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, 18 (5), 3991-4001

Histological Changes in Patients Who Developed Hepatocellular Carcinoma After Hepatitis C Virus Eradication by Interferon-Based Therapy

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Histological Changes in Patients Who Developed Hepatocellular Carcinoma After Hepatitis C Virus Eradication by Interferon-Based Therapy

Toshihiro Kawaguchi et al. Exp Ther Med.

Abstract

Although the incidence of hepatocellular carcinoma (HCC) occurring after hepatitis C virus (HCV) eradication has decreased, there are still reports of hepatocarcinogenesis. The present study investigated the histological changes of non-cancerous liver tissue obtained prior to interferon (IFN) therapy and after HCC development. A total of 669 HCV-infected Japanese patients who achieved sustained virological response (SVR) by IFN-based therapy were retrospectively enrolled. Of these, the present study investigated 18 patients who developed HCC after IFN-based SVR. Specimens from 9 of 18 patients were available for histological comparisons prior to IFN therapy and following HCC development. Of these 9 patients, the specimens of 5 individuals were compared via immunohistochemical staining [CD3, CD4, CD8, CD20, forkhead box P3 (FOXP3), transforming growth factor-β1 and granzyme B]. The current study included 6 control patients with HCV-associated chronic liver disease who subsequently developed HCC (non-SVR-HCC group). Mann-Whitney and Wilcoxon tests were used to compare groups. Bonferroni correction was used for multiple comparisons. P<0.05 was used as a critical P-value, and following Bonferroni's correction, P<0.017 was considered to indicate a statistically significant difference. In the 9 patients examined, continuous inflammation and fibrosis were observed after HCC development. There was also a significant decrease in the positive rate of FOXP3 in all 5 patients at the time of HCC development compared with that prior to IFN therapy (P=0.0084). Additionally, there was a significant difference in the positive rate of FOXP3 between the 5 patients after HCC development and the control individuals (P=0.0022). In patients who developed HCC after IFN-based SVR, the frequency of FOXP3 decreased, but inflammation and fibrosis remained. The extent of the reduction of FOXP3 differed in patients who developed HCC in the presence of HCV. Inflammation and fibrosis remained for a long duration after SVR, which may be associated with hepatocarcinogenesis.

Keywords: chronic hepatitis C; forkhead box P3; hepatocellular carcinoma; histological inflammation; sustained virological response.

Figures

Figure 1.
Figure 1.
Flow chart of the subjects included in the present study. IFN, interferon; HCV, hepatitis C virus; HCC, hepatocellular carcinoma.
Figure 2.
Figure 2.
Representative H&E stained specimens of 5 patients that developed HCC after achieving an IFN-based SVR. Specimens are presented from case 1 (A) before IFN therapy and (B) after HCC development; case 2 (C) before IFN therapy and (D) after HCC development; case 3 (E) before IFN therapy and (F) after HCC development; case 4 (G) before IFN therapy and (H) after HCC development; and case 5 (I) before IFN therapy and (J) after HCC development. Inflammation and fibrosis remained in all specimens (cases 1–5). HCC, hepatocellular carcinoma; IFN, interferon. SVR, sustained virological response.
Figure 2.
Figure 2.
Representative H&E stained specimens of 5 patients that developed HCC after achieving an IFN-based SVR. Specimens are presented from case 1 (A) before IFN therapy and (B) after HCC development; case 2 (C) before IFN therapy and (D) after HCC development; case 3 (E) before IFN therapy and (F) after HCC development; case 4 (G) before IFN therapy and (H) after HCC development; and case 5 (I) before IFN therapy and (J) after HCC development. Inflammation and fibrosis remained in all specimens (cases 1–5). HCC, hepatocellular carcinoma; IFN, interferon. SVR, sustained virological response.
Figure 2.
Figure 2.
Representative H&E stained specimens of 5 patients that developed HCC after achieving an IFN-based SVR. Specimens are presented from case 1 (A) before IFN therapy and (B) after HCC development; case 2 (C) before IFN therapy and (D) after HCC development; case 3 (E) before IFN therapy and (F) after HCC development; case 4 (G) before IFN therapy and (H) after HCC development; and case 5 (I) before IFN therapy and (J) after HCC development. Inflammation and fibrosis remained in all specimens (cases 1–5). HCC, hepatocellular carcinoma; IFN, interferon. SVR, sustained virological response.
Figure 3.
Figure 3.
Average frequency of CD3+, CD4+, CD8+ and CD20+ lymphocytes, and FOXP3, TGF-β1, and granzyme B-positive cells in cases 1–5 within the portal area. For the enumeration of positive mononuclear cells, all mononuclear cells were counted in two microscopic fields of the portal tract, twice. For each sample, the mean percentage of positive cells was used. In FOXP3, there was a significant difference before IFN therapy and after HCC development (P=0.0084), as determined by the Wilcoxon test. No significant differences were identified in levels of CD3 (P=0.46), CD4 (P=0.46), CD8 (P=0.60), CD20 (P=0.60), TGF-β1 (P=0.46) and granzyme B (P=0.60) before IFN therapy compared with after HCC development. FOXP3, forkhead box P3; TGF-β1, transforming growth factor-β1; IFN, interferon; HCC, hepatocellular carcinoma; SVR, sustained virological response.
Figure 4.
Figure 4.
Positive rates of intrahepatic FOXP3 in cases 1–5 and in control individuals. There was a significant difference in positive rates before IFN therapy and after HCC development (SVR-HCC) in cases 1–5 (P=0.0084), as determined via a Wilcoxon test with Bonferroni correction. A significant difference was identified after HCC development in cases 1–5 (SVR-HCC) when compared with control patients (non-SVR-HCC; P=0.0022), as determined via a Mann-Whitney test with Bonferroni correction. The results present the median positive rate of FOXP3 (interquartile range). FOXP3, forkhead box P3; IFN, interferon; HCC, hepatocellular carcinoma; SVR, sustained virological response.
Figure 5.
Figure 5.
Representative of liver specimens immunohistochemically stained for CD4, CD8, FOXP3 and TGF-β1 (case 2). Sections were immunohistochemically stained before IFN therapy for (A) CD4, (B) CD8, (C) FOXP3, and (D) TGF-β1. Sections were then stained for (E) CD4, (F) CD8, (G) FOXP3 and (H) TGF-β1 after hepatocellular carcinoma development. CD4 and CD8 exhibited distinct membrane staining. FOXP3 exhibited distinct nuclear staining. The distribution of TGF-β1-positive cells were observed in the periportal area. FOXP3, forkhead box P3; TGF-β1, transforming growth factor-β1.
Figure 5.
Figure 5.
Representative of liver specimens immunohistochemically stained for CD4, CD8, FOXP3 and TGF-β1 (case 2). Sections were immunohistochemically stained before IFN therapy for (A) CD4, (B) CD8, (C) FOXP3, and (D) TGF-β1. Sections were then stained for (E) CD4, (F) CD8, (G) FOXP3 and (H) TGF-β1 after hepatocellular carcinoma development. CD4 and CD8 exhibited distinct membrane staining. FOXP3 exhibited distinct nuclear staining. The distribution of TGF-β1-positive cells were observed in the periportal area. FOXP3, forkhead box P3; TGF-β1, transforming growth factor-β1.

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