Correlation of preoperative Gd-EOB-DTPA-contrast-enhanced MRI with TGF- β 1 expression in hepatocellular carcinoma

Purpose: To investigate the feasibility of preoperative gadolinium ethoxy-benzyl diethylenetriaminepentaacetic acid (Gd-EOB-DTPA) -enhanced magnetic resonance imaging (MRI) related signs in predicting transforming growth factor-β1(TGF-β1) gene expression in hepatocellular carcinoma (HCC). Materials and methods: Sixty patients with HCC (55 males, mean age 52.6±12 years) who underwent preoperative MRI enhancement were retrospectively analyzed. Qualitative and quantitative features of Gd-EOB-DTPA-enhanced MRI in these pathologically confirmed HCC patients were analyzed. Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC) were performed to determine the mRNA and protein levels of TGF-β1 in HCC. The relationship between these imaging features and TGF-β1 expression level in HCC was evaluated by rank sum test, correlation analysis and logistic regression analysis. Results: RT-PCR indicated that the expression level of TGF-β1 mRNA was statistically significant with the change of hepatobiliary signal (P=0.009). Immunohistochemical results indicated that the apparent diffusion coefficient (ADC) value of tumor was statistically significant at the protein level (P=0.038). There was no statistical significance in the distribution of other image features. Binary logistic regression analysis suggested that heterogeneous signals in hepatobiliary phase were independent risk factors for high TGF-β1 expression in HCC (P =0.012,OR=5.333,95%CI:1.437-19.801). Multivariate logistic regression analysis showed that ADC value was not an independent risk factor for TGF-β1 expression. Conclusions: heterogeneous signal performance in the hepatobiliary phase and/or ADC values of HCC are potential indicators of high expression of TGF-β1. Gd-EOB-DTPA- enhanced MRI may be useful in the selection of targeted therapies for patients with HCC. slightly negative correlation between the TGF-β1 gene expression and enhanced MRI features(ADC values) in HCC; (2) Heterogeneous in the hepatobiliary on enhanced independent indicator for high TGF-β1 expression in suggested the potential for as noninvasive procedure in an evaluation of TGF-β1 in patients with


Introduction
HCC is one of the leading reasons of cancer-related deaths worldwide [1].
For advanced HCC, targeted therapy is the main treatment or adjuvant method commonly used in clinical practice. However, due to the existence of tumor genetic heterogeneity, a large proportion of patients cannot benefit from targeted therapy. Most patients do not have a good prognosis due to recurrence or metastasis after surgery. Therefore, it is very important to identify markers or target gene expression that can predict the efficacy of targeted therapy. TGF-β1 is a multifunctional cytokine that regulates important cellular processes such as angiogenesis, proliferation and differentiation, as well as immunosuppression, tissue development, and extracellular matrix formation. TGF-β1 can promote the epithelial-to-mesenchymal transformation(EMT) of hepatocytes, leading to the progression of liver fibrosis [2].In addition, TGF-β1 has a dual role, inhibiting the occurrence of liver tumors by inducing cell proliferation and apoptosis in the early stage. However, TGF-β1 can promote the growth of late stage tumors and promote the invasion, metastasis and drug resistance of cancer cells by promoting epithelial-mesenchymal transformation, leading to malignant progression [3].TGF-β1 is an independent prognostic factor for hepatocellular carcinoma, and its up-regulated expression in cancer is associated with poor prognosis [4,5], and may be a potential target for adjuvant therapy [6].Currently, several inhibitors of the TGF-β signaling pathway, such as receptor kinase inhibitors, ligand traps, and neutralizing antibodies, have been undergoing clinical trials [3,7]. A phase 2 clinical trial of TGF-β showed elevated TGF-β levels in 35% of hepatocellular carcinomas, and patients with high levels of TGF-β and alpha-fetoprotein significantly improved overall survival by up to 21 months compared to the current 9-11 months survival rate in patients with advanced HCC [8,9].Other studies have shown that the combination of PD-1/PD-L1 with TGF-β inhibitor can improve the activity of tumor immunotherapy [10].
At present, preoperative genetic testing in HCC patients is mainly performed by biopsy. If TGF-β expression can be predicted by noninvasive methods, such as enhanced MRI related signs, it will be of great significance for the decision-making of drug therapy in clinical HCC patients. The aim of this study was to assess which enhanced MR image features were associated with TGF-β1 expression at the transcriptional and protein levels.

Study patients
We retrospectively collected and analyzed 60 patients with HCC who were diagnosed and treated in the First Affiliated Hospital of Guangxi  Figure 1.)

Immunohistochemical analysis
IHC was carried out to detect the protein expression level of TGF-β1 in HCC specimens. The primary antibody is rabbit monoclonal antibody (EPR21143 /TGF-Β1 antibody) used for immunohistochemical staining of all nodular specimens with TGF-β1， The second antibody is goat antimouse/rabbit IgG polymer (SP-9000). Intratumoral specimens were taken from 1-5 sites of the primary lesion to avoid necrosis and bleeding.
All specimens were stained with TGF-β1 immunohistochemistry and the TGF-β1 positive staining was defined as brown / yellow staining of the cell cytoplasm and membrane. In this study, after hematoxylin reverse staining, the percentage of positive cells for TGF-β1 was quantified microscopically by two independent examinators. The IHC staining of the TGF-β1 protein expression was categorized: Grade1with staining in <10%( -) , Grade 2 with staining in 10%-50%( +) , and Grade3 with staining in ≥50%( ++) for tumor cells [12] (Fig. 2).

RT-PCR
Trizol reagent (Invitrogen) were used to extract total RNA from freshly collected specimens in strict accordance with standard operating instructions. RT-PCR was performed to amplify target RNA, during which the following primers were used: TGF-β1: forward TTGACTTCCGCAAGGACCTC, reverse TCCAGGCTCCAAATGTAGGG.
The internal reference primer is GADPH. SYBR primers and miRNA reverse transcription polymerase chain reaction kits are used for polymerase chain reaction amplification. Then Ct value of the sample was obtained according to the analytical dissolution curve and the realtime amplification curve. TGF-β1 gene expression in HCC tissues relative to that in adjacent tissues was calculated using the following formula: 2 -△ △ C t . Where, △ △ C t= △ Ct ca-△ Ct caliv， △ Ct ca = Ct ca-Ct GADPH， △ Ct caliv = △ Ct caliv-Ct GADPH [13]. The relationship between TGF-β1 gene expression and MRI qualitative and quantitative features was analyzed.

Statistical analysis
All statistical analyses were performed using software IBMSPSS23.0(Chicago, IL, USA

Discussion
The FDA has approved more than 20 small molecule inhibitors and 65 polymetallic oxidase inhibitors for the clinical treatment of cancer, one of which contains TGF-βR [14].There is substantial evidence that TGF- There is growing evidence that differences in gene expression influence responses to targeted therapies, and predicting response rates may be interesting.
In patients with HCC, elevated plasma TGF-β1levels are associated with shorter survival [16].High concentrations of TGF-β1 are considered as a potential negative prognostic marker in patients with unresectable HCC disease [17].In hepatocytes, transforming growth factor β induces cell

Conclusion
Taken together, the findings of this study suggest that the heterogeneous signal in the hepatobiliary phase and the relatively low ADC value of the tumor during enhanced magnetic resonance imaging reflect the high expression of TGF-β1 in HCC. Considering that TGF-β1 is a target of small molecule targeted therapy for HCC and is an independent prognostic factor for HCC, enhanced magnetic resonance imaging is a potential approach for prognosis assessment and treatment selection and efficacy evaluation of targeted therapy in HCC patients.

Consent for publication
Not applicable.

Competing interests
The authors (Zhiqing Mo, Liling Long, Hao Ding, Xiaojiao Zhou) declare that they have no competing interests.

Funding
This study was supported by the National Natural Science Foundation of China (No. 82060310).

Authors' contributions
The First author: Zhiqingmo, who was responsible for the experimental process, specimen collection and manuscript writing. The corresponding author ： Liling Long, who was responsible for reviewing articles and supervising the experiment process. The second author： Hao Ding， responsible for chart processing. The third author ： Xiaojiao Zhou， provided experimental guidance and participated in the specimen collection.

Data Availability
The data supporting the conclusions of this study are available from the corresponding author upon a reasonable request.

Conflicts of Interests
The authors declare no competing interests.  Immunohistochemical staining for TGF-β1 was conducted in 60 HCC tissues.
Yellow/brown staining was observed on the nucleus, membrane, and cytoplasm.