Gut Microbiota Characterization in Patients with HCC Post Chronic HCV Infection


 Aim: Dysbiosis of gut microbiota favors chronic hepatic inflammation with subsequent hepatic carcinogenesis. The current study aimed to evaluate the role of gut dysbiosis in the development of hepatocellular carcinoma in patients with chronic HCV infection.Methods: This descriptive cross-sectional cohort study carried out on 400 subjects recruited from the Internal Medicine Department and Tropical Medicine and Gastroenterology Department of Helwan and South Valley University Hospitals in Egypt. The study period was from January 2017 till January 2020. The subjects were divided clinically into three groups. Group I: One hundred patients with HCC, evaluated by Child Pugh, TNM and BCLC scoring systems. Group II: 200 chronic hepatitis C virus-infected patients. All patients infected with hepatitis C virus genotype 4. Group III: One hundred healthy control subjects with negative hepatitis marker and normal abdominal ultrasound. PCR of stool Microbiota, complete blood counts, complete liver function tests, INR, HCV antibodies and HBsAg were done for all included subjects. HCV PCR assessment and alpha-fetoprotein (AFP) were done for all patients.Results: No statistically significant difference was detected between HCC patients and control (p-value > 0.05) as regard Bacteroides fragilis & Akkermansia muciniphila. Faecalibacterium prausnitzii was less detected in HCC patients (51%), opposite to 70% of healthy control. With Statistically significant difference (p-value < 0.05). Bifidobacterium was less detected in HCC patients (43%), opposite to (76%) of healthy control. With highly statistically significant difference (p-value < 0.001). Lactobacillus & Enterobacteriaceae was more detected in HCC patients (80%) and (81%), in. Opposite to (36%) and (58%) in healthy control, respectively. With highly statistically significant difference (p-value < 0.001). no significant difference was detected between gut-microbiota and HCC progression with respect to Child or TNM systems. However, a significant difference was detected between number of positive stool isolate of Bacteroid Fragilis and BCLC staging system; where it was isolated from 66.7% of patients with BCLC stage IV opposite to 10.7% of patients with BCLC stage I.Conclusion: A characteristic pattern of Bifidobacterium, Lactobacillus and Enterobacteriaceae species in patients with chronic HCV and HCC was detected. Alteration of gut microbiota may be accused as a predisposing factor for liver disease progression.

development. [4][5] HCV infection is a public health problem in Egypt, it has the highest prevalence according to WHO. [6] It's known that post-viral hepatitis HCC mainly related to hepatocellular in ammation, oxidative stress, abnormal signaling pathways with activation of oncogenic pathways and integration of virus DNA in host DNA. [7][8] Intact intestinal mucosal barrier, mucus layer, secretion of IgA and associated lymphoid tissues are the intestinal barrier that prevents the passage of microbes and its metabolites across the mucosa. [9] A close relationship exists between the gut microbiome and liver; mainly due to its anatomical position and its nutritional and blood supply from the gut via the portal vein, at the same time liver is the 1st organ vulnerable to gut-derived toxins, dangerous metabolites, bacteria, and its metabolites. [10][11] Growth and Alteration in gut microbiota cause damage to the intestinal wall that promotes the transmission of bacteria and its metabolites that elicit systemic endotoxemia and distant organ disease including the liver. [12][13] Dysbiosis is a loss of bene cial microorganisms with overgrowth of harmful microbes, and/or a loss of overall microbial pro le. [14] Dysbiosis of gut microbiota favors chronic hepatic in ammation with subsequent carcinogenesis; elicited by an interaction between the liver, abnormal gut microbiota, and its metabolites together with the immune system via macrophages and Kupffer cells that secrete IL-8, TNF-α, and IL1β in ammatory cytokines. [15][16][17] Logically, dysbiosis of gut microbiota augments the pathophysiology of viral hepatitis by inducing chronic in ammation of the liver with subsequent hepatic HCC. [18] Mechanisms by which viral hepatitis induce disturbance of gut microbiota are unknown; anyway, maintaining gut homeostasis can prevent viral hepatitis induced hepatic disease progression and HCC up-growth. Therefore, we herein in this study to nd out any change in gut microbiota between healthy volunteers, patients with chronic HCV infection and patients with HCV induced hepatocellular carcinoma.

Aim Of The Work:
This study aims to detect the role of dysbiosis in the development of hepatocellular carcinoma in patients with chronic HCV infection.

Patients And Methods:
This cross-sectional cohort study was carried out on 400 subjects with average body volume recruited from the Internal Medicine Department and Tropical Medicine and Gastroenterology Department of two dedicated centers (Helwan University Hospital and South Valley University Hospital) in Egypt. The study period was from January 2017 till January 2020. The subjects were divided clinically into three groups Group I: One hundred patients with HCC on top of liver cirrhosis; caused by chronic HCV infection. The diagnosis of HCC was done according to AASLD guidelines; based on clinical, laboratory, imaging data and liver biopsy when needed. (19) Group II: 200 chronic hepatitis C virus-infected patients. All patients infected with hepatitis C virus genotype 4.
Group III: One hundred healthy control subjects with negative hepatitis marker and normal abdominal ultrasound.
The exclusion criteria include age below 18 years, previous antibiotics therapy within the last two weeks, autoimmune hepatitis, HIV or HBV co-infection, alcohol intake, and patients with other etiologies of hepatic affection.

Clinical examination
Complete history taking and full clinical examination, laboratory and radiological investigations were done for each included subject. Serum alpha-fetoprotein protein and multislice abdominal CT (MSCT) were done for patients with suspected HCC. For Cirrhotic patients with hepatic focal lesion of > 1 cm on abdominal ultrasound and AFP > 20 ng/mL a multislice CT imaging is done, if the typical features of HCC not seen but HCC still suspected; liver biopsy was done to con rm the diagnosis (according to AASLD guidelines).
All Patients with HCC were evaluated by Child-Pugh score [22]. Tumor staging was assessed by following scoring systems: 1) TNM [20] 2) Barcelona Clinic Liver Cancer (BCLC) [21] Laboratory Investigation Blood samples Ten-milliliter blood samples obtained from each subject under sterile conditions and transported to the laboratory for routine laboratory investigations. The investigations include complete blood counts (CBC) by Sysmex, serum creatinine, complete liver function tests including alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, total bilirubin by the auto-analyzer (Dialab 450 system), prothrombin time, prothrombin concentration and INR. HCV genotype estimation and HCV PCR assessment using quantitative PCR (Roche COBAS Taq Man HCV assay version 2.0 with a lower limit of detection 15 IU/mL). HBsAg estimation using and HCV antibodies detection using the automated MiniVidas immunoassay system; Biomerieux, France. AFP (alpha-fetoprotein) assessment for patients with suspected HCC.

Stool Sample
Stool samples collected from each participant in a clean container and transported to the laboratory rapidly. At the laboratory, an aliquot was taken from stool to 1.5 ml Eppendorf and stored at -80 o C.

Polymerase Chain Reaction Analysis
The ampli cation of different bacterial species of microbiota performed by the use of the primers summarized in Table 1. Complete blood count (Mean ± SD) Total leucocyte count (x10 3 /cmm) (Mean ± SD) 6.9 ± 3.9 Hemoglobin (g/dl) 11.02 ± 1. The ampli cation reaction was carried out in a total volume of 20 µL and consisted of 4 mM MgCl2, 0.25 µM of each primer and 2 µL of DNA template. Ampli cation involved an initial denaturation at 95 °C for 10 min following by 45 cycles of denaturation at 95 °C for 10 s, annealing at the speci c annealing temperature (Table 1) for 5 s, and extension at 72 °C for 10 s. The products of the ampli ed PCR were analyzed by electrophoresis. For the primers determined in the study are shown on 7Table I. Figure 1. Abdominal Muttislice CT (HCC radiological hallmark) The typical feature for diagnosis of HCC is arterial phase hyperintensity (wash-in) followed portal venous washout to iso-or hypodensity (delayed phases). [23] Statistical analysis Data were analyzed using Statistical Program for Social Science (SPSS) version 24. Qualitative data were expressed as frequency and percentage. Quantitative data were expressed as mean ± standard deviation (SD). Mean (average): the central value of a discrete set of numbers, speci cally the sum of values divided by the number of values. Standard deviation (SD): is the measure of dispersion of a set of values. A low SD indicates that the values tend to be close to the mean of the set, while a high SD indicate that the values are spread out over a wider range. Chi-square test: was used when comparing between studied groups as regard gut micro-biota. Probability (P-value): P-value < 0.05 was considered signi cant, P-value < 0.001 was considered as highly signi cant & P-value > 0.05 was considered insigni cant. Results: This descriptive cross-sectional cohort study included 300 hundred patients and 100 healthy control. The rst group included 100 patients with hepatocellular carcinoma (HCC); Their basic demographic and laboratory data are shown in Table 1.
The second group included 200 Patients with chronic hepatitis C virus infection. Their basic demographic data are shown in Table 2.
The third group included one hundred healthy volunteers; their gut microbiota is evaluated to be compared with both HCC and HCV groups of patients. Their demographic data are shown in Table 3.   Figure 2 showed the characteristic pattern of gut micro-biota; that showed a statistically signi cant difference between all studied groups.
In HCC group, no correlation could be detected between gut micro-biota and Child-Pugh grade or TNM stage.
A correlation was detected between the presence of Bacteroid fragilis microbiota and the BCLC staging with the highest detection in stage IV (66.7%). Table 7  Table 7 Correlation between studied gut-microbiota and different scores in the HCC group.

Variables
Child staging P-value Discussion: Gut and liver are physiologically and anatomically connected; which known as the gut-liver axis.

Gut microbiota component and their metabolites affect both hepatocytes and stromal cells (hepatic stellate cells and Kupffer cells). [24]
Intestinal dysbiosis is observed in different chronic hepatic diseases; which increased concern about its role in the development and progression of hepatic diseases with aggravation of liver disease-related complications. [25][26] Few studies evaluated the role of gut dysbiosis in patients with viral hepatitis. So, in this study we try to compare changes that occur in gut microbiota in patients with chronic hepatitis C, HCC with normal healthy control.
The different species of Bi dobacterium has bene cial health effects, including the regulation of homeostasis of intestinal microbes, the suppression of pathogens and harmful bacteria that colonize the gut mucosa, the modi cation of local and systemic immune responses, the inhibition of procarcinogen enzymatic activities of the gut-microbiota and the mend of the gut mucosal barrier by lowering the level of lipopolysaccharides. [27][28] In our study less than half of patients with HCC and chronic HCV infection have detected Bi dobacterium in their stool sample compared to 76% of healthy volunteers. This come in agreement with Yu et al. who found that decrease Bi dobacterium species, led to accumulation of lipopolysaccharides; which act as pathological mediator of in ammation-associated HCC. [29] In our opinion also impairment of gut wall integrity due to absence of Bi dobacterium, may allow translocation of pathogenic bacteria and endotoxins which, may produce chronic hepatic in ammation with subsequent risk of development of HCC.
Although, its known that lactobacillus species is important for human health, by decreasing gastrointestinal PH, that protect the host against invasion by pathogens. [30] on the other hand, lactobacilli may be pathogenic in susceptible patients related to several mechanisms including, the ability of some strains to bind to intestinal wall with translocation to bloodstream leading to bacteremia, also they have ability to adhere to collagen in extracellular matrix with production of glycosidase enzyme leading to damage of affected tissues. [31][32][33] In this study against usual, most patients with HCC and chronic HCV infection have lactobacillus in their stool compared with healthy controls, with more detected in HCC patients than chronic HCV infected patients.
This come with Sherid et al. Who found in their case study, an involvement of lactobacillus bacteria in the development of bacteremia and liver abscess. [34] Against our results, Zhang et al. reported that disturbance of gut microbiota homeostasis with decrease lactobacilli, led to damage of mucosa with the development of endotoxemia, systemic in ammation and tumor formation. [35] In healthy individuals, Faecalibacterium prausnitzii represent (> 5%) of the gut ora, it plays an important role in improvement of the immune system; it is an anti-in ammatory commensal activate IL-10 secretion and inhibiting IL-12 and interferon-γ expression. [36][37] In this study, Faecalibacterium prausnitzii has been detected in about half of patients with HCC and (38%) of patients with chronic HCV infection, opposite to (70%) of healthy control.
This come in agreement with Munukka et al. who found that treatment of mice with Faecalibacterium prausnitzii improved hepatic ALT, AST, and decreases adipose tissue in ammation. [38] Also, Liu et al. found in his study on patients with different causes of HCC, decreased level of Faecalibacterium resulting in reduction of the level of anti-in ammatory short-chain fatty acids. [39] The family Enterobacteriaceae includes a medically important species such as Salmonella, Escherichia coli, Yersinia pestis, Klebsiella, Shigella, Proteus, Enterobacter, Serratia, and Citrobacter. Many members of this family are normally present in human as a gut microbiota. Some enterobacteria are pathogens, because they produce endotoxins; when released into the bloodstream, cause a systemic in ammatory and vasodilatory response. [40][41] In this study, Enterobacteriaceae was found in stool samples of about 80% of patients with HCC and 70% of chronic HCV infected patients.
This come in agreement with, Sanduzzi, Chen, Lax and Bajaj et al. who found in their studies that, increased Enterobacteriaceae was linked to progression of liver cirrhosis and development of cirrhosis related complications. [42][43][44][45] Bacteroides is the most predominant bacteria in the colon, with Bacteroides fragilis group is the most abundant one among Bacteroides species. [46] Bacteroides together with other gut commensal bacteria provide the human body with energy through Carbohydrate fermentation producing a pool of volatile fatty acids that are absorbed in the colon. [47] Akkermansia muciniphila is one of gut micro-biota known to have an anti-in ammatory effect in humans, improves hepatic in ammation and protect against liver cell damage through an immune-mediated mechanism. Also, it's believed to have a role in cancer response to immunotherapy. [48][49][50] In our study, we found no signi cant difference between HCC patients and chronic HCV infected patients, in comparison with control group, regarding the stool isolate of Bacteroides fragilis. Also, in this study, no signi cant difference has been detected between HCC patients and chronic HCV infected patients, compared with control group, as regard the stool isolate of Akkermansia muciniphila. Akkermansia muciniphila, was detected in the stool of less than half of patients with HCC and HCV groups.
To some extent different experimental studies on animal models; come in accordance with our results.
They demonstrated that, the presence gut Akkermansia muciniphila can enhance the anticancer effect of T-cell-based immunotherapies. [53][54][55][56] Though many staging systems for HCC were used worldwide, there is no system is considered the best in evaluating the suitable treatment and patient's prognosis. [58] Child-Pugh and TNM had a better predictive ability for overall survival than BCLC. [59] However, TNM fails to evaluate patient's prognosis accurately, because it only evaluates tumor extension and BCLC has a better prognostic ability than TNM staging system. [60] So, in this study we try to use different systems for evaluation of patients with HCC Conclusion A characteristic pattern of Bi dobacterium, Lactobacillus and Enterobacteriaceae species in patients with chronic HCV and HCC was detected. So, dysbiosis may be associated with the progression of liver disease and hepatic carcinogenesis. It also may act as a clue for a possible role probiotic to prevent the progression of liver disease. However, many debates are present regarding the role of dysbiosis in the development of liver disease and hepatic carcinogenesis. further studies are needed on a large number of patients.
Declarations Figure 1 Positive stool sample for Faecalibacterium prausnitzii Figure 2 The characteristic pattern of gut micro-biota; that showed a statistically signi cant difference between all studied groups.