Role of Autophagy in the Prognosis of Combined Hepatocellular Carcinoma and Cholangiocarcinoma after Surgical Resection

Background: Autophagy-related proteins may predict postresection overall survival (OS) and disease-free survival (DFS) in patients with combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-CC). Methods: We prospectively investigated how these proteins affect clinical prognosis in 40 patients who underwent hepatectomy for cHCC-CC from 2011 to 2019 at a Taiwanese hospital. Levels of autophagy-related proteins, namely LC3, Beclin-1, and p62, were immunohistochemically assessed in patient tumor and non-tumor tissues. Results: We noted that LC3 expression was signicantly correlated with mild clinicopathological characteristics, including macrovascular invasion, lymph node metastasis, American Joint Committee on Cancer and Barcelona Clinic Liver Cancer stages, recurrence, and mortality. Ten patient showed tumor recurrence, and 15 patients died. Postresection 5-year OS and DFS rates were 43.7% and 57.4%, respectively. Cox regression analysis showed that high intratumoral LC3 expression was signicantly associated with improved OS [hazard ratio (HR; 95% condence interval (CI)): (1.68–26.9), p = 0.007], but multiple tumors and microvascular invasion was signicantly correlated with poor OS [HR (95% CI): 0.03 (0.01–0.34), p = 0.004, and 0.07 (0.01–0.46), p = 0.006, respectively]. Furthermore, high LC3 expression and cirrhosis had improved DFS [HR (95% CI): 51.3 (2.85–922), p = 0.008, and 17.9 (1.05–306), p = 0.046, respectively]. The 5-year OS and DFS rates were respectively 61.2% and 74.6% in high LC3 expression patients and 0% and 0% in those with low LC3 expression. Conclusion: High LC3 expression in tumors is signicantly associated with mild clinicopathological characteristics and favorable clinical prognosis in patients with cHCC-CC after resection.


Background
Combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-CC) is a rare type of primary liver cancer that contains unequivocal, intimately mixed components of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) [1] . In 1949, Allen and Lisa classi ed cHCC-CC into three types: types A, B, and C [2]. In type A, HCC and CC are showed at different sites of the liver [3]; in type B, HCC and CC are showed at adjacent sites [3]; and in type C, HCC and CC are combined within the same tumor [3]. This few form of liver cancer is clinically quiescent until the advanced stages, which often manifest with abdominal pain, jaundice, hepatomegaly, and weight loss [3]. cHCC-CC is an extremely aggressive liver cancer that is often associated with poor long-term prognosis [1,2]. This is largely due to its misdiagnosis as either HCC or CC pre-operatively [4]. The predominant extrahepatic recurrence sites are lymph nodes, which are typically seen in patients with late stage CC [4]. Hence, identi cation of predictive biomarkers for cHCC-CC overall survival (OS) and disease-free survival (DFS) can help promote the clinical prognosis of patients with cHCC-CC undergoing surgical resection.
The hallmark of autophagy is the formation of autophagosomes, which engulf and break down cytosolic components by fusion with lysosomes [5]. Autophagy-related genes (ATG) encode proteins that tightly regulate the process of autophagy [5]. Of the many ATG proteins, ATG8/LC3 is the most studied and has been elucidated as a critical component in autophagosome development. As such, LC3 has been popularized as a marker for monitoring autophagy [6]. LC3 also plays a vital role in cellular differentiation, apoptosis, and cancer development and metastasis [7]. Our previous studies presented that intratumoral LC3 expression and the liver microenvironment are correlated with DFS and OS after surgical resection [7,8]. However, the expression of LC3 and its possible role in cHCC-CC remain poorly understood and unstudied in the literature.
Beclin-1, another ATG protein, has also been implicated as a biomarker in a variety of tumors. Silencing of the Beclin-1 gene resulted in autophagic dysfunction and ultimately induced spontaneous HCC in mice [9]. Beclin-1 was shown to be poorly expressed in CC, and its expression strongly correlated with lymph node metastasis [10]. Similarly, p62 promoted the selective degradation of deranged proteins by delivering them to autophagosomes [11]. Further, there is overwhelming evidence that p62 is involved in the early stage of cholangiocarcinogenesis [12,13]. Hence, understanding the expression patterns and alterations in LC3, Beclin-1, and p62 in cHCC-CC can provide new insights into the discovery, diagnosis, and targeting of many autophagy-related human diseases [5]. Finally, the OS and recurrence pattern of cHCC-CC was distinct from that of HCC and CC [14]. Therefore, the aim of this study is to explore the clinicopathological characteristics and risk factors of patients with cHCC-CC and the role of autophagyrelated biomarkers for DFS and OS after surgical resection.

Patients and follow-up
Initially, in this prospective cohort study, 608 patients with liver neoplasm who underwent resection from 2011 to 2019 at E-Da Hospital, Taiwan, were recruited. We excluded 568 patients because they had HCC, CC, or metastatic liver tumors. Finally, this prospective study enrolled 40 cHCC-CC patients, which was diagnosed by histology. Our study was approved by the Institutional Review Board of E-Da Hospital (EMRP32100N). Clinicopathological data including demographic features, etiology, cirrhotic liver, tumor behaviors, vascular involvement, metastasis, death, and recurrence were recorded.
Patients were regularly followed up every 3-6 months via abdominal ultrasound, magnetic resonance imaging, or computed tomography. OS was de ned as time from the date of cHCC-CC diagnosis to death, the last follow-up, or study completion in June 2019, whichever came rst. DFS was de ned as time from the date of cHCC-CC diagnosis to recurrence, the last follow-up, or study completion in June 2019, whichever came rst.

Immunohistochemical staining and scoring
Both tumor and non-tumor tissue samples obtained from the patients were formalin-xed and para nembedded and were con rmed on hematoxylin and eosin-stained sections. The tissues were built as previously described [7,8,15,16]. We stained the tissues with an anti-LC3 antibody (Novus Biologicals, CO, USA), anti-p62 antibody (Abnova, Taipei, Taiwan), and anti-Beclin-1 antibody (Abcam, Cambridge, UK). LC3, p62, and Beclin-1 expression was quantitated by the semiquantitative immunoreactive scoring system (IRS) as previously described [7,8,15,16], and the expression was classi ed as either negative (IRS < 2) or positive (IRS ≥ 2) according to the percentage and intensity scores ( Figure S1). All the slides were independently calculated by two investigators.

Data statistics
Categorical data are expressed as numbers and percentages. Continuous data are described as medians and ranges. Student's t test were applied to normally distributed continuous variables, and Wilcoxon ranksum test was used to compare two groups. The chi-squared test was applied to compare categorical variables. OS and DFS were evaluated by the Kaplan-Meier analysis. Statistically different OS and DFS among groups were done by the log-rank test. Median OS is shown as median and 95% con dence interval (CI). A p-value of <0.05 was regarded statistically signi cant. All statistical analyses were examined by SPSS version 23.0 (SPSS, Chicago, IL, USA).

Demographic features
Overall, 40 cHCC-CC patients were enrolled in our study. The clinicopathological characteristics are shown in Table 1. The median age was 57 years; most patients were male (80%), 40% of patients had hepatitis B virus, 27.5% of patients had hepatitis C virus, and 30% of patients had a history of heavy alcohol consumption. Around 32.5% of patients had cirrhotic liver. Several patients (67.5%) had tumors of ≥5 cm in diameter, and 7.5% of patients had multiple tumors. Approximately 17.5% of patients had macrovascular invasion, and 37.5% of patients had microvascular invasion. Several patients (82.5%) had an American Joint Committee on Cancer (AJCC) stage I/II, and 82.5% of patients had a Barcelona Clinic Liver Cancer (BCLC) stage A/B. LC3 expression is associated with mortality and recurrence Immunohistochemistry revealed that LC3, Beclin-1, and p62 expression levels were elevated in 82.5% (33 of 40), 62.5% (25 of 40), and 76.5% (27 of 40) of tumor specimens, respectively (Table 2). High intratumoral LC3 expression was remarkably associated with worse survival and low recurrence rate after surgical resection. LC3 expression in non-tumor parts and Beclin-1 and p62 expression in tumor and nontumor parts was not associated with OS or recurrence.

Prognostic factors correlated with OS in cHCC-CC patients underwent surgical resection
The median follow-up duration was 50 months, and 15 cases eventually died. The 1-, 3-, and 5-year OS rates after surgical resection were 87.2%, 61.7%, and 43.7%, respectively ( Figure 1A). According to the univariate analysis, high intratumoral LC3 expression, microvascular invasion, tumor number, and tumor size were remarkably associated with OS (Table 3).  Table 3.

Discussion
Although autophagy plays a signi cant role in HCC and CC development, its role in the clinical outcome of patients with cHCC-CC is not well-understood [7-10, 12, 15]. To the best of our knowledge, this is the rst report on the association between autophagy and the clinicopathological signi cance, prognosis, and clinical outcome of patients with cHCC-CC after surgical resection.
In our study, 40 patients with cHCC-CC who underwent hepatectomy were assessed to identify the predictive factors associated with OS and tumor recurrence. LC3, p62, and Beclin-1 expression levels were elevated in tumors. Moreover, we also noted that high intratumoral LC3 expression was remarkably correlated with mild clinicopathological characteristics and high OS and DFS rates. These results imply that LC3 expression confers protection and serves as a predictive factor of OS and DFS after hepatectomy for cHCC-CC.
Beclin-1 is poorly expressed in CC and is strongly correlated with lymph node metastasis [9,10]. We previously reported that high intratumoral LC3 expression and the liver microenvironment is associated with mild clinicopathological features in patients with HCC [7,8]. Here, we found that LC3 expression is signi cantly correlated with mild clinicopathological features, including macrovascular involvement, lymph node metastasis, AJCC stage, BCLC stage, recurrence, and mortality in cHCC-CC patients. In this study, the multivariate analysis presented that high intratumoral LC3 expression and the presence of multiple tumors and microvascular invasion is remarkably correlated with OS in cHCC-CC patients.
Tumor factors (multiple tumors and microvascular involvement) were predictors of poor OS in patients with cHCC-CC. The results are a little different from those in HCC patients, in whom cirrhosis, and tumor recurrence predicted poor OS in our previous study [8].
In the multivariate analysis, high LC3 expression and cirrhosis were found to be correlated with promoted DFS in cHCC-CC patients. Cirrhosis was associated with improved DFS in cHCC-CC patients different from that in HCC patients, who have poor DFS [7]. However, LC3 can be considered an independent predictive factor of DFS and OS in cHCC-CC patients.
This study has some limitations. First, only one-third of our cohort had cirrhosis; this low prevalence may have affected the clinical outcome. Second, the underlying mechanism of autophagy, particularly with respect to the role of LC3 in carcinogenesis and clinical prognosis in cHCC-CC patients, needs to be further explored in vivo and vitro.

Conclusions
High intratumoral LC3 expression is remarkably correlated with mild clinicopathological characteristics and improved OS and DFS in cHCC-CC patients after surgical resection. Furthermore, this study is the rst to show that LC3 expression plays a vital role in predicting OS and DFS in cHCC-CC patients. The analysis of intratumoral LC3 expression, in combination with clinicopathological characteristics, could serve as predictors of OS and DFS after hepatectomy. Our ndings indicated that high LC3 expression remarkably associated with mild clinicopathological features and improved OS and DFS in cHCC-CC patients and that LC3 may serve as a prognostic biomarker to predict OS and DFS in cHCC-CC patients after surgical resection.

Declarations
Ethics approval and consent to participate The study was conducted in accordance with the guidelines of the International Conference on Harmonization for Good Clinical Practice and was approved by the Ethics Committee of E-Da Hospital, I-Shou University (EMRP32100N).
The consent for study participation is informed and signed.