all outpatients at Kyoto University Hospital, and were followed up at least once a month from 1994. All patients tested positive for anti-HCV (Abbot Japan, Tokyo) and/or HCV RNA by RT-PCR. The diagnoses of CH, LC and HCC were made by conducting a comprehensive analysis of liver histology, image analysis and blood tests. Patients underwent abdominal ultrasonography every 3-6 mo and/or contrast enhanced computed tomography every 6-12 mo. The CH group included 27 men and 12 women (age, 48.9±12.2 years). The LC group included 11 men and 10 women (age, 64.0±7.5 yeas). The HCC group included 13 men and 9 women (age, 64.1±5.4 years). Patients with HCV infection had not received interferon therapy for at least 3 mo prior to the measurement of IFN production capacity. In the case of patients with HCC, the IFN production capacity values of patients used were those determined at the point of first diagnosis of HCC.

We selected age-matched healthy persons as controls for comparison with the patients with HCV infection. Healthy subjects, 30 men (age, 53.6±8.9 years) and 95 women (age, 53.2±7.6 years), were selected from the people receiving medical examinations at the clinic of the Louis Pasteur Center for Medical Research who were over 40 years of age and had no acute and/or chronic disease and no abnormal values in blood tests.

Measurement of IFN-α production capacity in human whole blood

Two milliliters of heparinized blood was cultured with 500 HA U/mL Sendai virus (HVJ) within 5 h after the withdrawal of blood. The blood-virus mixture was incubated at 37 °C for 20 h. Supernatants were harvested by centrifugation at 3 000 r/min for 10 min. IFN-α activity in the supernatants was determined by bioassay as mentioned in a previous paper[12].

Statistical analysis

Results were expressed as mean±SD. Statistical significance was tested by Student’s t-test. For multiple comparisons, one-way analysis of variance (ANOVA) or the χ² test was used, when appropriate.

RESULTS

IFN-α production capacity in patients with chronic HCV infection in the various stages

A previous study has reported that there was a significant difference in IFN-α production between men and women, but that the IFN-α production capacity per 1 000 leukocytes (WBC) did not differ significantly between men and women throughout various groups[12]. In this study, IFN-α production capacity in various stages is expressed as IFN-α production per 1 000 WBC for comparing each stage, normalized for any difference between men and women. We found that IFN-α production capacity per 1 000 WBC decreased with the progression of the disease (healthy control: 1.54±0.57, CH: 1.41±1.01; LC: 0.89±0.49; HCC: 0.87±0.45 IU/1 000 WBC) (Figure 1). Using ANOVA,we found that HCV groups showed a significantly lower IFN-α titer as compared with the control group.

Figure 1 IFN-α production capacity per 1 000 WBC in patients with persistent HCV infection in various stages.

IFN-α production capacity per 1 000 WBC and the duration from HCV infection by blood transfusion

It has been reported that a significant correlation exists between the incidence of HCC and the duration of HCV infection[1-3]. As the time of initial infection is precisely known for patients who were infected while undergoing transfusion, we chose HCV patients infected in this manner. We then looked at the relationship between the patient’s IFN-α production capacity and the duration of the HCV infection (Figure 2). A significant correlation between the duration of HCV infection and impaired IFN-α production capacity per 1 000 WBC was observed (n = 39, r = 0.48, P = 0.0019). We could not find any significant correlation between platelet number, which was a surrogate marker for liver fibrosis, and ALT and AST levels, which were markers for the activity grade.

Figure 2 IFN-α production capacity per 1 000 WBC and duration of HCV infection.

IFN-α production capacity and the risk of HCC development within 3 years of LC

We measured IFN-α production capacity in the LC group. After 3 years, we divided the original group into two, according to whether each patient developed HCC or not. We observed a significant difference (P<0.05) in the initial IFN-α production capacity per 1 000 WBC between the patients who developed HCC (Group A, n = 11) and those who remained in LC without developing HCC (Group B, n = 10). In addition, we observed significant differences in AST and ALT levels as well as IFN-α production capacity per 1 000 WBC between the two groups (Table 1 ).

Table 1 IFN-α production capacity and the risk of HCC development within 3 years of LC.

		Group A			Group B
n		11			10
Age	(yr)	63.6	±	8.3	64.6	±	6.7
IFN-α	(IU/mL	2 406	±	1 239	3 936	±	2 284
IFN-α/WBC	(IU/1 000 WBC)	0.66	±	0.32	1.14	±	0.53a
AST(GOT)	(IU/mL)	97.0	±	52.7	55.9	±	21.3a
ALT(GPT)	(IU/mL)	99.2	±	47.0	54.8	±	22.4b
Plt	(x103/μL)	79.8	±	44.8	93.6	±	39.2

^aP<0.05,

^bP<0.01

Since the mean IFN-α production value of the HCC patients was 0.87±0.45 IU/1 000 WBC, we divided the LC group into two sub-groups: patients with IFN-α production value greater or less than the mean value (0.87 IU/1 000 WBC) (Table 2). After 3 years, we determined whether each patient had developed HCC or not. Compared to the 22% of patients in the high titer group, 75% of patients in the low titer group developed HCC. The c2 test showed significant differences between the two groups (P<0.05). These results indicated that LC patients with a low IFN-α production capacity (<0.87 IU/1 000 WBC) had a high risk of developing HCC within 3 years. Although low IFN-α production and high AST and ALT values, HCV copies and genotypes were demonstrated as risk factors of HCC development, no correlations were found between IFN-α production and these factors.

Table 2 Relationship between IFN-α production capacity and incidence of HCC development of patients within 3 years.

IFN-α IU/1 000 WBC	No of patients	No of patients	%
		who developed HCC within 3 yr
≥0.87	9	2	22.2
＜0.87	12	9	75a

^aP<0.05

DISCUSSION

In this study, we have demonstrated that (1) HVJ-stimulated IFN-α production capacity decreased gradually with the progression of HCV infection from CH to LC to HCC; (2) the longer the duration of HCV infection was, the lower was the patient’s IFN-α production capacity; and (3) IFN-α production capacity was lower in patients who developed HCC within 3 years as compared to those who did not develop HCC. Based on these results, a patient with lower IFN-α production capacity might be considered to be at a high risk for the development of HCC.

The progress of chronic HCV infection is one of slow progression from the early stages of CH without fibrosis to LC and HCC[1-3]. The disease progresses to its advanced stages over a period of 10-30 years, unless the viral infection is terminated by antiviral therapy[14]. In this study, we have demonstrated that the IFN-α production capacity of patients with HCV infection gradually decreased as the disease progressed, resulting in the development of HCC, suggesting that extended dysfunction of the type I IFN system may lead to an increased risk of HCC development.

In our previous studies, we have reported that IFN-α production was somehow decreased in patients with lung cancer, MDS, pulmonary tuberculosis, HIV infection, diabetes mellitus and IgA nephritis[12,13], suggesting that IFN-α production capacity is impaired by various diseases, including cancer, infections, and metabolic disorder. In addition, periodic measurements of IFN-α production revealed decreased IFN-α production capacities in patients with lung[12]. Consistent with these results, we demonstrated that LC patients who developed HCC within 3 years possessed a decreased capacity to produce IFN-α, suggesting that a low level of IFN-α production is associated with a higher incidence of HCC development in LC patients. In this study, 75% of LC patients with IFN-α production less than 0.87 IU/1 000 WBC developed HCC within 3 years. In the previous study, we have reported the mean values of IFN-α production in lung cancer and MDS to be 0.88±0.80 and 0.35±0.28 IU/1 000 WBC, respectively. These results suggest that patients with IFN-α production less than 0.87 IU/1 000 WBC need to be cautioned that they are at particularly high risk of cancer development. Consistently, MDS patients with low IFN-α production capacity developed cancer within several years[12,15,16]. Moreover, DM patients with low IFN-α production had a higher risk of developing cancer[6]. To clarify the utility of this IFN-α production capacity in the early detection of various types of cancers, further large scale clinical surveys are needed.

Why is impaired IFN-α production a risk factor for developing HCC? In our previous study, we have demonstrated that impairment of IFN-α production capacity in whole blood mainly depends not only on a defect in the number of mononuclear lymphocytes but also on the lower capacity of HVJ-stimulated IFN-α production from mononuclear lymphocytes. Mononuclear lymphocytes included monocytes, B cells, NK cells and plasmacytoid dendritic cells, all of which were producers of IFN-α[17,18]. Recently, there has been a new interest in type I IFN as a “bridge system” linking innate and adaptive immunity stemming from the identification of natural IFN producing cells, characterized as plasmacytoid dendritic cells[19,20]. Since IFN-α/β participate in cancer immunosurveillance[21], IFN-α production capacity is considered to reflect a facet of individual cancer immunosurveillance potential. In fact, some studies have clearly demonstrated that metastatic tumor cells grow progressively in mice pre-treated with neutralizing antibodies to murine IFN-α/β, demonstrating the critical role of endogenous IFN-α/β in the inhibition of anti-tumor immune responses[20,22,23]. Furthermore, type I IFN is involved in the anti-tumor activation of NK cells and macrophages as well as the induction of anti-tumor CTLs[24-27].

IFN-α production capacity is determined by both the genetic background and by the physical condition of each individual, such as the presence/absence of infection, metabolic disorder and cancer[28]. It is therefore anticipated that periodic measurements of IFN-α production and the early detection of reduced IFN-α production capacity will contribute to the early detection of HCC as well as the early detection of cancer and infectious disease. Furthermore, intervention to increase and/or maintain IFN-α production capacity may be useful for the prevention of HCC development.