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Gut and Liver is an international journal of gastroenterology, focusing on the gastrointestinal tract, liver, biliary tree, pancreas, motility, and neurogastroenterology. Gut atnd Liver delivers up-to-date, authoritative papers on both clinical and research-based topics in gastroenterology. The Journal publishes original articles, case reports, brief communications, letters to the editor and invited review articles in the field of gastroenterology. The Journal is operated by internationally renowned editorial boards and designed to provide a global opportunity to promote academic developments in the field of gastroenterology and hepatology. +MORE
| Yong Chan Lee |
Professor of Medicine Director, Gastrointestinal Research Laboratory Veterans Affairs Medical Center, Univ. California San Francisco San Francisco, USA |
| Jong Pil Im | Seoul National University College of Medicine, Seoul, Korea |
| Robert S. Bresalier | University of Texas M. D. Anderson Cancer Center, Houston, USA |
| Steven H. Itzkowitz | Mount Sinai Medical Center, NY, USA |
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Yoon Jung Hwang1,2 
, Hyejung Lee1 , Suk Kyun Hong3 , Su Jong Yu4 , Haeryoung Kim1,5
Correspondence to: Haeryoung Kim
ORCID https://orcid.org/0000-0002-4205-9081
E-mail haeryoung.kim@snu.ac.kr
Su Jong Yu
ORCID https://orcid.org/0000-0001-8888-7977
E-mail ydoctor2@snu.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Gut Liver.
Published online January 8, 2025
Copyright © Gut and Liver.
Background/Aims: Fibronectin (FN) has recently been identified as being overexpressed in patients with hepatocellular carcinoma (HCC) and deemed a promising biomarker of vascular invasion. The aim of this study was to examine the patterns of FN expression in HCC cells and their clinicopathological significance, such as their association with vascular invasion and angiogenesis patterns.
Methods: Immunohistochemical analysis of FN was conducted using tissue microarrays from 258 surgically resected HCCs and matched nontumorous liver tissues. Three distinct FN expression patterns were observed: cytoplasmic, membranous, and sinusoidal. Moderate or strong expression was considered FN-positive.
Results: Cytoplasmic or sinusoidal FN expression was significantly more common in HCC cells than in the adjacent liver tissue (p<0.001). FN expression was detected in the membranes of HCC cells and absent in nonneoplastic hepatocytes (p<0.001). Overall survival and disease-free survival in patients with HCC cells with membranous FN expression were significantly shorter than those in patients without membranous FN expression. Membranous FN expression in HCC was significantly associated with high serum alpha-fetoprotein (AFP) and protein induced by vitamin K absence-II (PIVKA-II) levels, infiltrative gross type, poor Edmondson-Steiner grade, major vessel invasion, microvascular invasion, macrotrabecular massive subtype, advanced T stage, and vessel-encapsulating tumor cluster pattern. Sinusoidal pattern of FN expression in HCC was significantly associated with high serum AFP and PIVKA-II levels, infiltrative gross type, large tumor size, microvascular invasion, macrotrabecular massive subtype, and vessel-encapsulating tumor cluster patterns.
Conclusions: Evaluating FN expression in HCC cells may be useful for identifying aggressive cases of HCC with vascular invasion via biopsy.
Keywords: Fibronectins, Carcinoma, hepatocellular, Microvessels, Immunohistochemistry, Prognosis
Fibronectin (FN) is a multifunctional high-molecular-weight glycoprotein that exists in soluble plasma form and insoluble cellular form on the cell surface.1-5 Plasma FN, produced by hepatocytes, is a major protein component of blood plasma, and cellular FN is a major component of the extracellular matrix. It is secreted by various cells, including fibroblasts, hepatic stellate cells, and endothelial cells as a soluble dimer, and is then assembled into an insoluble matrix.1,3,6 FN binds to integrin, a transmembrane receptor, and other extracellular matrix proteins such as collagen, fibrin, actin, and heparan sulfate proteoglycans, which plays major roles in cell adhesion, migration, proliferation and differentiation.1,3,7,8 FN is also involved in embryonic development, wound healing, and the pathogenesis of cancer and fibrosis.1-3,5,7-10 Although the role of FN in tumorigenesis and malignant progression is controversial, the expression of FN in several types of cancer has been studied, and overexpression of FN has been associated with poor prognosis in esophageal, gastric, colorectal, pancreatic, and renal cancer.5,11-17 Abnormal and increased FN expression has also been reported in hepatocellular carcinoma (HCC)2,9 and has recently been associated with vascular invasion in HCC.18,19
Vascular invasion is a major prognostic factor for HCC. However, it is difficult to identify microvascular invasion (MVI) on preoperative imaging or biopsy specimens, which can only be detected in the peritumoral liver parenchyma under microscopic examination.20-25 Thus, the identification of biomarkers for MVI would be beneficial for preoperative risk stratification of patients with HCC. In this study, we evaluated the expression patterns of FN in HCCs and its clinicopathological implications, including vascular invasion status.
We retrospectively reviewed a cohort of 258 consecutive adult patients with surgically resected HCCs at Seoul National University Hospital between January 2009 and December 2011. This study was approved by the Institutional Review Board of Seoul National University Hospital (IRB number: 2305-013-1427). The requirement for patient consent was waived by the IRB because of the retrospective nature of the study. Clinical data, including patient age, sex, underlying etiology, preoperative locoregional treatment (including radiofrequency ablation and transarterial chemo/radioembolization), and preoperative laboratory findings (including serum alpha-fetoprotein [AFP] and protein induced by vitamin K absence-II [PIVKA-II] levels) were retrieved from the electronic medical records.
Pathology reports for all 258 cases and microscopy slides for 175 cases, for which glass or digital slides were available for assessment, were reviewed by two pathologists (Y.J.H. and H.K.). The following information was recorded: underlying cirrhosis, infiltrative gross type, tumor size, multiplicity, histological differentiation grade (according to the Edmondson-Steiner [E-S] system), presence of major vascular invasion or MVI, histologic subtype (especially macrotrabecular massive [MTM] subtype), pathological T stage according to the American Joint Committee on Cancer 8th edition, vessel-encapsulating tumor clusters (VETC) pattern and cytokeratin 19 positivity. The infiltrative gross type includes multinodular confluent, nodular with perinodular extension, and infiltrative type.26 Multiplicity was defined as the presence of two or more tumors, including intrahepatic metastases and multicentric occurrences. Major vascular invasion was defined as the invasion of the main portal vein and first-order branches; the right, middle, and left hepatic veins; or the right or left hepatic artery. MVI was defined as the invasion of microvessels, which were identifiable only under microscopic examination, located in the fibrous capsule surrounding the tumor or in the peritumoral hepatic parenchyme, and not in the portal vein, hepatic veins, or hepatic arteries. MTM subtype was defined as a tumor showing thick trabeculae with more than six cells in more than 50% of the tumor area.
Tissue microarray cores of 2 mm diameter, consisting of one to three cores from HCCs and matched nonneoplastic tissues, were obtained from 258 HCCs (SuperBioChips Laboratory, Seoul, Korea). A total of 818 cores with 543 HCC and 275 adjacent liver parenchymal cores were evaluated in this study. Immunohistochemical stain for FN (MAB1918; R&D Systems, Minneapolis, MN, USA) and CD34 (M716501-2; Agilent, Santa Clara, CA, USA) was performed on 4 μm-thick tissue microarray sections manually or by using the Ventana BenchMark GX automated platform (Ventana Medical Systems, Oro Valley, AZ, USA). FN expression was evaluated according to the staining intensity as faint (discernable at ×100), weak (discernable at ×40), moderate (discernable at ×12.5), or strong (as strong as in trophoblasts), and according to the location of expression as cytoplasmic (stained in the cytoplasm of hepatocytes or tumor cells), membranous (stained at the membrane of hepatocytes or tumor cells), and sinusoidal (stained at endothelial cells of hepatic sinusoids) (Fig. 1). CD34 staining was used to confirm the VETC pattern, and one or more foci of tumor cell clusters completely surrounded by CD34-positive endothelial cells were considered VETC-positive.
Figure 1.Fibronectin (FN) stain in nonneoplastic liver tissue (A) and hepatocellular carcinoma (B-E) (left: ×40, right: ×400). (A) Faint sinusoidal staining. (B) Weak sinusoidal staining. (C) Strong cytoplasmic staining. (D) Strong membranous and sinusoidal staining with vessels-encapsulating tumor cluster pattern. (E) Moderate sinusoidal staining.
Statistical analyses were performed using commercially available software (SPSS Statistics for Windows version 26.0, IBM Corp., Armonk, NY, USA; R Studio software for Windows version 2022.12.0+353, R Foundation for Statistical Computing, Vienna, Austria; GraphPad Prism Software version 7, GraphPad Software, San Diego, CA, USA). Categorical variables were analyzed using the chi-square test, linear-by-linear association, and Fisher exact test. Survival analyses for overall survival (OS) and disease-free survival (DFS) were performed using the Kaplan-Meier method, log-rank test, and Cox proportional hazards regression analysis. The multivariable analysis was performed using the stepwise backward selection method. OS was defined as the interval between the date of surgery and the date of the last follow-up or death. DFS was defined as the interval between the date of operation and the date of local recurrence or intrahepatic or distant metastasis. Statistical significance was set at p<0.05.
The clinicopathological features of the study population are summarized in Table 1. Two hundred and fourteen patients (83%) were male and 44 (17%) were female, and the median age at operation was 59 years (interquartile range, 51 to 64 years). The most common etiology was hepatitis B viral infection (n=213, 83%) with the mean viral load of 219,026 IU/mL (range, 0 to 29,800,000 IU/mL), followed by hepatitis C viral infection (n=18, 7%) and alcohol intake (n=8, 3%). The median tumor size was 4.0 cm (interquartile range, 2.5 to 8.0 cm), and 195 tumors (75%) were E-S grade III or IV. MVI and major vessel invasion were observed in 108 (42%) and 44 patients (17%), respectively. The MTM subtype was identified in 26 out of 175 cases (15%), and the VETC pattern was identified in 61 cases (24%).
Table 1. Clinicopathological Characteristics
| Characteristic | Total (n=258) |
|---|---|
| Clinical features | |
| Age, yr | 59 (51–64) |
| Sex | |
| Male | 214 (82.9) |
| Female | 44 (17.1) |
| Etiology | |
| Hepatitis B | 208 (80.6) |
| Hepatitis C | 15 (5.8) |
| Alcohol | 6 (2.3) |
| Hepatitis B+hepatitis C | 3 (1.2) |
| Hepatitis B+alcohol | 2 (0.8) |
| Unknown | 24 (9.3) |
| Preoperative locoregional treatment | 115 (44.6) |
| TACE | 101 (39.1) |
| RFA | 23 (8.9) |
| PEIT | 23 (8.9) |
| PT-INR | 1.10 (1.04–1.25) |
| Albumin, g/L | 3.8 (3.2–4.2) |
| Bilirubin, mg/dL | 1.1 (0.7–2.1) |
| AST, U/L | 39 (31–64) |
| ALT, U/L | 35 (25–57) |
| Child Pugh score | |
| A | 189 (73.3) |
| B | 50 (19.4) |
| C | 19 (7.4) |
| MELD score (n=238) | 6.7 (6.5–7.4) |
| AFP, ng/mL | 26 (6–430) |
| PIVKA-II, mAU/mL | 120 (28–993) |
| Pathological findings | |
| Underlying cirrhosis | 192 (74.4) |
| Infiltrative gross type | 151 (58.5) |
| Tumor size, cm | 4.0 (2.5–8.0) |
| Multiplicity | 135 (52.3) |
| Edmondson-Steiner grade | |
| I | 8 (3.1) |
| II | 54 (20.9) |
| III | 122 (47.3) |
| IV | 73 (28.3) |
| Microvascular invasion | 108 (41.9) |
| Major vessel invasion | 44 (17.1) |
| Macrotrabecular massive subtype (n=175) | 26 (14.9) |
| T stage | |
| 1a | 13 (5.0) |
| 1b | 53 (20.5) |
| 2 | 127 (49.2) |
| 3 | 31 (12.0) |
| 4 | 34 (13.2) |
| VETC pattern | 61 (23.6) |
| CK19 positivity | 18 (7.0) |
| Fibronectin expression | |
| Cytoplasmic | 36 (14.0) |
| Membranous | 50 (19.4) |
| Sinusoidal | 119 (46.1) |
Data are presented as median (interquartile range) or number (%).
TACE, transarterial chemoembolization; RFA, radiofrequency ablation; PEIT, percutaneous ethanol injection therapy; PT-INR, prothrombin time-international normalized ratio; AST, aspartate aminotransferase; ALT, alanine aminotransferase; MELD, Model for End-Stage Liver Disease; AFP, alpha-fetoprotein; PIVKA-II, protein induced by vitamin K absence-II; VETC, vessels-encapsulating tumor clusters; CK19, cytokeratin 19.
The staining intensity of FN was stronger in the HCCs than in the adjacent parenchyma (Fig. 2). In nonneoplastic livers, moderate cytoplasmic or sinusoidal staining was rarely observed (n=3, 0.6% and n=4, 0.7%, respectively), while strong cytoplasmic/sinusoidal staining or moderate-to-strong membranous staining were not identified. In contrast, a much greater proportion of HCCs exhibited moderate or strong FN staining (cytoplasmic, n=59, 10.8%; membranous, n=77, 14.2%; sinusoidal, n=198, 36.5%). FN expression in the nonneoplastic tissue was mostly negative in the cytoplasm (n=225, 81.8%) and membrane (n=248, 90.2%) of hepatocytes, and predominantly negative or faint in sinusoidal endothelial cells (n=212, 77.1%). On the other hand, negative cytoplasmic or membranous staining was observed in a much smaller proportion of tumor tissue (n=296, 54.5% and n=255, 47.0%, respectively), with only 8.5% of tumors showing negative sinusoidal staining (n=46). Based on these staining patterns, moderate or strong FN staining was considered indicative of FN expression (FN-positive).
Figure 2.Comparison of staining pattern and intensity of fibronectin between hepatocellular carcinoma and nonneoplastic livers. (A) Cytoplasmic staining intensity. (B) Membranous staining intensity. (C) Sinusoidal staining intensity. (D) Proportion of fibronectin expression pattern in tumor and nontumorous tissue. *p<0.001.
Cytoplasmic FN expression was more frequently observed in HCC (n=59, 10.9%) than in the adjacent parenchyma (n=3, 1.1%, p<0.001). Membranous FN expression was observed in tumor cells (n=77, 14.2%), but not in nonneoplastic hepatocytes (p<0.001). Sinusoidal FN expression was observed significantly more frequently in the tumors (n=198, 36.5%) than in the adjacent liver tissue (n=4, 1.5%, p<0.001). Among the 258 patients, cytoplasmic FN expression was observed in 36 patients (14%), membranous expression was identified in 50 (19%), and sinusoidal expression was observed in 119 (46%).
A comparison of the clinicopathological characteristics according to the FN expression patterns is presented in Table 2 and Supplementary Figs 1 and 2. Cytoplasmic FN expression was significantly associated with high serum PIVKA-II levels (p=0.021) and MVI (p=0.004) (Fig. 3). Membranous FN expression was significantly associated with high serum AFP (p<0.001) and PIVKA-II levels (p=0.045), infiltrative gross type (p=0.001), poor E-S grade (p=0.027), MVI (p=0.001), major vessel invasion (p=0.007), MTM subtype (p<0.001), high T stage (p<0.001), and VETC pattern (p=0.001). Sinusoidal FN expression was significantly associated with high serum AFP (p=0.029) and PIVKA-II levels (p=0.002), infiltrative gross type (p=0.034), large tumor size (p=0.013), MVI (p=0.038), MTM subtype (p-0.011), and VETC patterns (p<0.001).
Figure 3.Microvascular invasion (A), vessels-encapsulating tumor cluster (VETC) pattern (B) and macrotrabecular massive (MTM) subtype (C) according to fibronectin expression pattern. *p<0.05, †p<0.001.
Table 2. Clinicopathological Characteristics According to the Fibronectin Expression Pattern
| Variable | Cytoplasmic expression | Membranous expression | Sinusoidal expression | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Absent (n=222) | Present (n=36) | p-value | Absent (n=208) | Present (n=50) | p-value | Absent (n=139) | Present (n=119) | p-value | |||
| Clinical feature | |||||||||||
| Age ≥60 yr | 97 (43.7) | 15 (41.7) | 0.820 | 97 (46.6) | 15 (30.0) | 0.033* | 62 (44.6) | 50 (42.0) | 0.676 | ||
| Male sex | 187 (84.2) | 27 (75.0) | 0.172 | 172 (82.7) | 42 (84.0) | 0.825 | 119 (85.6) | 95 (79.8) | 0.219 | ||
| Underlying HBV infection | 184 (82.9) | 29 (80.6) | 0.733 | 170 (81.7) | 43 (86.0) | 0.475 | 119 (85.6) | 94 (79.0) | 0.162 | ||
| AFP ≥1,000 ng/mL | 43 (19.4) | 9 (25.0) | 0.435 | 32 (15.4) | 20 (40.0) | <0.001* | 21 (15.1) | 31 (26.1) | 0.029* | ||
| Pre-op PIVKA-II level ≥200 | 90 (40.5) | 22 (61.1) | 0.021* | 84 (40.4) | 28 (56.0) | 0.045* | 48 (34.5) | 64 (53.8) | 0.002* | ||
| Pathological findings | |||||||||||
| Underlying cirrhosis | 168 (75.7) | 24 (66.7) | 0.250 | 160 (76.9) | 32 (64.0) | 0.060 | 113 (81.3) | 79 (66.4) | 0.006* | ||
| Infiltrative gross type | 125 (56.3) | 26 (72.2) | 0.072 | 111 (53.4) | 40 (80.0) | 0.001* | 73 (52.5) | 78 (65.5) | 0.034* | ||
| Size >5 cm | 93 (41.9) | 18 (50.0) | 0.362 | 85 (40.9) | 26 (52.0) | 0.153 | 50 (36.0) | 61 (51.3) | 0.013* | ||
| Multiplicity | 115 (51.8) | 20 (55.6) | 0.676 | 105 (50.5) | 30 (60.0) | 0.226 | 79 (56.8) | 56 (47.1) | 0.117 | ||
| E-S grade III or IV | 165 (74.3) | 31 (86.1) | 0.125 | 152 (73.1) | 44 (88.0) | 0.027* | 99 (71.2) | 97 (81.5) | 0.054 | ||
| Microvascular invasion | 85 (38.3) | 23 (63.9) | 0.004* | 77 (37.0) | 31 (62.0) | 0.001* | 50 (36.0) | 58 (48.7) | 0.038* | ||
| Major vessel invasion | 37 (16.7) | 7 (19.4) | 0.681 | 29 (13.9) | 15 (30.0) | 0.007* | 21 (15.1) | 23 (19.3) | 0.369 | ||
| MTM subtype (n=175) | 19/147 (12.9) | 7/28 (25.0) | 0.142 | 13 (9.4) | 13 (35.1) | <0.001* | 8 (8.5) | 18 (22.2) | 0.011* | ||
| T stage | 0.050 | <0.001* | 0.152 | ||||||||
| T1a | 12 (5.4) | 1 (2.8) | 11 (5.3) | 2 (4.0) | 9 (6.5) | 4 (3.4) | |||||
| T1b | 52 (23.4) | 1 (2.8) | 50 (24.0) | 3 (6.0) | 27 (19.4) | 26 (21.8) | |||||
| T2 | 103 (46.4) | 24 (66.7) | 102 (49.0) | 25 (50.0) | 70 (50.4) | 57 (47.9) | |||||
| T3 | 28 (12.6) | 3 (8.3) | 25 (12.0) | 6 (12.0) | 22 (15.8) | 9 (7.6) | |||||
| T4 | 27 (12.2) | 7 (19.4) | 20 (9.6) | 14 (28.0) | 11 (7.9) | 23 (19.3) | |||||
| VETC pattern | 49 (22.1) | 12 (33.3) | 0.140 | 40 (19.2) | 21 (42.0) | 0.001* | 14 (10.1) | 47 (39.5) | <0.001* | ||
| CK19 positivity | 16 (7.2) | 2 (5.6) | 1.000 | 14 (6.7) | 4 (8.0) | 0.758 | 11 (7.9) | 7 (5.9) | 0.523 | ||
Data are presented as number (%).
HBV, hepatitis B virus; AFP, alpha-fetoprotein; Pre-op, preoperative; PIVKA-II, protein induced by vitamin K absence-II; E-S, Edmondson-Steiner; MTM, macrotrabecular massive; VETC, vessels-encapsulating tumor clusters; CK19, cytokeratin 19.
*Indicates p<0.05.
OS and DFS in patients with HCCs showing membranous FN expression were significantly shorter than those in patients without membranous expression (p=0.003 in both cases) (Fig. 4, Supplementary Fig. 3). There was no significant difference in OS and DFS between patients with cytoplasmic or sinusoidal FN-positive HCC and those without cytoplasmic or sinusoidal expression. DFS in patients with tumors showing sinusoidal FN expression tended to be shorter than that in those without sinusoidal expression (p=0.067). Univariable analysis showed that serum AFP ≥1,000 ng/mL, PIVKA-II ≥200 mAU/mL, infiltrative gross type, tumor size >5 cm, E-S grade III or IV, MVI, major vessel invasion, MTM subtype, cytokeratin 19 positivity, and membranous FN expression were significant prognostic factors for OS and DFS. In the multivariate analysis, tumor size >5 cm, E-S grade III or IV, and cytokeratin 19 positivity were significant independent factors for predicting OS and DFS (Table 3).
Figure 4.Kaplan-Meier survival curve for overall survival (A-C) and disease-free survival (D-F) according to fibronectin expression pattern.
Table 3. Univariable and Multivariable Analyses of Clinical and Histopathological Features for Overall and Disease-Free Survival
| Variable | Overall survival | Disease-free survival | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Univariable analysis | Multivariable analysis | Univariable analysis | Multivariable analysis | ||||||||
| HR (95% CI) | p-value | HR (95% CI) | p-value | HR (95% CI) | p-value | HR (95% CI) | p-value | ||||
| Clinical feature | |||||||||||
| Age ≥60 yr | 1.038 (0.723–1.490) | 0.841 | 0.831 (0.601–1.149) | 0.262 | |||||||
| Male sex | 1.015 (0.621–1.658) | 0.953 | 1.010 (0.658–1.550) | 0.965 | |||||||
| Underlying HBV infection | 0.997 (0.622–1.597) | 0.990 | 1.056 (0.697–1.601) | 0.797 | |||||||
| AFP ≥1,000 ng/mL | 2.148 (1.428–3.231) | <0.001* | 2.314 (1.609–3.327) | <0.001* | 1.590 (1.028–2.459) | 0.037* | |||||
| Pre-op PIVKA-II level ≥200 | 1.956 (1.362–2.810) | <0.001* | 2.067 (1.502–2.845) | <0.001* | |||||||
| Pathological findings | |||||||||||
| Underlying cirrhosis | 0.814 (0.544–1.218) | 0.317 | 0.776 (0.546–1.102) | 0.156 | |||||||
| Infiltrative gross type | 2.016 (1.362–2.983) | <0.001* | 1.516 (0.964–2.385) | 0.072 | 1.580 (1.135–2.200) | 0.007* | 1.445 (0.966–2.160) | 0.073 | |||
| Size >5 cm | 2.346 (1.629–3.378) | <0.001* | 3.020 (1.937–4.707) | <0.001* | 2.719 (1.968–3.756) | <0.001* | 3.237 (2.123–4.934) | <0.001* | |||
| Multiplicity | 1.546 (1.073–2.228) | 0.019* | 1.675 (1.056–2.659) | 0.028* | 0.914 (0.665–1.255) | 0.577 | |||||
| E-S grade III or IV | 3.067 (1.780–5.286) | <0.001* | 2.074 (1.135–3.789) | 0.018* | 2.208 (1.436–3.394) | <0.001* | 2.528 (1.464–4.364) | 0.001* | |||
| Microvascular invasion | 2.478 (1.715–3.580) | <0.001* | 2.390 (1.733–3.298) | <0.001* | |||||||
| Major vessel invasion | 2.817 (1.872–4.239) | <0.001* | 3.169 (2.186–4.596) | <0.001* | 1.697 (1.071–2.688) | 0.024* | |||||
| MTM subtype (n=175) | 2.108 (1.237–3.591) | 0.006* | 2.796 (1.755–4.453) | <0.001* | |||||||
| VETC pattern | 1.400 (0.936–2.095) | 0.101 | 1.317 (0.920–1.885) | 0.132 | |||||||
| CK19 positivity | 3.427 (1.947–6.030) | <0.001* | 2.771 (1.499–5.124) | 0.001* | 2.261 (1.301–3.931) | 0.004* | 1.870 (0.998–3.502) | 0.051 | |||
| Fibronectin expression | |||||||||||
| Cytoplasmic | 1.192 (0.730–1.947) | 0.483 | 1.255 (0.805–1.955) | 0.316 | |||||||
| Membranous | 1.852 (1.227–2.794) | 0.003* | 1.728 (1.192–2.504) | 0.004* | |||||||
| Sinusoidal | 1.341 (0.936–1.922) | 0.110 | 1.337 (0.974–1.836) | 0.073 | |||||||
HR, hazard ratio; CI, confidence interval; HBV, hepatitis B virus; AFP, alpha-fetoprotein; Pre-op, preoperative; PIVKA-II, protein induced by vitamin K absence-II; E-S, Edmondson-Steiner; MTM, macrotrabecular massive; VETC, vessels-encapsulating tumor clusters; CK19, cytokeratin 19.
*Indicates p<0.05.
We analyzed FN1 mRNA expression in The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort (Supplementary Fig. 4). On comparison between tumor and normal tissues from 49 HCC patients for whom matched normal tissue samples were available, we found slightly higher FN1 mRNA expression levels in HCCs, although not statistically significant (p=0.249). For survival analysis, patients were divided into high and low FN1 groups based on the median value of FN1 mRNA expression, and we found that OS was significantly decreased in the high FN1 expression group (p=0.027). In addition, we compared FN1 mRNA expression levels according to the vascular invasion status; however, there was no significant difference in FN1 expression levels between cases with and without vascular invasion.
This study revealed that FN was overexpressed in HCC compared to nontumorous liver tissue in three patterns: (1) cytoplasmic, (2) membranous, and (3) sinusoidal. FN expression was significantly associated with MVI and other aggressive clinicopathological features including high serum AFP and PIVKA-II levels, infiltrative gross type, large tumor size, poor histological differentiation, and major vessel invasion. Membranous and sinusoidal FN expression was significantly associated with the MTM subtype and VETC pattern of vascularization. Membranous FN expression was a significant predictive factor for poor OS and DFS.
HCC is an aggressive tumor, and vascular invasion is an important prognostic predictor in several staging systems. However, aggressive HCC with MVI may be missed because most HCCs are diagnosed based on clinical and imaging findings without biopsy, and MVI cannot be accurately evaluated based on imaging findings.27 The VETC pattern, a distinct vascularization pattern of HCC, has recently been reported to be associated with poor prognostic factors, including MVI, and is enriched in the MTM subtype, which is an aggressive subtype of HCC.28-33 Thus, the importance of evaluating VETC and macrotrabecular patterns in a biopsy, which are unique and relatively easily identifiable but still challenging to detect in a small specimen, has been emerging. A biomarker beneficial for identifying these patterns would help triage the aggressive HCC group with poor prognosis.
FN is an extracellular matrix glycoprotein involved in various functions. FN has been shown to significantly impact disease pathogenesis, particularly by promoting proliferation, invasion, and metastasis through interactions with integrins and other cell surface receptors.5,34-36 Several studies have revealed the poor prognostic effect of FN expression in various types of cancer.5,11-17 In HCC, a few studies demonstrated that serum FN levels increased in patients with early HCC and decreased after treatment,6,37 and cellular FN was upregulated in HCC tumor cells.19 In this study, we demonstrated FN overexpression in HCC, and highlighted its expression patterns and significance. Among the three FN expression patterns, the membranous pattern showed a broader range of associations with the aggressive parameters compared to the cytoplasmic and sinusoidal patterns. FN overexpression on the cell membrane can more easily facilitate the interactions with cell surface receptors, including integrins, leading to the activation of downstream signaling pathways involved in tumor growth, angiogenesis, and metastasis. In consequence, it might contribute to a stronger association with aggressive clinicopathological parameters. This suggests that the localization of FN is crucial for its functional role, and further research is needed to elucidate the exact mechanisms and pathways through which FN exerts its effects.
FN is recently proposed as a potential biomarker for vascular invasion in HCC.18 Cellular FN is a well-known structural element of angiogenesis in embryogenesis and wound healing and is involved in the formation of tumor vessels as well.38-43 Several studies have indicated that cellular FN upregulation is associated with MVI.19 This study revealed the significant association between vascular invasion and FN expression. The mechanism of FN in tumor angiogenesis provides a ridged structure for neovascular lumen formation and the binding of vascular endothelial growth factor to maintain a directional concentration gradient for blood vessel formation.38,44,45 This may explain why the FN is related to the vascularization pattern in HCC.
There have been studies on FN as a molecular target for therapy, although its usefulness is still unclear. The method of conjugating a drug to extra-domain A or extra-domain B antibodies for drug delivery to malignant cells, which are contained in cellular FN, is expected to be promising given that extra-domain A or extra-domain B is limitedly expressed in malignancy.39,46,47
To validate our results, we analyzed FN expression using the external bulk RNA sequencing data of the TCGA-LIHC cohort. Although we found slightly higher FN levels in HCCs compared to paired non-neoplastic livers and decreased survival for the high FN group, there was no significant difference in vascular invasion status according to FN expression status. The discrepancy between our data and the TCGA analysis results could be explained by the fact that RNA sequencing measures the average RNA levels across the entire tissue, while immunohistochemistry reveals the distribution and localization of proteins within tissue. On immunohistochemistry, the expression in membranes of hepatocytes or in sinusoidal endothelial cells was most significant, and this suggests that for FN, the localization/distribution of protein expression could be more relevant than the level of expression. In addition, it may be possible that the post-transcriptional protein expression levels could exert a more significant influence than the RNA expression levels. To confirm this, further research analyzing the correlation between immunohistochemistry and RNA expression in the same tissue samples is necessary.
A limitation of this study is that this is a retrospective cohort study performed on archival formalin-fixed paraffin-embedded tissues from resected HCC specimens, and serum samples were not available for serum FN analysis. A prospective study would be necessary in order to correlate the tissue FN expression with the serum FN levels.
In conclusion, FN expression was associated with MVI and aggressive clinicopathological parameters in HCC; thus, FN may be a potential biomarker for an aggressive group of HCC with MVI, especially in the biopsy setting, and a potential molecular target for therapy.
This was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2022R1A2C2010348).
S.J.Y. is an editorial board member of the journal but was not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.
Study concept and design: H.K., S.J.Y. Data acquisition: S.J.Y., S.K.H. Data analysis and interpretation: Y.J.H., H.L., H.K. Drafting of the manuscript: Y.J.H. Critical revision of the manuscript for important intellectual content: H.K., S.J.Y. Statistical analysis: Y.J.H. Obtained funding: H.K. Administrative, technical, or material support; study supervision: H.K., S.J.Y. Approval of final manuscript: all authors.
Supplementary materials can be accessed at https://doi.org/10.5009/gnl240254.
Gut and Liver
Published online January 8, 2025
Copyright © Gut and Liver.
Yoon Jung Hwang1,2 , Hyejung Lee1 , Suk Kyun Hong3 , Su Jong Yu4 , Haeryoung Kim1,5
1Department of Pathology, Seoul National University College of Medicine, Seoul, Korea; 2Department of Pathology, Seoul National University Bundang Hospital, Seongnam, Korea; 3Department of Surgery, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea; 4Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine and Biomedical Research Institute, Center for Medical Innovation, Seoul National University Hospital, Seoul, Korea; 5Department of Pathology, Seoul National University Hospital, Seoul, Korea
Correspondence to:Haeryoung Kim
ORCID https://orcid.org/0000-0002-4205-9081
E-mail haeryoung.kim@snu.ac.kr
Su Jong Yu
ORCID https://orcid.org/0000-0001-8888-7977
E-mail ydoctor2@snu.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background/Aims: Fibronectin (FN) has recently been identified as being overexpressed in patients with hepatocellular carcinoma (HCC) and deemed a promising biomarker of vascular invasion. The aim of this study was to examine the patterns of FN expression in HCC cells and their clinicopathological significance, such as their association with vascular invasion and angiogenesis patterns.
Methods: Immunohistochemical analysis of FN was conducted using tissue microarrays from 258 surgically resected HCCs and matched nontumorous liver tissues. Three distinct FN expression patterns were observed: cytoplasmic, membranous, and sinusoidal. Moderate or strong expression was considered FN-positive.
Results: Cytoplasmic or sinusoidal FN expression was significantly more common in HCC cells than in the adjacent liver tissue (p<0.001). FN expression was detected in the membranes of HCC cells and absent in nonneoplastic hepatocytes (p<0.001). Overall survival and disease-free survival in patients with HCC cells with membranous FN expression were significantly shorter than those in patients without membranous FN expression. Membranous FN expression in HCC was significantly associated with high serum alpha-fetoprotein (AFP) and protein induced by vitamin K absence-II (PIVKA-II) levels, infiltrative gross type, poor Edmondson-Steiner grade, major vessel invasion, microvascular invasion, macrotrabecular massive subtype, advanced T stage, and vessel-encapsulating tumor cluster pattern. Sinusoidal pattern of FN expression in HCC was significantly associated with high serum AFP and PIVKA-II levels, infiltrative gross type, large tumor size, microvascular invasion, macrotrabecular massive subtype, and vessel-encapsulating tumor cluster patterns.
Conclusions: Evaluating FN expression in HCC cells may be useful for identifying aggressive cases of HCC with vascular invasion via biopsy.
Keywords: Fibronectins, Carcinoma, hepatocellular, Microvessels, Immunohistochemistry, Prognosis
Fibronectin (FN) is a multifunctional high-molecular-weight glycoprotein that exists in soluble plasma form and insoluble cellular form on the cell surface.1-5 Plasma FN, produced by hepatocytes, is a major protein component of blood plasma, and cellular FN is a major component of the extracellular matrix. It is secreted by various cells, including fibroblasts, hepatic stellate cells, and endothelial cells as a soluble dimer, and is then assembled into an insoluble matrix.1,3,6 FN binds to integrin, a transmembrane receptor, and other extracellular matrix proteins such as collagen, fibrin, actin, and heparan sulfate proteoglycans, which plays major roles in cell adhesion, migration, proliferation and differentiation.1,3,7,8 FN is also involved in embryonic development, wound healing, and the pathogenesis of cancer and fibrosis.1-3,5,7-10 Although the role of FN in tumorigenesis and malignant progression is controversial, the expression of FN in several types of cancer has been studied, and overexpression of FN has been associated with poor prognosis in esophageal, gastric, colorectal, pancreatic, and renal cancer.5,11-17 Abnormal and increased FN expression has also been reported in hepatocellular carcinoma (HCC)2,9 and has recently been associated with vascular invasion in HCC.18,19
Vascular invasion is a major prognostic factor for HCC. However, it is difficult to identify microvascular invasion (MVI) on preoperative imaging or biopsy specimens, which can only be detected in the peritumoral liver parenchyma under microscopic examination.20-25 Thus, the identification of biomarkers for MVI would be beneficial for preoperative risk stratification of patients with HCC. In this study, we evaluated the expression patterns of FN in HCCs and its clinicopathological implications, including vascular invasion status.
We retrospectively reviewed a cohort of 258 consecutive adult patients with surgically resected HCCs at Seoul National University Hospital between January 2009 and December 2011. This study was approved by the Institutional Review Board of Seoul National University Hospital (IRB number: 2305-013-1427). The requirement for patient consent was waived by the IRB because of the retrospective nature of the study. Clinical data, including patient age, sex, underlying etiology, preoperative locoregional treatment (including radiofrequency ablation and transarterial chemo/radioembolization), and preoperative laboratory findings (including serum alpha-fetoprotein [AFP] and protein induced by vitamin K absence-II [PIVKA-II] levels) were retrieved from the electronic medical records.
Pathology reports for all 258 cases and microscopy slides for 175 cases, for which glass or digital slides were available for assessment, were reviewed by two pathologists (Y.J.H. and H.K.). The following information was recorded: underlying cirrhosis, infiltrative gross type, tumor size, multiplicity, histological differentiation grade (according to the Edmondson-Steiner [E-S] system), presence of major vascular invasion or MVI, histologic subtype (especially macrotrabecular massive [MTM] subtype), pathological T stage according to the American Joint Committee on Cancer 8th edition, vessel-encapsulating tumor clusters (VETC) pattern and cytokeratin 19 positivity. The infiltrative gross type includes multinodular confluent, nodular with perinodular extension, and infiltrative type.26 Multiplicity was defined as the presence of two or more tumors, including intrahepatic metastases and multicentric occurrences. Major vascular invasion was defined as the invasion of the main portal vein and first-order branches; the right, middle, and left hepatic veins; or the right or left hepatic artery. MVI was defined as the invasion of microvessels, which were identifiable only under microscopic examination, located in the fibrous capsule surrounding the tumor or in the peritumoral hepatic parenchyme, and not in the portal vein, hepatic veins, or hepatic arteries. MTM subtype was defined as a tumor showing thick trabeculae with more than six cells in more than 50% of the tumor area.
Tissue microarray cores of 2 mm diameter, consisting of one to three cores from HCCs and matched nonneoplastic tissues, were obtained from 258 HCCs (SuperBioChips Laboratory, Seoul, Korea). A total of 818 cores with 543 HCC and 275 adjacent liver parenchymal cores were evaluated in this study. Immunohistochemical stain for FN (MAB1918; R&D Systems, Minneapolis, MN, USA) and CD34 (M716501-2; Agilent, Santa Clara, CA, USA) was performed on 4 μm-thick tissue microarray sections manually or by using the Ventana BenchMark GX automated platform (Ventana Medical Systems, Oro Valley, AZ, USA). FN expression was evaluated according to the staining intensity as faint (discernable at ×100), weak (discernable at ×40), moderate (discernable at ×12.5), or strong (as strong as in trophoblasts), and according to the location of expression as cytoplasmic (stained in the cytoplasm of hepatocytes or tumor cells), membranous (stained at the membrane of hepatocytes or tumor cells), and sinusoidal (stained at endothelial cells of hepatic sinusoids) (Fig. 1). CD34 staining was used to confirm the VETC pattern, and one or more foci of tumor cell clusters completely surrounded by CD34-positive endothelial cells were considered VETC-positive.
Figure 1. Fibronectin (FN) stain in nonneoplastic liver tissue (A) and hepatocellular carcinoma (B-E) (left: ×40, right: ×400). (A) Faint sinusoidal staining. (B) Weak sinusoidal staining. (C) Strong cytoplasmic staining. (D) Strong membranous and sinusoidal staining with vessels-encapsulating tumor cluster pattern. (E) Moderate sinusoidal staining.
Statistical analyses were performed using commercially available software (SPSS Statistics for Windows version 26.0, IBM Corp., Armonk, NY, USA; R Studio software for Windows version 2022.12.0+353, R Foundation for Statistical Computing, Vienna, Austria; GraphPad Prism Software version 7, GraphPad Software, San Diego, CA, USA). Categorical variables were analyzed using the chi-square test, linear-by-linear association, and Fisher exact test. Survival analyses for overall survival (OS) and disease-free survival (DFS) were performed using the Kaplan-Meier method, log-rank test, and Cox proportional hazards regression analysis. The multivariable analysis was performed using the stepwise backward selection method. OS was defined as the interval between the date of surgery and the date of the last follow-up or death. DFS was defined as the interval between the date of operation and the date of local recurrence or intrahepatic or distant metastasis. Statistical significance was set at p<0.05.
The clinicopathological features of the study population are summarized in Table 1. Two hundred and fourteen patients (83%) were male and 44 (17%) were female, and the median age at operation was 59 years (interquartile range, 51 to 64 years). The most common etiology was hepatitis B viral infection (n=213, 83%) with the mean viral load of 219,026 IU/mL (range, 0 to 29,800,000 IU/mL), followed by hepatitis C viral infection (n=18, 7%) and alcohol intake (n=8, 3%). The median tumor size was 4.0 cm (interquartile range, 2.5 to 8.0 cm), and 195 tumors (75%) were E-S grade III or IV. MVI and major vessel invasion were observed in 108 (42%) and 44 patients (17%), respectively. The MTM subtype was identified in 26 out of 175 cases (15%), and the VETC pattern was identified in 61 cases (24%).
Table 1 . Clinicopathological Characteristics.
| Characteristic | Total (n=258) |
|---|---|
| Clinical features | |
| Age, yr | 59 (51–64) |
| Sex | |
| Male | 214 (82.9) |
| Female | 44 (17.1) |
| Etiology | |
| Hepatitis B | 208 (80.6) |
| Hepatitis C | 15 (5.8) |
| Alcohol | 6 (2.3) |
| Hepatitis B+hepatitis C | 3 (1.2) |
| Hepatitis B+alcohol | 2 (0.8) |
| Unknown | 24 (9.3) |
| Preoperative locoregional treatment | 115 (44.6) |
| TACE | 101 (39.1) |
| RFA | 23 (8.9) |
| PEIT | 23 (8.9) |
| PT-INR | 1.10 (1.04–1.25) |
| Albumin, g/L | 3.8 (3.2–4.2) |
| Bilirubin, mg/dL | 1.1 (0.7–2.1) |
| AST, U/L | 39 (31–64) |
| ALT, U/L | 35 (25–57) |
| Child Pugh score | |
| A | 189 (73.3) |
| B | 50 (19.4) |
| C | 19 (7.4) |
| MELD score (n=238) | 6.7 (6.5–7.4) |
| AFP, ng/mL | 26 (6–430) |
| PIVKA-II, mAU/mL | 120 (28–993) |
| Pathological findings | |
| Underlying cirrhosis | 192 (74.4) |
| Infiltrative gross type | 151 (58.5) |
| Tumor size, cm | 4.0 (2.5–8.0) |
| Multiplicity | 135 (52.3) |
| Edmondson-Steiner grade | |
| I | 8 (3.1) |
| II | 54 (20.9) |
| III | 122 (47.3) |
| IV | 73 (28.3) |
| Microvascular invasion | 108 (41.9) |
| Major vessel invasion | 44 (17.1) |
| Macrotrabecular massive subtype (n=175) | 26 (14.9) |
| T stage | |
| 1a | 13 (5.0) |
| 1b | 53 (20.5) |
| 2 | 127 (49.2) |
| 3 | 31 (12.0) |
| 4 | 34 (13.2) |
| VETC pattern | 61 (23.6) |
| CK19 positivity | 18 (7.0) |
| Fibronectin expression | |
| Cytoplasmic | 36 (14.0) |
| Membranous | 50 (19.4) |
| Sinusoidal | 119 (46.1) |
Data are presented as median (interquartile range) or number (%)..
TACE, transarterial chemoembolization; RFA, radiofrequency ablation; PEIT, percutaneous ethanol injection therapy; PT-INR, prothrombin time-international normalized ratio; AST, aspartate aminotransferase; ALT, alanine aminotransferase; MELD, Model for End-Stage Liver Disease; AFP, alpha-fetoprotein; PIVKA-II, protein induced by vitamin K absence-II; VETC, vessels-encapsulating tumor clusters; CK19, cytokeratin 19..
The staining intensity of FN was stronger in the HCCs than in the adjacent parenchyma (Fig. 2). In nonneoplastic livers, moderate cytoplasmic or sinusoidal staining was rarely observed (n=3, 0.6% and n=4, 0.7%, respectively), while strong cytoplasmic/sinusoidal staining or moderate-to-strong membranous staining were not identified. In contrast, a much greater proportion of HCCs exhibited moderate or strong FN staining (cytoplasmic, n=59, 10.8%; membranous, n=77, 14.2%; sinusoidal, n=198, 36.5%). FN expression in the nonneoplastic tissue was mostly negative in the cytoplasm (n=225, 81.8%) and membrane (n=248, 90.2%) of hepatocytes, and predominantly negative or faint in sinusoidal endothelial cells (n=212, 77.1%). On the other hand, negative cytoplasmic or membranous staining was observed in a much smaller proportion of tumor tissue (n=296, 54.5% and n=255, 47.0%, respectively), with only 8.5% of tumors showing negative sinusoidal staining (n=46). Based on these staining patterns, moderate or strong FN staining was considered indicative of FN expression (FN-positive).
Figure 2. Comparison of staining pattern and intensity of fibronectin between hepatocellular carcinoma and nonneoplastic livers. (A) Cytoplasmic staining intensity. (B) Membranous staining intensity. (C) Sinusoidal staining intensity. (D) Proportion of fibronectin expression pattern in tumor and nontumorous tissue. *p<0.001.
Cytoplasmic FN expression was more frequently observed in HCC (n=59, 10.9%) than in the adjacent parenchyma (n=3, 1.1%, p<0.001). Membranous FN expression was observed in tumor cells (n=77, 14.2%), but not in nonneoplastic hepatocytes (p<0.001). Sinusoidal FN expression was observed significantly more frequently in the tumors (n=198, 36.5%) than in the adjacent liver tissue (n=4, 1.5%, p<0.001). Among the 258 patients, cytoplasmic FN expression was observed in 36 patients (14%), membranous expression was identified in 50 (19%), and sinusoidal expression was observed in 119 (46%).
A comparison of the clinicopathological characteristics according to the FN expression patterns is presented in Table 2 and Supplementary Figs 1 and 2. Cytoplasmic FN expression was significantly associated with high serum PIVKA-II levels (p=0.021) and MVI (p=0.004) (Fig. 3). Membranous FN expression was significantly associated with high serum AFP (p<0.001) and PIVKA-II levels (p=0.045), infiltrative gross type (p=0.001), poor E-S grade (p=0.027), MVI (p=0.001), major vessel invasion (p=0.007), MTM subtype (p<0.001), high T stage (p<0.001), and VETC pattern (p=0.001). Sinusoidal FN expression was significantly associated with high serum AFP (p=0.029) and PIVKA-II levels (p=0.002), infiltrative gross type (p=0.034), large tumor size (p=0.013), MVI (p=0.038), MTM subtype (p-0.011), and VETC patterns (p<0.001).
Figure 3. Microvascular invasion (A), vessels-encapsulating tumor cluster (VETC) pattern (B) and macrotrabecular massive (MTM) subtype (C) according to fibronectin expression pattern. *p<0.05, †p<0.001.
Table 2 . Clinicopathological Characteristics According to the Fibronectin Expression Pattern.
| Variable | Cytoplasmic expression | Membranous expression | Sinusoidal expression | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Absent (n=222) | Present (n=36) | p-value | Absent (n=208) | Present (n=50) | p-value | Absent (n=139) | Present (n=119) | p-value | |||
| Clinical feature | |||||||||||
| Age ≥60 yr | 97 (43.7) | 15 (41.7) | 0.820 | 97 (46.6) | 15 (30.0) | 0.033* | 62 (44.6) | 50 (42.0) | 0.676 | ||
| Male sex | 187 (84.2) | 27 (75.0) | 0.172 | 172 (82.7) | 42 (84.0) | 0.825 | 119 (85.6) | 95 (79.8) | 0.219 | ||
| Underlying HBV infection | 184 (82.9) | 29 (80.6) | 0.733 | 170 (81.7) | 43 (86.0) | 0.475 | 119 (85.6) | 94 (79.0) | 0.162 | ||
| AFP ≥1,000 ng/mL | 43 (19.4) | 9 (25.0) | 0.435 | 32 (15.4) | 20 (40.0) | <0.001* | 21 (15.1) | 31 (26.1) | 0.029* | ||
| Pre-op PIVKA-II level ≥200 | 90 (40.5) | 22 (61.1) | 0.021* | 84 (40.4) | 28 (56.0) | 0.045* | 48 (34.5) | 64 (53.8) | 0.002* | ||
| Pathological findings | |||||||||||
| Underlying cirrhosis | 168 (75.7) | 24 (66.7) | 0.250 | 160 (76.9) | 32 (64.0) | 0.060 | 113 (81.3) | 79 (66.4) | 0.006* | ||
| Infiltrative gross type | 125 (56.3) | 26 (72.2) | 0.072 | 111 (53.4) | 40 (80.0) | 0.001* | 73 (52.5) | 78 (65.5) | 0.034* | ||
| Size >5 cm | 93 (41.9) | 18 (50.0) | 0.362 | 85 (40.9) | 26 (52.0) | 0.153 | 50 (36.0) | 61 (51.3) | 0.013* | ||
| Multiplicity | 115 (51.8) | 20 (55.6) | 0.676 | 105 (50.5) | 30 (60.0) | 0.226 | 79 (56.8) | 56 (47.1) | 0.117 | ||
| E-S grade III or IV | 165 (74.3) | 31 (86.1) | 0.125 | 152 (73.1) | 44 (88.0) | 0.027* | 99 (71.2) | 97 (81.5) | 0.054 | ||
| Microvascular invasion | 85 (38.3) | 23 (63.9) | 0.004* | 77 (37.0) | 31 (62.0) | 0.001* | 50 (36.0) | 58 (48.7) | 0.038* | ||
| Major vessel invasion | 37 (16.7) | 7 (19.4) | 0.681 | 29 (13.9) | 15 (30.0) | 0.007* | 21 (15.1) | 23 (19.3) | 0.369 | ||
| MTM subtype (n=175) | 19/147 (12.9) | 7/28 (25.0) | 0.142 | 13 (9.4) | 13 (35.1) | <0.001* | 8 (8.5) | 18 (22.2) | 0.011* | ||
| T stage | 0.050 | <0.001* | 0.152 | ||||||||
| T1a | 12 (5.4) | 1 (2.8) | 11 (5.3) | 2 (4.0) | 9 (6.5) | 4 (3.4) | |||||
| T1b | 52 (23.4) | 1 (2.8) | 50 (24.0) | 3 (6.0) | 27 (19.4) | 26 (21.8) | |||||
| T2 | 103 (46.4) | 24 (66.7) | 102 (49.0) | 25 (50.0) | 70 (50.4) | 57 (47.9) | |||||
| T3 | 28 (12.6) | 3 (8.3) | 25 (12.0) | 6 (12.0) | 22 (15.8) | 9 (7.6) | |||||
| T4 | 27 (12.2) | 7 (19.4) | 20 (9.6) | 14 (28.0) | 11 (7.9) | 23 (19.3) | |||||
| VETC pattern | 49 (22.1) | 12 (33.3) | 0.140 | 40 (19.2) | 21 (42.0) | 0.001* | 14 (10.1) | 47 (39.5) | <0.001* | ||
| CK19 positivity | 16 (7.2) | 2 (5.6) | 1.000 | 14 (6.7) | 4 (8.0) | 0.758 | 11 (7.9) | 7 (5.9) | 0.523 | ||
Data are presented as number (%)..
HBV, hepatitis B virus; AFP, alpha-fetoprotein; Pre-op, preoperative; PIVKA-II, protein induced by vitamin K absence-II; E-S, Edmondson-Steiner; MTM, macrotrabecular massive; VETC, vessels-encapsulating tumor clusters; CK19, cytokeratin 19..
*Indicates p<0.05..
OS and DFS in patients with HCCs showing membranous FN expression were significantly shorter than those in patients without membranous expression (p=0.003 in both cases) (Fig. 4, Supplementary Fig. 3). There was no significant difference in OS and DFS between patients with cytoplasmic or sinusoidal FN-positive HCC and those without cytoplasmic or sinusoidal expression. DFS in patients with tumors showing sinusoidal FN expression tended to be shorter than that in those without sinusoidal expression (p=0.067). Univariable analysis showed that serum AFP ≥1,000 ng/mL, PIVKA-II ≥200 mAU/mL, infiltrative gross type, tumor size >5 cm, E-S grade III or IV, MVI, major vessel invasion, MTM subtype, cytokeratin 19 positivity, and membranous FN expression were significant prognostic factors for OS and DFS. In the multivariate analysis, tumor size >5 cm, E-S grade III or IV, and cytokeratin 19 positivity were significant independent factors for predicting OS and DFS (Table 3).
Figure 4. Kaplan-Meier survival curve for overall survival (A-C) and disease-free survival (D-F) according to fibronectin expression pattern.
Table 3 . Univariable and Multivariable Analyses of Clinical and Histopathological Features for Overall and Disease-Free Survival.
| Variable | Overall survival | Disease-free survival | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Univariable analysis | Multivariable analysis | Univariable analysis | Multivariable analysis | ||||||||
| HR (95% CI) | p-value | HR (95% CI) | p-value | HR (95% CI) | p-value | HR (95% CI) | p-value | ||||
| Clinical feature | |||||||||||
| Age ≥60 yr | 1.038 (0.723–1.490) | 0.841 | 0.831 (0.601–1.149) | 0.262 | |||||||
| Male sex | 1.015 (0.621–1.658) | 0.953 | 1.010 (0.658–1.550) | 0.965 | |||||||
| Underlying HBV infection | 0.997 (0.622–1.597) | 0.990 | 1.056 (0.697–1.601) | 0.797 | |||||||
| AFP ≥1,000 ng/mL | 2.148 (1.428–3.231) | <0.001* | 2.314 (1.609–3.327) | <0.001* | 1.590 (1.028–2.459) | 0.037* | |||||
| Pre-op PIVKA-II level ≥200 | 1.956 (1.362–2.810) | <0.001* | 2.067 (1.502–2.845) | <0.001* | |||||||
| Pathological findings | |||||||||||
| Underlying cirrhosis | 0.814 (0.544–1.218) | 0.317 | 0.776 (0.546–1.102) | 0.156 | |||||||
| Infiltrative gross type | 2.016 (1.362–2.983) | <0.001* | 1.516 (0.964–2.385) | 0.072 | 1.580 (1.135–2.200) | 0.007* | 1.445 (0.966–2.160) | 0.073 | |||
| Size >5 cm | 2.346 (1.629–3.378) | <0.001* | 3.020 (1.937–4.707) | <0.001* | 2.719 (1.968–3.756) | <0.001* | 3.237 (2.123–4.934) | <0.001* | |||
| Multiplicity | 1.546 (1.073–2.228) | 0.019* | 1.675 (1.056–2.659) | 0.028* | 0.914 (0.665–1.255) | 0.577 | |||||
| E-S grade III or IV | 3.067 (1.780–5.286) | <0.001* | 2.074 (1.135–3.789) | 0.018* | 2.208 (1.436–3.394) | <0.001* | 2.528 (1.464–4.364) | 0.001* | |||
| Microvascular invasion | 2.478 (1.715–3.580) | <0.001* | 2.390 (1.733–3.298) | <0.001* | |||||||
| Major vessel invasion | 2.817 (1.872–4.239) | <0.001* | 3.169 (2.186–4.596) | <0.001* | 1.697 (1.071–2.688) | 0.024* | |||||
| MTM subtype (n=175) | 2.108 (1.237–3.591) | 0.006* | 2.796 (1.755–4.453) | <0.001* | |||||||
| VETC pattern | 1.400 (0.936–2.095) | 0.101 | 1.317 (0.920–1.885) | 0.132 | |||||||
| CK19 positivity | 3.427 (1.947–6.030) | <0.001* | 2.771 (1.499–5.124) | 0.001* | 2.261 (1.301–3.931) | 0.004* | 1.870 (0.998–3.502) | 0.051 | |||
| Fibronectin expression | |||||||||||
| Cytoplasmic | 1.192 (0.730–1.947) | 0.483 | 1.255 (0.805–1.955) | 0.316 | |||||||
| Membranous | 1.852 (1.227–2.794) | 0.003* | 1.728 (1.192–2.504) | 0.004* | |||||||
| Sinusoidal | 1.341 (0.936–1.922) | 0.110 | 1.337 (0.974–1.836) | 0.073 | |||||||
HR, hazard ratio; CI, confidence interval; HBV, hepatitis B virus; AFP, alpha-fetoprotein; Pre-op, preoperative; PIVKA-II, protein induced by vitamin K absence-II; E-S, Edmondson-Steiner; MTM, macrotrabecular massive; VETC, vessels-encapsulating tumor clusters; CK19, cytokeratin 19..
*Indicates p<0.05..
We analyzed FN1 mRNA expression in The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) cohort (Supplementary Fig. 4). On comparison between tumor and normal tissues from 49 HCC patients for whom matched normal tissue samples were available, we found slightly higher FN1 mRNA expression levels in HCCs, although not statistically significant (p=0.249). For survival analysis, patients were divided into high and low FN1 groups based on the median value of FN1 mRNA expression, and we found that OS was significantly decreased in the high FN1 expression group (p=0.027). In addition, we compared FN1 mRNA expression levels according to the vascular invasion status; however, there was no significant difference in FN1 expression levels between cases with and without vascular invasion.
This study revealed that FN was overexpressed in HCC compared to nontumorous liver tissue in three patterns: (1) cytoplasmic, (2) membranous, and (3) sinusoidal. FN expression was significantly associated with MVI and other aggressive clinicopathological features including high serum AFP and PIVKA-II levels, infiltrative gross type, large tumor size, poor histological differentiation, and major vessel invasion. Membranous and sinusoidal FN expression was significantly associated with the MTM subtype and VETC pattern of vascularization. Membranous FN expression was a significant predictive factor for poor OS and DFS.
HCC is an aggressive tumor, and vascular invasion is an important prognostic predictor in several staging systems. However, aggressive HCC with MVI may be missed because most HCCs are diagnosed based on clinical and imaging findings without biopsy, and MVI cannot be accurately evaluated based on imaging findings.27 The VETC pattern, a distinct vascularization pattern of HCC, has recently been reported to be associated with poor prognostic factors, including MVI, and is enriched in the MTM subtype, which is an aggressive subtype of HCC.28-33 Thus, the importance of evaluating VETC and macrotrabecular patterns in a biopsy, which are unique and relatively easily identifiable but still challenging to detect in a small specimen, has been emerging. A biomarker beneficial for identifying these patterns would help triage the aggressive HCC group with poor prognosis.
FN is an extracellular matrix glycoprotein involved in various functions. FN has been shown to significantly impact disease pathogenesis, particularly by promoting proliferation, invasion, and metastasis through interactions with integrins and other cell surface receptors.5,34-36 Several studies have revealed the poor prognostic effect of FN expression in various types of cancer.5,11-17 In HCC, a few studies demonstrated that serum FN levels increased in patients with early HCC and decreased after treatment,6,37 and cellular FN was upregulated in HCC tumor cells.19 In this study, we demonstrated FN overexpression in HCC, and highlighted its expression patterns and significance. Among the three FN expression patterns, the membranous pattern showed a broader range of associations with the aggressive parameters compared to the cytoplasmic and sinusoidal patterns. FN overexpression on the cell membrane can more easily facilitate the interactions with cell surface receptors, including integrins, leading to the activation of downstream signaling pathways involved in tumor growth, angiogenesis, and metastasis. In consequence, it might contribute to a stronger association with aggressive clinicopathological parameters. This suggests that the localization of FN is crucial for its functional role, and further research is needed to elucidate the exact mechanisms and pathways through which FN exerts its effects.
FN is recently proposed as a potential biomarker for vascular invasion in HCC.18 Cellular FN is a well-known structural element of angiogenesis in embryogenesis and wound healing and is involved in the formation of tumor vessels as well.38-43 Several studies have indicated that cellular FN upregulation is associated with MVI.19 This study revealed the significant association between vascular invasion and FN expression. The mechanism of FN in tumor angiogenesis provides a ridged structure for neovascular lumen formation and the binding of vascular endothelial growth factor to maintain a directional concentration gradient for blood vessel formation.38,44,45 This may explain why the FN is related to the vascularization pattern in HCC.
There have been studies on FN as a molecular target for therapy, although its usefulness is still unclear. The method of conjugating a drug to extra-domain A or extra-domain B antibodies for drug delivery to malignant cells, which are contained in cellular FN, is expected to be promising given that extra-domain A or extra-domain B is limitedly expressed in malignancy.39,46,47
To validate our results, we analyzed FN expression using the external bulk RNA sequencing data of the TCGA-LIHC cohort. Although we found slightly higher FN levels in HCCs compared to paired non-neoplastic livers and decreased survival for the high FN group, there was no significant difference in vascular invasion status according to FN expression status. The discrepancy between our data and the TCGA analysis results could be explained by the fact that RNA sequencing measures the average RNA levels across the entire tissue, while immunohistochemistry reveals the distribution and localization of proteins within tissue. On immunohistochemistry, the expression in membranes of hepatocytes or in sinusoidal endothelial cells was most significant, and this suggests that for FN, the localization/distribution of protein expression could be more relevant than the level of expression. In addition, it may be possible that the post-transcriptional protein expression levels could exert a more significant influence than the RNA expression levels. To confirm this, further research analyzing the correlation between immunohistochemistry and RNA expression in the same tissue samples is necessary.
A limitation of this study is that this is a retrospective cohort study performed on archival formalin-fixed paraffin-embedded tissues from resected HCC specimens, and serum samples were not available for serum FN analysis. A prospective study would be necessary in order to correlate the tissue FN expression with the serum FN levels.
In conclusion, FN expression was associated with MVI and aggressive clinicopathological parameters in HCC; thus, FN may be a potential biomarker for an aggressive group of HCC with MVI, especially in the biopsy setting, and a potential molecular target for therapy.
This was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2022R1A2C2010348).
S.J.Y. is an editorial board member of the journal but was not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.
Study concept and design: H.K., S.J.Y. Data acquisition: S.J.Y., S.K.H. Data analysis and interpretation: Y.J.H., H.L., H.K. Drafting of the manuscript: Y.J.H. Critical revision of the manuscript for important intellectual content: H.K., S.J.Y. Statistical analysis: Y.J.H. Obtained funding: H.K. Administrative, technical, or material support; study supervision: H.K., S.J.Y. Approval of final manuscript: all authors.
Supplementary materials can be accessed at https://doi.org/10.5009/gnl240254.
Table 1 Clinicopathological Characteristics
| Characteristic | Total (n=258) |
|---|---|
| Clinical features | |
| Age, yr | 59 (51–64) |
| Sex | |
| Male | 214 (82.9) |
| Female | 44 (17.1) |
| Etiology | |
| Hepatitis B | 208 (80.6) |
| Hepatitis C | 15 (5.8) |
| Alcohol | 6 (2.3) |
| Hepatitis B+hepatitis C | 3 (1.2) |
| Hepatitis B+alcohol | 2 (0.8) |
| Unknown | 24 (9.3) |
| Preoperative locoregional treatment | 115 (44.6) |
| TACE | 101 (39.1) |
| RFA | 23 (8.9) |
| PEIT | 23 (8.9) |
| PT-INR | 1.10 (1.04–1.25) |
| Albumin, g/L | 3.8 (3.2–4.2) |
| Bilirubin, mg/dL | 1.1 (0.7–2.1) |
| AST, U/L | 39 (31–64) |
| ALT, U/L | 35 (25–57) |
| Child Pugh score | |
| A | 189 (73.3) |
| B | 50 (19.4) |
| C | 19 (7.4) |
| MELD score (n=238) | 6.7 (6.5–7.4) |
| AFP, ng/mL | 26 (6–430) |
| PIVKA-II, mAU/mL | 120 (28–993) |
| Pathological findings | |
| Underlying cirrhosis | 192 (74.4) |
| Infiltrative gross type | 151 (58.5) |
| Tumor size, cm | 4.0 (2.5–8.0) |
| Multiplicity | 135 (52.3) |
| Edmondson-Steiner grade | |
| I | 8 (3.1) |
| II | 54 (20.9) |
| III | 122 (47.3) |
| IV | 73 (28.3) |
| Microvascular invasion | 108 (41.9) |
| Major vessel invasion | 44 (17.1) |
| Macrotrabecular massive subtype (n=175) | 26 (14.9) |
| T stage | |
| 1a | 13 (5.0) |
| 1b | 53 (20.5) |
| 2 | 127 (49.2) |
| 3 | 31 (12.0) |
| 4 | 34 (13.2) |
| VETC pattern | 61 (23.6) |
| CK19 positivity | 18 (7.0) |
| Fibronectin expression | |
| Cytoplasmic | 36 (14.0) |
| Membranous | 50 (19.4) |
| Sinusoidal | 119 (46.1) |
Data are presented as median (interquartile range) or number (%).
TACE, transarterial chemoembolization; RFA, radiofrequency ablation; PEIT, percutaneous ethanol injection therapy; PT-INR, prothrombin time-international normalized ratio; AST, aspartate aminotransferase; ALT, alanine aminotransferase; MELD, Model for End-Stage Liver Disease; AFP, alpha-fetoprotein; PIVKA-II, protein induced by vitamin K absence-II; VETC, vessels-encapsulating tumor clusters; CK19, cytokeratin 19.
Table 2 Clinicopathological Characteristics According to the Fibronectin Expression Pattern
| Variable | Cytoplasmic expression | Membranous expression | Sinusoidal expression | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Absent (n=222) | Present (n=36) | p-value | Absent (n=208) | Present (n=50) | p-value | Absent (n=139) | Present (n=119) | p-value | |||
| Clinical feature | |||||||||||
| Age ≥60 yr | 97 (43.7) | 15 (41.7) | 0.820 | 97 (46.6) | 15 (30.0) | 0.033* | 62 (44.6) | 50 (42.0) | 0.676 | ||
| Male sex | 187 (84.2) | 27 (75.0) | 0.172 | 172 (82.7) | 42 (84.0) | 0.825 | 119 (85.6) | 95 (79.8) | 0.219 | ||
| Underlying HBV infection | 184 (82.9) | 29 (80.6) | 0.733 | 170 (81.7) | 43 (86.0) | 0.475 | 119 (85.6) | 94 (79.0) | 0.162 | ||
| AFP ≥1,000 ng/mL | 43 (19.4) | 9 (25.0) | 0.435 | 32 (15.4) | 20 (40.0) | <0.001* | 21 (15.1) | 31 (26.1) | 0.029* | ||
| Pre-op PIVKA-II level ≥200 | 90 (40.5) | 22 (61.1) | 0.021* | 84 (40.4) | 28 (56.0) | 0.045* | 48 (34.5) | 64 (53.8) | 0.002* | ||
| Pathological findings | |||||||||||
| Underlying cirrhosis | 168 (75.7) | 24 (66.7) | 0.250 | 160 (76.9) | 32 (64.0) | 0.060 | 113 (81.3) | 79 (66.4) | 0.006* | ||
| Infiltrative gross type | 125 (56.3) | 26 (72.2) | 0.072 | 111 (53.4) | 40 (80.0) | 0.001* | 73 (52.5) | 78 (65.5) | 0.034* | ||
| Size >5 cm | 93 (41.9) | 18 (50.0) | 0.362 | 85 (40.9) | 26 (52.0) | 0.153 | 50 (36.0) | 61 (51.3) | 0.013* | ||
| Multiplicity | 115 (51.8) | 20 (55.6) | 0.676 | 105 (50.5) | 30 (60.0) | 0.226 | 79 (56.8) | 56 (47.1) | 0.117 | ||
| E-S grade III or IV | 165 (74.3) | 31 (86.1) | 0.125 | 152 (73.1) | 44 (88.0) | 0.027* | 99 (71.2) | 97 (81.5) | 0.054 | ||
| Microvascular invasion | 85 (38.3) | 23 (63.9) | 0.004* | 77 (37.0) | 31 (62.0) | 0.001* | 50 (36.0) | 58 (48.7) | 0.038* | ||
| Major vessel invasion | 37 (16.7) | 7 (19.4) | 0.681 | 29 (13.9) | 15 (30.0) | 0.007* | 21 (15.1) | 23 (19.3) | 0.369 | ||
| MTM subtype (n=175) | 19/147 (12.9) | 7/28 (25.0) | 0.142 | 13 (9.4) | 13 (35.1) | <0.001* | 8 (8.5) | 18 (22.2) | 0.011* | ||
| T stage | 0.050 | <0.001* | 0.152 | ||||||||
| T1a | 12 (5.4) | 1 (2.8) | 11 (5.3) | 2 (4.0) | 9 (6.5) | 4 (3.4) | |||||
| T1b | 52 (23.4) | 1 (2.8) | 50 (24.0) | 3 (6.0) | 27 (19.4) | 26 (21.8) | |||||
| T2 | 103 (46.4) | 24 (66.7) | 102 (49.0) | 25 (50.0) | 70 (50.4) | 57 (47.9) | |||||
| T3 | 28 (12.6) | 3 (8.3) | 25 (12.0) | 6 (12.0) | 22 (15.8) | 9 (7.6) | |||||
| T4 | 27 (12.2) | 7 (19.4) | 20 (9.6) | 14 (28.0) | 11 (7.9) | 23 (19.3) | |||||
| VETC pattern | 49 (22.1) | 12 (33.3) | 0.140 | 40 (19.2) | 21 (42.0) | 0.001* | 14 (10.1) | 47 (39.5) | <0.001* | ||
| CK19 positivity | 16 (7.2) | 2 (5.6) | 1.000 | 14 (6.7) | 4 (8.0) | 0.758 | 11 (7.9) | 7 (5.9) | 0.523 | ||
Data are presented as number (%).
HBV, hepatitis B virus; AFP, alpha-fetoprotein; Pre-op, preoperative; PIVKA-II, protein induced by vitamin K absence-II; E-S, Edmondson-Steiner; MTM, macrotrabecular massive; VETC, vessels-encapsulating tumor clusters; CK19, cytokeratin 19.
*Indicates p<0.05.
Table 3 Univariable and Multivariable Analyses of Clinical and Histopathological Features for Overall and Disease-Free Survival
| Variable | Overall survival | Disease-free survival | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Univariable analysis | Multivariable analysis | Univariable analysis | Multivariable analysis | ||||||||
| HR (95% CI) | p-value | HR (95% CI) | p-value | HR (95% CI) | p-value | HR (95% CI) | p-value | ||||
| Clinical feature | |||||||||||
| Age ≥60 yr | 1.038 (0.723–1.490) | 0.841 | 0.831 (0.601–1.149) | 0.262 | |||||||
| Male sex | 1.015 (0.621–1.658) | 0.953 | 1.010 (0.658–1.550) | 0.965 | |||||||
| Underlying HBV infection | 0.997 (0.622–1.597) | 0.990 | 1.056 (0.697–1.601) | 0.797 | |||||||
| AFP ≥1,000 ng/mL | 2.148 (1.428–3.231) | <0.001* | 2.314 (1.609–3.327) | <0.001* | 1.590 (1.028–2.459) | 0.037* | |||||
| Pre-op PIVKA-II level ≥200 | 1.956 (1.362–2.810) | <0.001* | 2.067 (1.502–2.845) | <0.001* | |||||||
| Pathological findings | |||||||||||
| Underlying cirrhosis | 0.814 (0.544–1.218) | 0.317 | 0.776 (0.546–1.102) | 0.156 | |||||||
| Infiltrative gross type | 2.016 (1.362–2.983) | <0.001* | 1.516 (0.964–2.385) | 0.072 | 1.580 (1.135–2.200) | 0.007* | 1.445 (0.966–2.160) | 0.073 | |||
| Size >5 cm | 2.346 (1.629–3.378) | <0.001* | 3.020 (1.937–4.707) | <0.001* | 2.719 (1.968–3.756) | <0.001* | 3.237 (2.123–4.934) | <0.001* | |||
| Multiplicity | 1.546 (1.073–2.228) | 0.019* | 1.675 (1.056–2.659) | 0.028* | 0.914 (0.665–1.255) | 0.577 | |||||
| E-S grade III or IV | 3.067 (1.780–5.286) | <0.001* | 2.074 (1.135–3.789) | 0.018* | 2.208 (1.436–3.394) | <0.001* | 2.528 (1.464–4.364) | 0.001* | |||
| Microvascular invasion | 2.478 (1.715–3.580) | <0.001* | 2.390 (1.733–3.298) | <0.001* | |||||||
| Major vessel invasion | 2.817 (1.872–4.239) | <0.001* | 3.169 (2.186–4.596) | <0.001* | 1.697 (1.071–2.688) | 0.024* | |||||
| MTM subtype (n=175) | 2.108 (1.237–3.591) | 0.006* | 2.796 (1.755–4.453) | <0.001* | |||||||
| VETC pattern | 1.400 (0.936–2.095) | 0.101 | 1.317 (0.920–1.885) | 0.132 | |||||||
| CK19 positivity | 3.427 (1.947–6.030) | <0.001* | 2.771 (1.499–5.124) | 0.001* | 2.261 (1.301–3.931) | 0.004* | 1.870 (0.998–3.502) | 0.051 | |||
| Fibronectin expression | |||||||||||
| Cytoplasmic | 1.192 (0.730–1.947) | 0.483 | 1.255 (0.805–1.955) | 0.316 | |||||||
| Membranous | 1.852 (1.227–2.794) | 0.003* | 1.728 (1.192–2.504) | 0.004* | |||||||
| Sinusoidal | 1.341 (0.936–1.922) | 0.110 | 1.337 (0.974–1.836) | 0.073 | |||||||
HR, hazard ratio; CI, confidence interval; HBV, hepatitis B virus; AFP, alpha-fetoprotein; Pre-op, preoperative; PIVKA-II, protein induced by vitamin K absence-II; E-S, Edmondson-Steiner; MTM, macrotrabecular massive; VETC, vessels-encapsulating tumor clusters; CK19, cytokeratin 19.
*Indicates p<0.05.
pISSN 1976-2283
eISSN 2005-1212
Gut and Liver is an international journal of gastroenterology, focusing on the gastrointestinal tract, liver, biliary tree, pancreas, motility, and neurogastroenterology. Gut atnd Liver delivers up-to-date, authoritative papers on both clinical and research-based topics in gastroenterology. The Journal publishes original articles, case reports, brief communications, letters to the editor and invited review articles in the field of gastroenterology. The Journal is operated by internationally renowned editorial boards and designed to provide a global opportunity to promote academic developments in the field of gastroenterology and hepatology. +MORE
| Yong Chan Lee |
Professor of Medicine Director, Gastrointestinal Research Laboratory Veterans Affairs Medical Center, Univ. California San Francisco San Francisco, USA |
| Jong Pil Im | Seoul National University College of Medicine, Seoul, Korea |
| Robert S. Bresalier | University of Texas M. D. Anderson Cancer Center, Houston, USA |
| Steven H. Itzkowitz | Mount Sinai Medical Center, NY, USA |
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Supplementary 1
