Duodenal Microbiome and Serum Metabolites Predict Hepatocellular Carcinoma in a Multicenter Cohort of Patients with Cirrhosis

Hepatocellular carcinoma (HCC) is a highly lethal cancer with a significant increasing incidence and mortality [1]. For a person with cirrhosis, the 5-year risk of developing could be as high as 30% [2, 3]. Therefore, given the high burden of disease, early detection of HCC is critical and is associated with improved survival [4]. Unfortunately, there is currently a lack of accurate and easily accessible biomarkers for the early detection of HCC.

There has been growing evidence that the intestinal microbiome contributes to the progression of cirrhosis and cancer pathogenesis [5‐7]. While most human studies have focused on fecal analyses, fecal analysis alone may miss important taxa and functional changes limited to the small intestine (SI). The microbial population, structure, and function vary considerably throughout the gut [8], and prior research suggests that the microbiome of the SI, the site of key physiological functions including bile acid uptake and nutrient absorption [9, 10], is distinct from the fecal microbiome of the colon [8]. The liver is the end point of all portal circulation with the majority of its tributaries arising from the upper and middle gastrointestinal tract. While the SI microbiome has been associated with significant clinical outcomes in other diseases [11], it has not been assessed in HCC.

In addition to the microbiome, there is also a growing body of literature that has shown microbe-associated metabolites to be important biomarkers and affecters of liver disease and cancer [12, 13]. Microbe-associated metabolites such as bile acids, damage-associated molecular patterns (DAMPs), amino acids, and short-chain fatty acids can be released into the portal circulation and absorbed in the liver causing changes in immune function, cytokine release, and cell signaling [13‐15]. Therefore, the study of these microbe-associated metabolites is just as important as the study of the microbes themselves.

This study examines for the first time the association between the duodenal microbiome and related metabolites in association with HCC risk in a multicenter prospective cohort of participants with cirrhosis. We report a novel microbial and metabolite signature that is associated with HCC risk.

In our base clinical and demographic time-to-event model, we found that male sex and HE were significantly associated with increased risk of HCC. Research has shown that males have a higher HCC risk compared to females for a multitude of reasons [22]. Men are more likely to be exposed to environmental risk factors for HCC such as alcohol and smoking [23]. Men are also more likely to have metabolic derangements, such as diabetes and metabolic syndrome, which are among the most dominant risk factors for HCC [24]. There is also recent evidence that estrogen may play a protective role in the development of HCC [22]. Therefore, our finding that males have a significantly higher risk of developing HCC is consistent with the previous published works.

HE, which is typically a late complication of cirrhosis, has a strong link to dysbiosis and small intestinal bacterial overgrowth [29, 30]. The observed association between HE and HCC risk may be related to common associations with alterations in the microbiome. Additionally, HE may be a marker for participants with more advanced cirrhosis who have increased cumulative risk for HCC [23].

Our observation that Alloprevotella was associated with HCC risk adds to a growing body of literature demonstrating an important role for the gut microbiome in HCC development [5]. Liu et al. showed an increase in fecal Alloprevotella in patients with hepatitis B-related HCC as compared to healthy controls [25]. Our study suggests that the SI microbiome may also be an important compartment for HCC risk. The exact role of Alloprevotella in the development of HCC is still unknown, but some studies have suggested an association of Alloprevotella with gut barrier function [26]. This suggests that Alloprevotella in the SI may affect gut permeability and increase bacterial translocation, metabolite transport, or bile acid exposure to the liver.

There is a growing body of evidence stating the importance of metabolomics profiling in both liver disease and cancer [12, 13]. Given the growing recognition for the importance of metabolic activity of gut microbiota in HCC pathogenesis, we examined biogenic amines and bile acid concentration in relation to HCC risk. We observed that participants with higher levels of serum methionine, methioninesulfoxide, and TCA were at increased risk for developing HCC. Methionine is an essential amino acid that plays a role in several key pathways such as protein synthesis and DNA methylation [27]. In normal everyday processes, methionine is either used for protein synthesis or converted to S-adenosylmethionine (SAM) where it is further used for DNA methylation, protein methylation, or as a component of homocysteine, cysteine, taurine, and adenosine monophosphate [27]. Several cancer types have been shown to be dependent on methionine or have deficiency in methionine conversion. Cancer cells that are deficient in these pathways are unable to regenerate methionine in either the de novo or salvage pathways, making them dependent on methionine [27]. Several animal studies have shown a diet that is restricted in methionine is protective against cancer growth [28]. In regards to HCC, studies have shown that HCC harbors defects that lead to an inability to convert methionine to SAM [29]. SAM has several anti-tumor properties which include antioxidative effects, stabilization of DNA repair, and changes in DNA methylation [29]. Additionally, studies have shown that addition of SAM can lead to reduced tumorigenesis and metastasis [30, 31]. Therefore, the increase in serum methionine in patients who developed HCC may be secondary to their inability to covert methionine to SAM effectively. Consistent with these findings, epidemiological human studies have shown that patients on vegetable-based diets, which are low in methionine, have a reduce risk of HCC development [32]

Serum TCA was also seen as a key risk factor for the development of HCC. TCA is a primary bile acid and studies have consistently shown that high levels of bile acids can induce liver injury, hepatocyte DNA damage, and hepatocyte apoptosis [33]. Elevated levels of TCA in the systemic circulatory system likely represent elevated levels of TCA in the liver and portal circulation. TCA in the colon can be converted to deoxycholic acid and hydrogen sulfide by bacterial communities, a tumor promoter and carcinogen, respectively [34, 35]. In studies of colorectal cancer and liver cancer, the level of TCA was associated with an increased risk of development of both cancer types [34, 36]. In the liver, TCA leads to upregulation of Toll-like receptor four signaling and an increase in inflammation and stellate cell activation [36].

Despite the novelty of this study, a few caveats must be noted. The study assessed the SI microbiome by testing duodenal biopsy samples, resulting in microbiome profiles that are more reflective of the mucosa-associated microbiome rather than the luminal microbiome. However, given the importance of mucosal adherent bacteria in gut permeability, we believe that the mucosal microbiome has pathophysiological relevance to downstream effects in the liver. Additionally, the duodenal microbiome may not be completely representative of the entire microbiome of the SI. While there are no human studies comparing duodenal samples to jejunal or ileal samples, mouse studies have shown that duodenal composition is similar in alpha diversity, beta diversity, and bacterial composition as the rest of the SI [37]. As this is a relatively young cohort, we observed a low number of incident HCC diagnoses. Based on longitudinal power calculation, 227 patients with 14 incident events had a power to detect large differences (i.e., hazard ratios of 7.68 or higher). However, despite the low number of participants with incident HCC, we were able to identify several metabolites and microbial biomarkers that were significantly associated with HCC development after adjusting for false discovery. But we are aware that the more subtle markers may have been missed as the study was underpowered to capture small differences. Validation of these findings will be needed in an independent cohort.