Fig. S1. Flow chart of patient recruitment.
CHC, chronic hepatitis C; HBV, hepatitis B virus; HCC, hepatocellular
carcinoma; IHC, immunohistochemistry; SVR, sustained virological response.
Patients with CHC and liver biopsy
(n=2250)
Patients with an SVR and paired biopsies (n=130)
Patients with IHC staining (n=102)
Patients with no IHC staining (n=28)
Patients with HBV coinfection (n=201); HCC
development identified before biopsies (n=31); other
ineligible reasons (n=37);
without paired biopsies or without an SVR (n=1851)
Fig. S2. ROC curve analysis using the CPA changes to dichotomize changes in the METAVIR fibrosis stages: “no regression” (n = 56) versus “regression” (≥ 1 decline in fibrosis stages) (n = 74).
CPA change = the follow-up value (%) minus the baseline value (%). The optimal cutoff value of the CPA changes = −3.53%. The area under the ROC curve was 0.681 (95% confidence interval = 0.581–0.781) (P < 0.001). CPA, collagen proportionate area; ROC, receiver operating characteristic.
Fig. S3. Isotype negative control staining in a 52-year-old female patient.
(a), (b) the negative control staining for the present study using UltraVision™
Quanto Detection System HRP DAB with hematoxylin counterstain alone (ThermoFisher, MA, USA) demonstrating no signal; (c) CPA, 26.69% (F3A1) (S0); (d) CPA, 14.29% (F3A0) (S0); (e) CD34, 3.424%; (f) CD34, 1.426%; (g) α-SMA, 17.117%; (h) α-SMA, 9.186%; (i) eNOS, grade 3; (j) eNOS, grade 2 (baseline and post-SVR-- 4 years and 5 months apart). Diaminobenzidine reaction; hematoxylin counterstain. The biopsies were acquired from the 52-year-old male patient (not shown in the main manuscript) with chronic hepatitis C (genotype 1b) who achieved an SVR to a 12-month pegylated interferon (α-2b)-based therapy. No hepatocellular carcinoma developed during the follow-up period. A, METAVIR activity grade; α-SMA, α-smooth muscle actin; CPA, collagen proportionate area; eNOS, endothelial nitric oxide synthase; F, METAVIR fibrosis stage; S, steatosis grade; SVR, sustained virological response.
Fig. S4. Intermediate process of the segment quantitation of stained CD34 positivity and tissue areas at the baseline for the 25-year-old female patient demonstrated in Fig. 3.
(b)
(c)
(a) pretreatment CD34-positive histological image; (b) demonstration of CD34
quantitation process; (c) demonstration of tissue area quantification.
NIS-Elements software (Nikon corp. , Tokyo, Japan) detected the stained objects (pixels) in an automated manner. Both the numerator and denominator excluded (manually) the areas (in pixels) of defects, artifacts, and lumens using the NIS-Elements software. The area proportion was finalized and calculated by dividing the Σ numerator segments by Σ denominator segments (see Tables S10, S11, Supplemental Digital Content 1, which demonstrate the patient data and process of tissue quantitation).
Fig. S5. Liver tissue sections of the paired baseline (a, c, e, g) and post-SVR (b, d, f, h) picrosirius red staining for collagen types 1 and 3 and
immunohistochemical staining for CD 34, α-SMA, and eNOS, respectively (4 years apart) for the 25-year-old female patient demonstrated in Fig. 3.
(a) CPA, 8.66% (F2A3) (S0); (b) CPA, 3.23% (F1A0) (S0); (c) CD34, 2.337%;
(d) CD34, 0.985%; (e) α-SMA, 4.833%; (f) α-SMA, 2.980%; (g) eNOS, grade 3;
(h) eNOS, grade 2. Diaminobenzidine reaction; hematoxylin counterstain.
Original magnification: 200x. The consecutive liver sections were acquired from a 25-year-old female patient (same as Figure S4, S5) with chronic hepatitis C achieving an SVR to a 6-month pegylated interferon (α-2a)-based therapy (see Figures S3, S4, S5, Supplemental Digital Content 2, which demonstrate the process of digital image analyses and histology images) (see Tables S10, S11, Supplemental Digital Content 1, which demonstrate the patient data and tissue quantifications).
A, METAVIR activity grade; α-SMA, α-smooth muscle actin; CPA, collagen proportionate area; eNOS, endothelial nitric oxide synthase; F, METAVIR fibrosis stage; L, lobular (extraportal tissue area); P, portal tissue area; S,
steatosis grade; SVR, sustained virological response.
Fig. S6. ROC analysis using the CPAs at the baseline and follow-up, respectively, to predict the hepatocellular carcinoma development.
The optimal cutoff value of the posttreatment CPAs = 6.57%. The areas under the ROC curves were 0.729 (95% confidence interval = 0.549–0.908) (P = 0.012) for the posttreatment CPAs and 0.650 (0.479–0.821) (P = 0.100) for the pretreatment CPAs. CPA, collagen proportionate area; ROC, receiver
operating characteristic.
Fig. S7. Kaplan-Meier curves stratifying the cumulative incidence rates of post-SVR HCC development by the post-SVR CPA of 6.57%.
Log rank P = 0.001 over 258684 person-days (709 person-years) (median follow-up = 1996 days or 5.5 years). The posttreatment CPA cutoff value of 6.57 (%) was acquired through receiver operating characteristic curve analysis dichotomizing the post-SVR de novo HCC development. CPA, collagen proportionate area; HCC, hepatocellular carcinoma; SVR, sustained virological response.
Fig S8. Correlations between the overall CD34 area expressions and concurrent METAVIR F stages
At (a) the pretreatment baseline (Spearmann’s ρ = 0.559, P < 0.001), and (b) the post-SVR follow-up (Spearmann’s ρ = 0.369, P < 0.001), respectively.
Numbers under the boxes indicate the case n. F, METAVIR fibrosis stage;
SVR, sustained virological response.