1. IntroductionHepatocellular carcinoma (HCC) is a devastating cancer that accounts for the sixth largest number of cancer deaths worldwide [1], and rising HCC incidence places a heavy burden on healthcare systems [2]. The pathogenesis of HCC is complex and unclear [3], but the disease, in most patients in China, can be linked to chronic liver disease, especially chronic infection with hepatitis B virus (HBV) [4]. After HBV infects hepatocytes, its covalently closed circular genome can persist there for the individual’s lifetime, permanently increasing risk of liver injury and progression to HCC [5,6,7]. In addition, the superposition of external factors can also lead to more severe liver injury. Previous studies have shown that ongoing alcohol consumption was associated with liver fibrosis progression, and that ethanol intake was also a risk factor for liver injury after HBV infection [8].Chronic HBV-associated hepatitis involves not only inflammation but also fibrosis, and the two reinforce each other causing progression to liver cirrhosis and HCC in a significant proportion of patients [9,10,11]. In contrast, HCC precedes cirrhosis in 7–54% of patients [12,13,14], and the mechanism by which liver injury following HBV infection affects the prognosis of such non-cirrhosis HCC patients needs to be further investigated.Most previous studies of such patients have considered how inflammation or fibrosis on their own influence prognosis [15,16]. We wondered, instead, whether the interaction between inflammation and fibrosis in para-cancerous tissues might affect prognosis. Therefore, we compared survival between non-cirrhotic HBV-associated HCC patients who had more or less severe inflammation and fibrosis based on the Scheuer system. We also used RNA sequencing and time-of-flight cytometry (CyTOF) to begin to elucidate how inflammation and fibrosis might influence prognosis. 4. Discussion

In this study, we analyzed the effects of inflammation and fibrosis on the survival and transcriptome of patients with HBV-associated HCC who underwent hepatic resection, and whose para-carcinoma tissues did not meet the diagnostic criteria for cirrhosis. In our sample, univariate and multivariate regression identified moderate-to-severe inflammation and fibrosis as an independent risk factor for shorter RFS and OS. At the same time, our transcriptomic analysis linked moderate-to-severe inflammation and fibrosis to an increase in gene transcription, as well as signaling via PI3K-Akt, MAPK, and Ras in tumor tissues, which may drive tumor cell proliferation, invasion, and metastasis. Worse inflammation and fibrosis were also linked to an immunosuppressed tumor microenvironment, which in turn may facilitate tumor growth and spread.

Hepatis virus C (HCV) is one of the important causes of viral hepatitis, and chronic HCV infection displayed a high rate of progression to liver cirrhosis over a long-term follow-up compared with HBV infection [28]. However, China is a region with a high prevalence of HBV infection, so this study focuses on HBV-associated HCC.Our results are consistent with the idea that inflammation drives HCC and many other cancers [29]. Inflammation can induce the production of chemokines and cytokines, which can alter cell proliferation and apoptosis, immune responses, and growth of new blood vessels [30,31], ultimately promoting tumor onset, development and recurrence.The presence and extent of fibrosis after HBV infection marks the severity and duration of the disease [16]. As fibrosis progresses to cirrhosis, other events that are positively associated with fibrosis, such as chronic inflammation, hepatocellular injury, regeneration, etc., are also present for an equally long period of time and act synergistically, which becomes a driving factor in the development of HCC. In HCC patients with cirrhosis, although the primary tumor was surgically removed, it did not change the background of cirrhosis in their liver, and their para-carcinoma tissues still had drivers that induced HCC formation. These issues explain the prevalence of poorer prognosis for HCC patients with cirrhosis than those without cirrhosis.Other studies, like the present one, have concluded that the degree of liver fibrosis is an independent prognostic factor for patients with hepatis virus-associated HCC [32,33], though some work calls this idea into question [13,34]. This discrepancy likely reflects differences in patient samples and study methods. If our results can be replicated in other populations, they suggest that people with moderate-to-severe inflammation and fibrosis of liver cancer with chronic HBV infection could benefit from antiviral and anti-fibrosis therapies to prevent progression to liver cancer. Such therapies have already been shown to improve prognosis of patients with HBV-associated HCC [35,36,37,38].Patients with moderate-to-severe inflammation and fibrosis may also benefit from adjuvant treatment. Various studies are underway to explore the benefits of antiviral therapy with nucleoside/nucleotide analogues [21], anti-fibrosis therapy, and traditional Chinese medicine [39] for patients with HBV-associated HCC. Accurate assessment of liver inflammation and fibrosis may help optimize the type, dose, and timing of such treatments to prevent recurrence.In our sample, AFP was not an independent prognostic factor for OS or RFS. This contrasts with several studies linking higher AFP in HCC patients to more aggressive tumors, which in turn are associated with greater recurrence and worse survival [40,41,42]. The reason for this discrepancy may be that our patients generally had milder liver injury and were in earlier stages of HCC than the patients in those previous studies, since we excluded those who were ineligible for radical resection and those with cirrhosis.Many studies have shown MVI to predict RFS and OS in HCC patients after surgical resection [43,44]. In our sample, MVI approached, but did not achieve, statistical significance as an independent risk factor for OS and RFS. We still consider it to be clinically important in our daily practice, so further studies should continue to explore its prognostic significance.Based on RNA sequencing, we found that signaling pathways involving PI3K-Akt, MAPK, and Rap1 were activated in the tumors of patients with moderate-to-severe inflammation and fibrosis, relative to the tumors of patients with mild inflammation and fibrosis. These pathways not only promote tumor progression, but they interact with each other to promote tumor invasion and metastasis [45,46,47,48]. Worse inflammation and fibrosis in our sample was associated with activation of growth and adhesion pathways, and inhibition of tumor-inhibiting pathways, consistent with previous studies [49,50,51,52,53,54,55]. Additionally, within HCC tumors, we found a higher proportion of Th17 cells among patients with moderate-to-severe inflammation and fibrosis, and a larger number of Th17 cells correlates with tumor growth and progression [56,57,58,59]. In para-carcinoma tissues, we found that the proportions of activated mast cells and monocytes were significantly lower among patients with moderate-to-severe inflammation and fibrosis than among patients with mild inflammation and fibrosis, suggesting weakened anti-tumor immune responses [60,61]. And the high proportion of regulatory T cells suggests an immunosuppressive tumor microenvironment [62,63,64]. These findings suggest that worse inflammation and fibrosis in our patients was associated with greater malignancy, which could help explain their worse prognosis.Consistent with this idea, we found that CD8+ T cells in carcinoma tissues expressed higher levels of PD-L1 in patients with moderate-to-severe inflammation and fibrosis than in patients with mild inflammation and fibrosis. Higher levels of PD-L1 inhibit T cell migration and proliferation, induce T cell apoptosis, and help tumor cells resist apoptosis induced by immune cells, all of which promote tumor progression [65,66,67] and worsen prognosis [68,69]. An immunosuppressed tumor microenvironment makes it easier for tumor cells to escape from surveillance and metastasize. These considerations imply that monoclonal antibody against PD-L1 may be effective immunotherapy for HCC patients with moderate-to-severe inflammation and fibrosis.

RNA sequencing and time-of-flight cytometry of para-cancerous tissue suggested upregulation of cell adhesion, proliferation, differentiation, extracellular matrix formation, cell growth, and angiogenesis in patients with moderate-to-severe inflammation and fibrosis, relative to the corresponding tissues from patients with mild inflammation and fibrosis. This suggests that greater inflammation and fibrosis correlate with a para-tumor microenvironment more conducive to tumor cell colonization and growth. At the same time, our analysis suggests that such a microenvironment involves weakened innate and mucosal immune responses, and chemotaxis of neutrophils and monocytes. These findings may also help to explain the worse prognosis of patients with moderate-to-severe inflammation and fibrosis.

Our study presents several limitations. Our sample was small and came from a single center, so our findings should be verified and extended in larger, preferably prospective and multicenter studies. We did not consider how genetic differences may influence patient survival or the transcriptome in tumor and para-cancerous tissues. Genotype should be considered when individualizing HCC treatment. It is thus of interest to include genotype-based stratification of non-cirrhotic HCC patients.

Despite these limitations, our study provides evidence that the combination of inflammation and fibrosis can effectively predict the prognosis of patients with HBV-associated HCC without cirrhosis. Reducing liver inflammation and fibrosis may prevent recurrence and improve survival of such patients after hepatectomy.