Higher Inflammatory Response in Hepatocellular Carcinoma is Associated with Immune Cell Infiltration and a Better Outcome

Background & Aims: Hepatocellular carcinoma (HCC) often develops from chronic liver inflammation. Inflammation within a tumor can either promote cancer progression or activate an immune response against it. This study aims to determine the clinical significance of enhanced inflammation in HCC. Methods: Data from 655 HCC patients across four cohorts (TCGA, GSE6764, GSE76427, GSE89377) were examined. Inflammatory response was quantified using a scoring system derived from the gene set variation analysis of the “INFLAMMATORY_RESPONSE” gene set. Results: A stepwise increase in inflammatory response was noted from normal liver to cirrhosis, with consistently lower levels in HCC across both GSE6764 and GSE89377 cohorts (both p<0.001). Similar trends were observed in interferon response, pathways such as IL6/JAK/STAT3 and complement signaling, coagulation cascade, and allograft rejection (all p<0.02). HCCs with high inflammatory response were associated with increased immune cell infiltrations (p<0.01) and cytolytic activity (p<0.001). Interestingly, these HCCs had reduced mutation rates, no relationship with cell proliferation, and displayed both immune responses and pro-cancerous signals including epithelial-mesenchymal transition, KRAS, and hypoxia. Further, a high inflammatory score correlated with improved disease-free survival in TCGA (p=0.034) and overall survival in GSE76427 (p=0.008). Conclusion: HCC with higher levels of inflammatory response demonstrated increased immune cell infiltration, enhanced immune-related and other pro-cancerous-related signaling, and better patient prognosis.


Introduction
Liver cancer is the eighth most common cancer and the third leading cause of cancer-related mortality globally, and 80% of them are hepatocellular carcinoma (HCC) [1].The majority of HCC cases stem from chronic liver in ammation, primarily associated with conditions such as fatty liver disease, hepatitis virus infections, and exposure to toxic substances, including alcohol.Chronic liver damage by necroin ammation has been shown to lead to persistent hepatocyte regeneration, resulting in genetic mutations in hepatocytes and the proliferation of abnormal cells that become HCC [2].These can progress to liver cirrhosis and, in some cases, develop into HCC.Chronic in ammation is widely recognized for its signi cant role in carcinogenesis across various cancers.Chemicals and cytokines released by in amed cells, including reactive oxygen species, can damage nearby cells, accelerating their genetic evolution toward more malignant states.Rudolf Virchow observed the relationship between in ammation and carcinogenesis as early as the nineteenth century [3] and described leukocyte in ltrates within tumors, which are now considered one of the Hallmarks of Cancer [4].Currently chronic in ammation is estimated to contribute to 15 to 25% of human cancers [5].
For instance, infections by helicobacter pylori, papillomaviruses, hepatitis viruses, in ammatory conditions of uncertain origin or some autoimmune diseases, are known to cause chronic in ammation, thereby increasing the risk of speci c cancers like that increases the risk of cancer such as hepatitis for HCC, prostatitis for prostate cancer, and in ammatory bowel disease for colon cancer [6].Further, chronic in ammation has been shown to promote cancer progression, in several type of cancers.In ammatory mediators can directly interact with cancer cells and stromal cells and promote epithelial-to-mesenchymal transition (EMT) that leads to cancer cell metastasis [7].Chronic in ammation has been shown to contribute to multiple Hallmarks of Cancer, including genomic instability, proliferative signaling, and immune system evasion [4].
It is essential to comprehend the factors in uencing carcinogenesis and cancer progression, as some of these factors may exert divergent effects during development and advancement of cancer [8].Carcinogenesis is the initial phase of cancer, characterized by mutations that lead to abnormal cellular proliferation and the formation of a tumor cluster.In contrast, cancer progression signi es the exacerbation of an existing cancer, marked by gene mutations that contribute to intratumoral genomic heterogeneity and occurrence of metastasis [8].Notably, certain cellular pathways exert opposing effects during these two processes.DNA repair mechanisms, for instance, are crucial inhibiting carcinogenesis, while the same processes can give established cancer cells an advantage by enabling them to resist cytotoxic therapies and perpetuate genetic instability.While chronic in ammation is well-known to be carcinogenic, its effects on the progression of already established HCCs remain unclear.
Previously, we utilized gene set variation analysis (GSVA) on the transcriptome of bulk tumors to explore the impact of signaling networks in various malignancy types including HCC [9][10][11].GSVA scores, estimating pathway activation levels by analyzing numerous genes associated with a given pathway [12], were calculated.We speci ed the in ammatory response score as the GSVA score derived from the "INFLAMMATORY_RESPONSE" gene set in the molecular signatures database (MSigDB.Our results revealed that in ammation was associated with adverse outcomes within the entire breast cancer cohort.Interestingly, this association was inverted for patients with triple-negative breast cancer, where in ammation was linked to improved outcomes [13,14].Therefore, we extended our investigation to evaluate the clinical relevance of high in ammation in patients with HCC using this score.

In ammatory response signaling score
GSVA scoring [12] with the "HALLMARK_INFLAMMATORY_RESPONSE" gene set that includes 200 in ammatory-related genes in the MSigDB gene sets collection [14] was used to quantify the in ammatory response, which is the same score we used to analyze breast cancer cohort [13].We employed the median as a cut-off to categorize the cohorts into groups with low and high in ammatory scores.

Biological function analysis
In order to investigate the biological functions related to Hallmarks of cancer associated with a high in ammatory response in HCC within each cohort, we employed a gene set enrichment analysis (GSEA) algorithm [19].The statistical signi cance was de ned as a false discovery rate (FDR) of less than 25%, as recommended by GSEA [20].As reported earlier in our prior articles [21,22], we obtained the Hallmark cancer gene sets from the hallmark gene set collection within MSigDB [14]

Other scores
We used the CYT score to assess the cytolytic activity, which is calculated as the geometric mean of granzyme A (GZMA) and perforin (PRF1) expression in Transcripts Per Million (TPM) [23].An xCell algorithm was then employed to estimate the fraction of several immune and stromal cell types in tumor tissues, evaluating intra-tumor microenvironment composition [24].Thorsson et al. provided additional score values for TCGA samples, including intratumor heterogeneity, homologous recombination defects, fraction altered, insertions and deletions (indel) neoantigens, single-nucleotide variant (SNV) neoantigens, silent and non-silent mutations [25].

Statistical analysis
This study was analyzed using R software (version 4.1.0).Boxplots depict medians and interquartile range (IQR).For group comparison analysis, we employed the Kruskal-Wallis and Mann-Whitney U tests.Survival analysis was performed used log-rank test and Cox proportional hazards.

Results
3.1.In ammatory response showed an increase from dysplastic nodule to cirrhosis, but was lower in HCC within the carcinogenic sequence Since chronic in ammation plays a critical role in the carcinogenesis of HCC, we rst investigated the levels of in ammatory response in liver tissues; normal, dysplastic nodule, chronic hepatitis, cirrhosis and HCC, also known as HCC carcinogenic sequence.We found that the in ammatory response level increased during the stepwise carcinogenic process until cirrhosis (Fig. 1).Interestingly, in ammatory response was decreased in HCC compared to other states constantly in both the GSE6764 and GSE89377 cohorts (Fig. 1, both p < 0.001).This result implies that while the in ammatory response was heightened leading up to the onset of cancer, its activity wanes once the cancer was established.

Multiple immune response-related signaling follow the same trend as the in ammatory response in the HCC carcinogenesis sequence
Given that in ammatory response elevated with the stepwise carcinogenic process, we then investigated the association of carcinogenic process with several immune response-related pathways de ned by the MSigDB [14], including interferon (IFN)-α and IFN-γ responses, complement signaling, IL6/JAK/STAT3 signaling, coagulation cascade, and allograft rejection.We found that all these immune response-related pathways exhibited similar trends to the in ammatory response, increasing during the stepwise carcinogenic process until cirrhosis but decreasing in HCC, often reaching levels lower than or close to those in normal liver consistently in GSE6764 and GSE89377 cohorts (Fig. 2, all p < 0.02).These results suggest that multiple immune response-related signaling pathways follow a comparable trend to the in ammatory response within the carcinogenesis sequence of HCC.

Correlation of immune cell in ltration and in ammatory response levels in HCC tumor millieu
Given the notably lower in ammatory response observed in HCC compared to other liver tissues within the carcinogenesis sequence, we were intrigued to explore the relationship of in ammatory response and immune cell in ltration in HCC.First we analyzed the immunity-related scores precalculated on TCGA samples by Thorsson et al. [25], which included richness of T-cell and B-cell receptors (TCR and BCR), leukocyte fraction, IFN-γ response, and lymphocyte in ltration signature scores.High in ammatory response HCC was found to be linked with higher levels of all immunity-related scores (Fig. 3A, all p ≦ 0.003), as well as a signi cantly high level of cytolytic activity (CYT) in TCGA (Fig. 3B, p < 0.001).Next, we compared the in ltrating fractions of immune cells by the in ammatory response and found that in the TCGA and GSE6427 cohorts, high in ammatory response HCC was associated with signi cantly high in ltrations of many immune cells, including CD8 + , CD4 + memory, M1 and M2macrophages, dendritic cells, regulatory T cells (Tregs), and as well as B cells, and with less in ltrations of helper T type 1 cells (Fig. 3C).There were no consistent validated ndings in the other cells.These results suggest that although the overall levels of in ammatory response are low in HCC compared to the other conditions within the carcinogenesis sequence, higher in ammatory response among HCC is linked with higher in ltrations of multiple immune cellular components compared to the lower in ammatory response HCC groups.
3.4.HCC exhibiting a high in ammatory response showed a signi cant correlation with low mutation rates and neoantigen levels, while no relationship was found with cell proliferation Through neoantigens resulting from high tumor mutational burden, cancer cells are known to attract tumor-in ltrating immune cells [26].Simultaneously, highly mutated cancers are known to be proliferative and aggressive [26,27].Therefore, we investigated the association of in ammatory response with mutation rate and cell proliferation.Interestingly, we observed that high in ammatory response was linked with signi cantly low levels of silent and non-silent mutation rate, fraction altered, homologous recombination defects (HRD), intratumor heterogeneity, and single nucleotide variation (SNV) and indel neoantigens (Fig. 4A).This result was somewhat opposite from what we expected from immune cell in ltrations.Next, we investigated the relationship between in ammatory response and cell proliferation in HCC, using proliferation score and Ki67 gene (MKI67) expression.Despite the signi cantly lower mutation rate in HCC with high in ammatory response, there was no signi cant difference in the marker of cell proliferation (Fig. 4B and 4C, p = 0.905 and 0.436, respectively).In agreement, E2F target, G2M checkpoints, and MYC target v1, which are the cell proliferation gene sets, did not demonstrate any enrichment to in ammatory response (Fig. 4D).These results suggest that in ammatory response was inversely related to mutation rates and neoantigens but showed no relationship with cell proliferation in HCC.

High in ammatory response HCC enriched not only immune response-related but also several pro-cancerous signaling
We then investigated which other pathways could be associated with the in ammatory response in HCC.In our analysis, we observed a signi cant enrichment of all immune-related gene sets, including interferon (IFN)-α, IFN-γ response, IL6/JAK/STAT3 signaling, Complement, Coagulation, and allograft rejection, in HCC with a high in ammatory response.This enrichment was consistent in both TCGA and GSE76427 cohorts (Fig. 5A).Additionally, we found an enrichment of multiple pro-cancerous gene sets, such as KRAS signaling up, Epithelial Mesenchymal Transition (EMT), hypoxia, and angiogenesis, consistently in these cohorts.This denotes that HCC with a high in ammatory response exhibit an enhanced immune response as well as activation of cancer-promoting pathways.
3.6.HCC with a high in ammatory response showed a favorable correlation with survival compared to the group with a low score Finally, we assessed the clinical importance of in ammatory response in HCC patients.In our ndings, the high in ammatory response group exhibited a signi cant association with improved disease-free survival (DFS) in TCGA (p = 0.034) and overall survival (OS) in the GSE76427 cohort (p = 0.008) (Fig. 6).Although no signi cant differences were observed between the two groups, the high in ammatory response group tended to exhibit a more favourable patient prognosis than the low-in ammatory group for disease-speci c survival (DSS) in the TCGA cohort (Fig. 6, p = 0.361).These results suggest that HCC patients with high in ammatory response score trend to have a better prognosis compared to patients with low score.

Discussion
In our study, we explored the clinical signi cance of a heightened in ammatory response in HCC, leveraging the in ammatory response score we had previously computed [13].We observed that HCC exhibited a decreased level of in ammatory response in comparison with the other liver diseases, even though it increased throughout the carcinogenic process from normal liver to cirrhosis.This trend was mirrored in immune response-related signaling.Within HCC, high in ammatory response tumors had greater immune cell in ltrations.HCCs with high in ammatory response, demonstrated lower mutation rates, neoantigens, HRD and fraction altered.However, there was no relationship observed with cells proliferation gene sets.Moreover, high in ammatory response HCC enriched not only immune responserelated pathways but also several pro-cancerous pathways, such as epithelial mesenchymal transition (EMT), KRAS signaling, hypoxia, and angiogenesis.Ultimately, HCC characterized by a robust in ammatory response exhibited superior survival outcomes when contrasted with cases demonstrating a lower score.
Infection and in ammation are well known to promote carcinogenesis and increase cancer risk in certain settings.In HCC, repeated tissue injury caused by chronic exposure to a pathogen can be a primary mechanism for the development of chronic diseases that will eventually promote the development of malignant tumors.There is evidence that in ammation can promote the development of adenoma/neoplasia into invasive cancer [28].Consequently, in ammation can be viewed as an mediator that adds characteristics of the Hallmarks of Cancer to a normal cell, facilitating its development into cancer [4].Our study demonstrated that the level of in ammatory response quanti ed by transcriptomics, increases over time during the carcinogenesis process from normal liver to cirrhosis.Interesting observation is that there was a marked decrease in in ammatory response once HCC is established.Given our approach, we lack data to determine whether our nding suggests that the transformation of cirrhotic tissue to cancer alter the microenvironment in a way that reduces the likelihood of in ammation, or if this is simply because HCC bulk tumor is densely populated with cancer cells allowing relatively less immune cell in ltration that result in less in ammation.On the other hand, among the HCC cases, various immune response-related gene sets were enhanced in high in ammatory response HCCs and both antiand pro-cancerous immune cells were highly in ltrated compared to with low in ammatory response HCCs.These ndings suggest that immune responses may play an important role even in HCC, a status in which in ammatory response level was lower compared to the other liver conditions.
In ammation and cancer are intricately linked with complicated pathological processes controlled by numerous factors.Nuclear factor kappa B (NF-kB), a transcription factor, has been identi ed as a key modulator in driving in ammation toward cancer.In addition, an in ammatory microenvironment containing in ammatory cells and a signaling network is essential for the malignant progression of transformed cells [29].Tumor cells may grow, invade, and metastasize more readily when in amed tissues are stimulated by in ammatory cells and regulatory molecules.To date, research on in ammation-associated cancer development has predominantly focused on cytokines and chemokines, along with their downstream targets.Moreover, it's not just NF-kB that plays a role; various signaling pathways, including Janus kinase/signal transducers and activators of transcription (JAK-STAT), toll-like receptor (TLR) pathways, cGAS/STING, and mitogen-activated protein kinase (MAPK), are crucial [30].Similarly, in ammatory factors such as cytokines, chemokines, growth factors, and in ammasome, along with in ammatory metabolites like prostaglandins, leukotrienes, thromboxane, and specialized resolving mediators (SPM), are essential in regulating the onset and resolution of in ammation [30].Thus, while numerous hallmark signaling pathways have been identi ed in the relationship between cancer and in ammation, obtaining a comprehensive understanding of the extent to which each signaling pathway is activated within tumor tissues has proven challenging.Hence, the current study utilized GSVA to examine the entire in ammatory pathway rather than focusing on individual factors.This approach has shown that other immune-related signaling was also enhanced in HCC with high in ammatory response, and that enhanced levels of other pro-cancerous pathway, such as EMT, KRAS, and angiogenesis, were also correlated in the patient's tumor.HCC in ammation dynamics and their relationship to disease progression may be better understood using this approach.
Cancer progression is the process by which genes mutate and tumor heterogeneity develops [8].In this study, the tumor mutation burden and neoantigens were examined in depth in order to determine the association of in ammation and immune response in HCC.Our study ndings, however, revealed a surprising twist to this general observation.High levels of in ammatory response in HCC were associated with low mutation rates, neoantigens, and homologous recombination defects.This pattern of immune response deviates from the conventional mechanism, suggesting that in ammation and immune responses play an integral part in the process of cancer progression.
Various research studies have demonstrated that chronic in ammation plays an important role in promoting, initiating, and progressing cancer [29].However, we demonstrate that once HCC develops, the in ammatory response appears to decrease.Conversely, high in ammation is associated with a better prognosis among HCC patients, adding complexity to this relationship.This suggests that the in ammatory response pathway plays a different role in carcinogenesis and cancer progression in HCC.Various factors, such as the tumor's ability to manipulate the immune response or changes in the tumor microenvironment, along with other immune-related and pro-cancerous signaling, are involved in in ammation and their counterbalance may affect patient outcomes.Such complexities in cancer has been reported in other pathways.For example, certain cellular pathways, like DNA repair, exert opposing effects during these two phases.While DNA repair mechanisms inhibit cancer initiation, they can also enable established cancer cells to resist cytotoxic therapies and perpetuate genetic instability.It is crucial to understand that in ammation plays an important role in both carcinogenesis and cancer progression.These two conditions are distinct from each other, and our method may prove to be very useful in comprehending this complex in ammatory status in cancer.
Our study is not without limitations.Given that this is a retrospective study utilizing various public patient data cohorts, some important clinical data, such as treatment details, were not accessible.The relevance of in ammatory response to drug treatment in HCC needs to be investigated in future studies.Furthermore, further studies are needed to understand the causal relationships and detailed mechanisms, as this study represents a single, "snapshot" observation.

Conclusions
In ammatory response was associated with the HCC carcinogenesis sequence, and HCCs with a high in ammatory response correlated with immune cell in ltrations in the tumor microenvironment.These HCCs enriched immune responses as well as EMT, KRAS signaling, hypoxia and angiogenesis, and were associated with better patient outcomes.signaling; KRAS signaling up, apoptosis, epithelial mesenchymal transition, hypoxia, angiogenesis, which were enriched signi cantly in the HCC with enhanced in ammatory response score.This enrichment was observed consistently in both cohorts through GSEA.As recommended by the GSEA software, FDR < 0.25 de ned statistical signi cance.NES, normalized enrichment score; FDR, false discovery rate.

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