Contributed equally
Tumor budding (TB) has become a crucial factor for predicting the malignancy grade and prognostic outcome for multiple types of solid cancer. Studies have investigated the prognostic value of TB in hepatocellular carcinoma (HCC). However, its molecular mechanism in HCC remains unclear. To the best of our knowledge, the present study was the first to compare the expression of differentially expressed genes (DEGs) between TB-positive (TB-pos) and TB-negative HCC tissues. In the present study, total RNA was extracted from 40 HCC tissue specimens and then sequenced. According to Gene Ontology (GO) functional annotation, upregulated DEGs were markedly associated with embryonic kidney development-related GO terms, which suggested that the TB process may at least partly mimic the process of embryonic kidney development. Subsequently, two genes, a disintegrin and metalloproteinase domain with thrombospondin motifs 16 (ADAMTS16) and bone morphogenetic protein 2 (BMP2), were screened and verified through immunohistochemical analysis of HCC tissue microarrays. According to the immunohistochemical results, ADAMTS16 and BMP2 were upregulated in TB-pos HCC samples, and BMP2 expression was increased in budding cells compared with the tumor center. Additionally, through cell culture experiments, it was demonstrated that ADAMTS16 and BMP2 may promote TB of liver cancer, thus promoting the malignant progression of liver cancer. Further analysis revealed that ADAMTS16 expression was associated with necrosis and cholestasis, and BMP2 expression was associated with the Barcelona Clinic Liver Cancer stage and the vessels encapsulating tumor clusters. Overall, the findings of the present study provided insights into the possible mechanisms of TB in HCC and revealed potential anti-HCC therapeutic targets.
According to the 2020 Global Cancer Statistics, liver cancer ranks 6th in terms of global morbidity and 3rd among causes of cancer-associated mortality worldwide (
A disintegrin and metalloproteinase domain with thrombospondin motifs 16 (ADAMTS16) belongs to the ADAMTS family. Its role has been investigated in esophageal squamous cell carcinoma (
The aim of this study was to identify differentially expressed genes (DEGs) in TB-positive (TB-pos) HCC tissue samples relative to TB-negative (TB-neg) HCC tissue samples through bioinformatics analysis, and then explore the potential mechanism of TB in HCC. In the present study, it was demonstrated that ADAMTS16 and BMP2 levels were significantly increased in the TB-pos HCC samples, and BMP2 expression was significantly increased in budding cells compared with the tumor center. Additionally, it was demonstrated that overexpression (OE) of ADAMTS16 and BMP2 promoted invasion of HepG2 cells, which implied that ADAMTS16 and BMP2 may regulate TB in liver cancer. The association of ADAMTS16 and BMP2 expression with clinicopathological characteristics and patient survival time were also analyzed. The findings of the present study provide novel mechanistic insights into the role of ADAMTS16 and BMP2 in HCC.
Paraffin-embedded tumor specimens from patients with HCC (n=308) were retrospectively collected from The Affiliated Hospital of Jining Medical University (Jining, China) between June 2013 and August 2019. The clinical and pathological details of the patients were retrospectively reviewed by accessing the hospital's existing electronic medical record system. Ultimately, 266/308 (86%) of patients with HCC with complete clinical information were selected for the subsequent clinical significance analysis. In addition, of the 308 patients, 66 were excluded from the survival analysis due to a lack of follow-up. And additional 40 HCC tissue samples were prospectively collected from The Affiliated Hospital of Jining Medical University between February 2020 and June 2021 for transcriptome sequencing. All samples were histopathologically and clinically confirmed as HCC tissues. The histology, diagnostic methods, and α-fetoprotein (AFP) levels were reviewed for each case of HCC identified to eliminate cases of metastatic cancer or other primary liver cancer types. Microscopically, the HCC cells were arranged in solid nests, trabeculae, acinar or pseudoglandular structures. Papillary structures were occasionally seen. Abundant sinusoidal capillaries could be seen between the tumor alveolus, with steatosis and eosinophilic bodies. Ethics approval was provided by The Ethics Committee of The Affiliated Hospital of Jining Medical University (Jining, China; ethical approval no. 2021C145). Each participant (>18 years of age) in the study provided written informed consent and signed informed consent forms.
The human hepatoblastoma cell line, HepG2, was purchased from ATCC via the Shanghai Huiying Biological Technology Co., Ltd. The cell line was authenticated by human STR profiling and confirmed to be mycoplasma free. HepG2 cells were cultured in DMEM (Nanjing KeyGen Biotech Co., Ltd.) containing 10% fetal bovine serum (FBS; Biological Industries) and 1% penicillin-streptomycin (Nanjing KeyGen Biotech Co., Ltd.), and incubated in a humidified 5% CO2 incubator at 37°C.
The lentiviral vectors, GV513-ADAMTS16 (Ubi-MCS-CBh-gcGFP-IRES-puromycin) and GV358-BMP2 (Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin), and the corresponding control lentiviruses containing the empty vector, CON335 and CON238, respectively, were purchased from Shanghai GeneChem Co., Ltd. Accession numbers for the two genes used in the present study are as follows: ADAMTS16, NM139056; BMP2, NM001200. For lentiviral infection, HepG2 cells were incubated with lentivirus at a MOI of 10 for 16 h, and the stable cell line was selected using 2 µg/ml puromycin for a week, followed by 2 µg/ml puromycin maintenance. The overexpression efficiency of ADAMTS16 and BMP2 were assessed through reverse transcription-quantitative PCR (RT-qPCR).
In total, 40 HCC tissue samples were divided into TB-pos and TB-neg groups by two independent pathologists on the basis of hematoxylin-eosin (HE) staining for transcriptome sequencing, which was conducted by Berry Genomics Co., Ltd. Briefly, using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (E7770S, New England Biolabs, Inc.), total RNA (≥1 µg) from each sample was used for generating sequencing libraries in-line with the manufacturer's instructions. Oligo (dT) magnetic beads were used to enrich and purify poly A-containing mRNA, followed by random interruption of mRNA into short fragments, which served as templates for random-primed cDNA synthesis performed using reverse transcriptase. Subsequently, purified double-stranded cDNA was subjected to end-repair, A-tailing and adapter ligation. Moreover, AMPure XP beads (Beckman Coulter, Beverly, USA) were used to purify cDNA library fragments for selecting the 250-300 bp cDNA fragments. Lastly, PCR amplification was performed by ABI Q3TMReal-Time PCR System, followed by purification of PCR products using the AMPure XP beads for obtaining the cDNA library.
Following library construction, the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Inc.) was used to quantify the library before diluting to 1.5 ng/µl. Then, the Agilent 2100 Bioanalyzer was used to assess the library insert size. When the expected insert size was obtained, qPCR was conducted to precisely quantify library effective concentration (>2 nM) for ensuing library quality. After the library quality was confirmed, the Illumina platform was used for sequencing to generate 150 bp paired end reads.
The edgeR software package (version 3.28.1;
Total RNA from the HepG2 cells was extracted using TRIzol reagent (Ambion; Thermo Fisher Scientific, Inc.). Reverse transcription was conducted using the HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd.) according to the manufacturer's protocol. The reverse transcription reactions were performed at 42°C for 2 min, followed by 37°C for 15 min and 85°C for 5 sec. qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.) in a CFX Connect Real-time System (Bio-Rad Laboratories Inc.). GAPDH was employed as the internal reference and the 2−∆∆Cq method was used for quantification (
The inverse Matrigel invasion assays were performed as reported previously (
Spheroid-based sprouting assays were performed as previously described (
In total, 308 formalin-fixed, paraffin-embedded tissue samples from patients with HCC were used to establish HCC tissue microarrays (TMAs) with a 2.0-mm diameter per core for IHC. In brief, TMAs were dewaxed, hydrated and antigen repaired by EDTA antigen repair buffer (PH 8.0), and the endogenous peroxidase activity was then blocked. TMAs were blocked in goat serum (OriGene Technologies, Inc.) at room temperature for 30 min to block non-specific staining. Primary antibodies were incubated with the TMAs overnight at 4°C, including ADAMTS16 (1:200; cat. no. DF9173; Affinity Biosciences) and BMP2 (1:200; cat. no. A0231; ABclonal Biotech Co., Ltd.). Subsequently, a horseradish peroxidase-labeled secondary antibody (KIT-5020; Maxim Co., Ltd., Fuzhou, China) was incubated with the TMAs for 30 min at room temperature. Finally, a chromogenic reaction was developed with DAB kit (DAB-0031 (20×); Fuzhou Maixin Biotech Co., Ltd.), followed by counterstaining with hematoxylin (G1120; Beijing Solarbio Science & Technology Co., Ltd.) for 10-30 sec at room temperature. All IHC staining analyses were assessed using a semi-quantitative scoring approach by two senior pathologists. IHC scores were calculated by multiplying staining intensity with the stained area, in which the staining intensity was scored as follows: 0 (no staining), 1 (light yellow staining), 2 (yellow-brown staining) and 3 (brown staining). The staining area was scored as follows: 1 (1–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%), according to the percentage of stained area in the field of vision. The median IHC score of ADAMTS16 or BMP2 was used as a cut-off to divide the samples into the low expression and high expression groups. ‘High’ was defined an IHC score higher than the cut-off value, and ‘low’ was defined as an IHC score lower than or equal the cut-off value.
SPSS 25.0 (IBM Corp.) was used for statistical analyses. Differences between two groups were analyzed using the Wilcoxon rank-sum test or unpaired student's t-test. The association of ADAMTS16 and BMP2 expressions with clinicopathological features was analyzed using the chi-square test or Fisher's exact test as appropriate. Kaplan-Meier (KM) curves were generated using the ‘survfit’ function in the survival package of R software (version 3.5.3), while significant differences in survival were compared using the log-rank test or Cramer-von Mises test as appropriate. Cramer-von Mises test was performed to generate the P-values when KM curves crossed over. P<0.05 was considered to indicate a statistically significant difference.
A total of 40 surgical HCC specimens were divided into two groups: TB-pos group (n=21) and TB-neg group (n=19), followed by transcriptome sequencing analysis. As shown in the volcano plot in
Results of GO (BP) enrichment analysis for downregulated genes are shown in
The details of BP enrichment of upregulated genes in the GO analysis are provided in
To identify ADAMTS16 and BMP2 expression profiles within HCC tissues with different TB statuses, IHC staining was performed using TMAs of 308 paraffin-embedded HCC tissues. In addition, the DEGs of the budding cells and cancer center were compared. The statistical analysis results for the IHC staining scores of ADAMTS16 and BMP2 expression are summarized in
To explore whether ADAMTS16 and BMP2 play a role in the TB of liver cancer, HepG2 cells with stable ADAMTS16-OE or BMP2-OE were constructed using lentiviral vectors. The overexpression efficiency was assessed through RT-qPCR assays (
To analyze the association of ADAMTS16 and BMP2 expression levels with HCC clinicopathological characteristics, 266 HCC cases with complete available clinicopathological information for analysis were enrolled. These 266 cases were classified into the low- or high-expression groups by considering the respective median IHC scores for ADAMTS16 and BMP2 expression as the cut-off. The relationship between the clinicopathological characteristics of patients and gene expression was assessed using the chi-square test and Fisher's exact test. Consequently, ADAMTS16 expression was found to be significantly associated with necrosis (P=0.023) and cholestasis (P=0.011) (
A total of 242 HCC cases with available follow-up data were enrolled for survival analysis. These 242 HCC cases were divided into two groups (low or high expression) based on the median IHC scores for ADAMTS16 or BMP2. As shown in
TB, a histological phenomenon observed in the tumor invasive edge, has been recognized as the initial stage of cancer invasion and metastasis (
In the present study, the expression profiles of 40 HCC tissues were analyzed using transcriptome sequencing technology. Functional annotation indicated that upregulated DEGs, obtained by comparing the gene expression of TB-pos and TB-neg HCC tissues, were mainly involved in embryonic kidney development, including nephron tubule development, kidney morphogenesis, renal tubule development and ureteric bud development. With advancements in developmental and cancer biology, numerous studies have suggested that cancer invasion and metastasis are similar to normal embryonic development (
The IHC results in the present study indicated an upregulation of ADAMTS16 and BMP2 expression in TB-pos HCC tissues compared with TB-neg HCC tissues. BMP2 expression in budding cells was also increased compared with that in the tumor center. Moreover, through the inverse Matrigel invasion and spheroid-based sprouting assays, the present study revealed that ADAMTS16 and BMP2 may regulate the TB process of liver cancer. Therefore, we hypothesize that these two genes may be associated with the malignant progression of liver cancer. Furthermore, the association of ADAMTS16 and BMP2 expression with clinicopathological characteristics of patients with HCC was analyzed, and an association between ADAMTS16 expression and necrosis and cholestasis was confirmed in. In addition, BMP2 expression was significantly associated with the BCLC stage and VETC.
It is well known that rapidly growing tumor cells, which represent an important malignant behavior of tumors, require an adequate supply of oxygen and nutrients, and the blood supply cannot meet this need of rapid tumor growth, eventually resulting in tumor necrosis (
As for BMP2, in the present study, patients with BCLC stage B-C had significantly higher BMP2 expression levels than patients with BCLC stage 0-A, suggesting the involvement of BMP2 in the progression and metastasis of HCC. Previous studies have demonstrated that BMP2 may promote HCC cell proliferation and invasion, thereby promoting malignant progression of HCC (
The overall survival analysis of 242 patients with HCC indicated that ADAMTS16 expression was associated with HCC prognosis whereas BMP2 expression was not associated with HCC prognosis. In addition, the survival data obtained in the present study is inconsistent with the results predicted using the GSE76427 dataset and the results predicted using GEPIA, which is a The Cancer Genome Atlas (TCGA)-based online tool. The results of the GEPIA analysis demonstrated that BMP2 upregulation was significantly associated with poor OS. The results of the GSE76427 dataset showed that neither ADAMTS16 expression nor BMP2 expression was associated with HCC prognosis. The possible reasons for this discrepancy are as follows: Firstly, sample size varied widely across these three cohorts. which may cause the inconsistent results. And compared with the large sample size within the TCGA cohort, the retrospective cohort of the present study and the GSE76427 cohort have relatively small sample sizes. Secondly, the sources of the tumor samples in the three cohorts were different. The majority of patients from TCGA database are white. In GSE76427 cohort, all of the patients were derived from Singapore. And our study is based on data collected from a single center (Affiliated Hospital of Jining Medical University, China). Thirdly, as patient characteristics, surgical skills and treatment regimens are different among countries, the final outcome of patients with HCC could be affected. The absence of animal models of HCC is also a limitation of this study, animal models of HCC will be constructed for further study validation in the future.
Regardless of these limitations, to the best of our knowledge, the present study was the first to investigate the TB-related molecular mechanism in HCC. The findings of the present study provide evidence for mechanism studies of TB. Moreover, the present study provides a basis for the potential application of ADAMTS16 and BMP2 as predictive diagnosis markers and treatment targets for HCC.
Not applicable.
The RNA-Seq datasets generated and/or analyzed during the current study are available in the GEO repository, the accession number is GSE227335. All other data generated or analyzed during this study are included in this published article.
DJ performed the experiments, data analysis and collection of clinical specimens, and drafted the manuscript. SX assisted with the data analysis and reverse transcription-quantitative PCR. CZ performed the bioinformatics analysis of the GEO database. CZ, CH and LL contributed to the analysis and interpretation of the data. MZ and HW contributed to the acquisition and interpretation of the data. DY and YL conceived and designed the study. YL revised the manuscript. DJ and SX confirm the authenticity of all the raw data. All authors have read and approved the final manuscript.
Written informed consent was obtained from all patients. The study was approved by The Ethics Committee of the Affiliated Hospital of Jining Medical University (ethical approval no. 2021C145; Jining, China).
Not applicable.
The authors declare that they have no competing interests.
Transcriptome sequencing of hepatocellular carcinoma tissues with different tumor budding statuses. (A) Volcano plot analysis of 95 upregulated DEGs and 150 downregulated DEGs. (B) Top 20 GO-BP terms for upregulated DEGs in TB-positive hepatocellular carcinoma tissues. (C) The top 20 GO-BP terms for downregulated DEGs in TB-positive hepatocellular carcinoma tissues. BPs, biological processes; DEGs, differentially expressed genes; FC, fold change; GO, Gene Ontology.
Immunohistochemical analysis of ADAMTS16 and BMP2 in patients with HCC with different TB statuses. (A) Representative IHC staining images of ADAMTS16 and BMP2 in TB-neg and TB-pos HCC tissues (magnification, ×100). Red boxes represent the tumor center and black arrows indicate budding tumor cells. (B) Representative IHC images of ADAMTS16 and BMP2 levels in the tumor center and budding cells (magnification, ×400), which are magnified views of the areas indicated by the red boxes and black arrows in (A), respectively. ADAMTS16, a metalloproteinase domain with thrombospondin motifs 16; BMP2, bone morphogenetic protein 2; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; neg, negative; pos, positive; TB, tumor budding.
ADAMTS16 and BMP2 promote the TB of liver cancer
KM survival curves for ADAMTS16 and BMP2 expression. (A) KM curves (Cramer-von Mises test) for the overall survival of patients with HCC in the ADAMTS16 high-expression group (n=93) vs. the ADAMTS16 low-expression group (n=149). (B) KM curves (Cramer-von Mises test) for overall survival of patients with HCC in the BMP2 high-expression group (n=82) vs. the BMP2 low-expression group (n=160). (C) KM curves (log-rank test) for overall survival of patients with HCC with high/low ADAMTS16 expression using The Cancer Genome Atlas data. (D) KM curves (log-rank test) for overall survival of patients with HCC with high/low BMP2 expression using The Cancer Genome Atlas data. (E) KM curves (log-rank test) for overall survival of patients with HCC with high/low ADAMTS16 expression using the GSE76427 dataset. (F) KM curves (Cramer-von Mises test) for overall survival of patients with HCC with high/low BMP2 expression. ADAMTS16, a metalloproteinase domain with thrombospondin motifs 16; BMP2, bone morphogenetic protein 2; HCC, hepatocellular carcinoma; KM, Kaplan-Meier; TPM, transcripts per million.
Association between a metalloproteinase domain with thrombospondin motifs 16 expression and TB in hepatocellular carcinoma tissues (n=308).
Two-sample Wilcoxon rank-sum test | ||||
---|---|---|---|---|
Group | Median (P25, P75) | Median difference (95% CI) | Z value | P-value |
TB | −1.000 (−1.000-0.000) | −2.802 | 0.005 |
|
TB-neg | 2 (1, 4) | |||
TB-pos | 3 (1, 6) | |||
Area | 0.000 (−2.000-0.000) | −1.359 | 0.174 | |
Tumor center | 3 (2, 6) | |||
Budding cells | 4 (3, 8) |
P<0.05. neg, negative; pos, positive; TB, tumor budding; P25, lower quartile; P75, upper quartile.
Association between bone morphogenetic protein 2 expression and TB in hepatocellular carcinoma tissues (n=308).
Two-sample Wilcoxon rank-sum test | ||||
---|---|---|---|---|
Group | Median (P25, P75) | Median difference (95% CI) | Z value | P-value |
TB | 0.000 (0.000-0.000) | −2.434 | 0.015 |
|
TB-neg | 8 (8, 9) | |||
TB-pos | 8 (8, 12) | |||
Area | 0.000 (−2.000-0.000) | −2.038 | 0.042 |
|
Tumor center | 8 (8, 12) | |||
Budding cells | 12 (8, 12) |
P<0.05. neg, negative; pos, positive; TB, tumor budding, P25, lower quartile; P75, upper quartile.
Association between ADAMTS16 expression and the clinicopathological characteristics in patients with hepatocellular carcinoma (n=266).
ADAMTS16 | |||
---|---|---|---|
Clinicopathological characteristics | Low, n (n=131) | High, n (n=135) | P-value |
Age, years | 0.848 | ||
>60 | 48 | 51 | |
≤60 | 83 | 84 | |
Sex | 0.359 | ||
Male | 105 | 114 | |
Female | 26 | 21 | |
HBV infection | 0.193 | ||
Positive | 116 | 112 | |
Negative | 15 | 23 | |
HCV infection | 0.122 | ||
Positive | 0 | 4 | |
Negative | 131 | 131 | |
AFP serum level, ng/ml | 0.407 | ||
>400 | 43 | 38 | |
≤400 | 88 | 97 | |
Liver cirrhosis (Yes vs. No) | 0.359 | ||
Yes | 106 | 103 | |
No | 25 | 32 | |
BCLC stage | 0.325 | ||
B-C | 21 | 16 | |
0-A | 110 | 119 | |
Tumor size, cm | 0.969 | ||
>5 | 55 | 57 | |
≤5 | 76 | 78 | |
Tumor number | 0.114 | ||
Multiple | 12 | 21 | |
Single | 119 | 114 | |
Intrahepatic metastasis | 0.114 | ||
Yes | 12 | 21 | |
No | 119 | 114 | |
Collective invasion | 0.693 | ||
Yes | 9 | 11 | |
No | 122 | 124 | |
Ki-67, % | 0.236 | ||
>30 | 31 | 24 | |
≤30 | 100 | 111 | |
Necrosis | 0.023 |
||
Yes | 28 | 15 | |
No | 103 | 120 | |
Vessel carcinoma embolus | 0.285 | ||
Yes | 17 | 12 | |
No | 114 | 123 | |
Microtrabecular pattern | 0.735 | ||
Yes | 106 | 107 | |
No | 25 | 28 | |
Macrotrabecular pattern | 0.780 | ||
Yes | 75 | 75 | |
No | 56 | 60 | |
Pseudoglandular pattern | 0.130 | ||
Yes | 23 | 34 | |
No | 108 | 101 | |
Compact pattern | 0.865 | ||
Yes | 53 | 56 | |
No | 78 | 79 | |
Cholestasis | 0.011 |
||
Yes | 17 | 34 | |
No | 114 | 101 | |
Hyaline bodies | 0.273 | ||
Yes | 23 | 31 | |
No | 108 | 104 | |
Steatosis | 0.908 | ||
Yes | 24 | 24 | |
No | 107 | 111 | |
Edmondson grade | 0.136 | ||
III–IV | 46 | 36 | |
I–II | 85 | 99 | |
VETC | 0.497 | ||
Yes | 33 | 39 | |
No | 98 | 96 |
P<0.05. ADAMTS16, a metalloproteinase domain with thrombospondin motifs 16; HBV, hepatitis B virus; HCV, hepatitis C virus; AFP, α-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; VETC, vessels-encapsulate tumor cluster.
Association between BMP2 expression and the clinicopathological characteristics in patients with hepatocellular carcinoma (n=266).
BMP2 | |||
---|---|---|---|
Clinicopathological characteristics | Low, n (n=172) | High, n (n=94) | P-value |
Age, years | 0.290 | ||
>60 | 68 | 31 | |
≤60 | 104 | 63 | |
Sex | 0.588 | ||
Male | 140 | 79 | |
Female | 32 | 15 | |
HBV infection | 0.105 | ||
Positive | 143 | 85 | |
Negative | 29 | 9 | |
HCV infection | 0.301 | ||
Positive | 4 | 0 | |
Negative | 168 | 94 | |
AFP serum level, ng/ml | 0.508 | ||
>400 | 50 | 31 | |
≤400 | 122 | 63 | |
Liver cirrhosis (Yes vs. No) | 0.789 | ||
Yes | 136 | 73 | |
No | 36 | 21 | |
BCLC stage | 0.003 |
||
B-C | 16 | 21 | |
0-A | 156 | 73 | |
Tumor size, cm | 0.054 | ||
>5 | 65 | 47 | |
≤5 | 107 | 47 | |
Tumor number | 0.091 | ||
Multiple | 17 | 16 | |
Single | 155 | 78 | |
Intrahepatic metastasis | 0.091 | ||
Yes | 17 | 16 | |
No | 155 | 78 | |
Collective invasion | 0.604 | ||
Yes | 14 | 6 | |
No | 158 | 88 | |
Ki-67, % | 0.417 | ||
>30 | 33 | 22 | |
≤30 | 139 | 72 | |
Necrosis | 0.530 | ||
Yes | 26 | 17 | |
No | 146 | 77 | |
Vessel carcinoma embolus | 0.123 | ||
Yes | 15 | 14 | |
No | 157 | 80 | |
Microtrabecular pattern | 0.683 | ||
Yes | 139 | 74 | |
No | 33 | 20 | |
Macrotrabecular pattern | 0.302 | ||
Yes | 93 | 57 | |
No | 79 | 37 | |
Pseudoglandular pattern | 0.964 | ||
Yes | 37 | 20 | |
No | 135 | 74 | |
Compact pattern | 0.511 | ||
Yes | 73 | 36 | |
No | 99 | 58 | |
Cholestasis | 0.750 | ||
Yes | 32 | 19 | |
No | 140 | 75 | |
Hyaline bodies | 0.352 | ||
Yes | 32 | 22 | |
No | 140 | 72 | |
Steatosis | 0.186 | ||
Yes | 35 | 13 | |
No | 137 | 81 | |
Edmondson grade | 0.264 | ||
III–IV | 49 | 33 | |
I–II | 123 | 61 | |
VETC | 0.014 |
||
Yes | 38 | 34 | |
No | 134 | 60 |
P<0.05. BMP2, bone morphogenetic protein 2; HBV, hepatitis B virus; HCV, hepatitis C virus; AFP, α-fetoprotein; BCLC, Barcelona Clinic Liver Cancer; VETC, vessels-encapsulate tumor cluster.