Intestinal Microbiome Associated with Efficacy of Atezolizumab and Bevacizumab Therapy for Hepatocellular Carcinoma

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However, no studies have reported on specific intestinal microbiota associated with the efficacy of Atezolizumab and Bevacizumab. In this study, we analyzed fecal samples collected before treatment to investigate the relationship between the intestinal microbiome and the efficacy of Atezolizumab and Bevacizumab. Methods: A total of 37 patients with advanced HCC who were treated with Atezolizumab and Bevacizumab were enrolled. Fecal samples were collected from the patients, and they were divided into responder (n = 28) and non-responder (n = 9) groups. We compared the intestinal microbiota of the two groups and analyzed the intestinal bacteria associated with prognosis using QIIME2. Results: The alpha and beta diversities were not significantly different between both groups, and the proportion of microbiota was similar. The relative abundance of Bacteroides stercoris and Parabacteroides merdae was higher in the responder group than in the non-responder group. When the prognosis was analyzed by the presence or absence of those bacteria, patients without both had a significantly poorer prognosis. Conclusion: Differences in intestinal microbiome are involved in the therapeutic effect of Atezolizumab and Bevacizumab. Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Hepatocellular carcinoma (HCC) is a leading cause of death worldwide, despite advancements in Hepatitis B virus (HBV) and Hepatitis C virus (HCV) treatments[ 1 ]. Various drugs, including lenvatinib, regorafenib, ramucirumab, and cabozantinib, have been developed for unresectable HCC [ 2 ]. However, atezolizumab and bevacizumab, a combination of immune checkpoint inhibitors (ICIs) and angiogenesis inhibitors, have been shown to be more effective than molecular target agents and are widely used as a first-line treatment[ 3 ]. ICIs are thought to have an indirect anti-tumor effect by inducing T cell activation mechanisms[ 4 ]. The administration of ICIs augments T cell-mediated immune responses against tumor cells and has been found to improve overall survival in patients with various cancer types [ 5 ]. However, the response to these treatments varies, and markers for treatment efficacy have not yet been identified[ 6 ]. Several studies have shown that the gut microbiome influences the antitumor immune response and that modulation of the microbiome may improve therapeutic response [ 7 ]. Thus, the gut microbiome is thought to be closely related to the function of ICIs. Although several reports have been published on the association between ICI-treatment for HCC and the gut microbiome[ 8 , 9 ], no reports have investigated whether the gut microbiome is directly related to the efficacy of atezolizumab and bevacizumab for HCC. Therefore, this study aimed to investigate the relationship between the efficacy of combination therapy of atezolizumab and bevacizumab in unresectable HCC and the gut microbiome by analyzing the gut microbiome of patients prior to treatment. Materials and Methods Study design This study was designed as a single-center cohort study to investigate the relationship between the gut microbiome and the efficacy of atezolizumab and bevacizumab therapy in patients with unresectable HCC. Patients who received this combination therapy at Nagoya University Hospital between December 2020 and October 2022 and who provided written informed consent were included in the study. Treatment response and patient groups Treatment response was assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 [ 10 ] during the treatment period, with responses classified as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Treatment efficacy was evaluated every 6 to 8 weeks from the start of treatment, and patients were divided into two groups based on the best response achieved during treatment: a responder group with treatment response classified as either SD, PR, or CR and a non-responder group with no treatment response, or PD. Survival analysis Overall survival (OS) was defined as the time from the first day of treatment to the date of death or last follow-up. Progression-free survival (PFS) was defined as the time from the first day of treatment to the date of PD onset or death. Ethics approval This study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of Nagoya University Hospital (approval number: 2015 − 0420). All patients provided written informed consent before enrollment. Patients Patient data, including sex, age, body mass index (BMI), cause of chronic hepatitis, blood test data, previous treatment, use of proton pump inhibitors (PPIs) and antibiotics use in the month prior to administration were collected from medical records. Hepatocellular carcinoma (HCC) stage was classified using the Barcelona Clinic Liver Cancer Classification (BCLC) [ 11 ], and liver function was classified using the Child–Pugh score [ 12 ]. Diabetes mellitus was diagnosed in accordance with the criteria of the American Diabetes Association, with a random plasma glucose level of ≥ 200 mg/dL, fasting plasma glucose level of ≥ 126 mg/dL, or use of any anti-hyperglycemic medication [ 13 ]. Alcohol consumption of more than 80 g/day was considered heavy drinking [ 14 ]. Before treatment, patients were screened for varices by endoscopy to assess the risk of bleeding. Patients with high-risk varices received endoscopic treatment before administration of atezolizumab and bevacizumab. In addition, patients were screened for endocrine, neovascular, and respiratory abnormalities before administration of combination therapy. Treatment protocol Patients received a combination therapy of atezolizumab and bevacizumab (Chugai Pharmaceutical Co. LTD, Tokyo, Japan) via intravenous administration. The dosage consisted of 1200 mg of atezolizumab and 15 mg/kg of bevacizumab, given every 3 weeks until either tumor progression or unacceptable adverse events occurred. If clinical benefits were observed, treatment was continued even beyond tumor progression. In the event of adverse events, patients were allowed to receive a monotherapy of either atezolizumab or bevacizumab, depending on the type of adverse event experienced. Sample collection and DNA isolation Sample collection and DNA isolation were performed as previously reported [ 15 ]. In brief, fecal samples were collected from patients before treatment and stored at − 80°C. DNA was extracted using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) and stored at − 80°C until further analysis. The V3-4 regions of the bacterial 16S rRNA gene were amplified using universal primers, and the PCR products were purified with AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). Samples were barcoded and pooled for sequencing on an Illumina MiSeq platform using the MiSeq Reagent Kit v3 with 2×300 reads and 600 cycles (Illumina, San Diego, CA, USA). 16S rRNA gene sequencing The 16S rRNA sequencing data was processed using QIIME2 (version 2021.2) [ 16 ]. After demultiplexing, the paired-end reads for 37 microbiome samples were imported into QIIME2. Sequence quality control and feature table construction was performed using the Divisive Amplicon Denoising Algorithm 2 (DADA2) QIIME2 plugin. After denoising, a pre-trained naive Bayes classifier based on the SILVA database version 132 was used to explore the taxonomic distribution of the samples [ 17 ]. Statistical analyses Statistical analyses were conducted using EZR, a graphical user interface for R [ 18 ]. The significance level was set at P < 0.05. Continuous variables were expressed as medians with interquartile ranges and analyzed using the Mann–Whitney U test. Categorical variables were analyzed using chi-squared or Fisher's exact tests. Microbiome data were analyzed and visualized using the MicrobiomeAnalyst online platform[ 19 ]. Alpha diversity was calculated using the Chao 1, Shannon index, and observed genuses, and beta diversity was estimated using the Bray–Curtis index and visualized using principal coordinate analysis (PCoA). The significance of the PCoA plot was determined using PERMANOVA. Differential taxonomy analysis was performed using linear discriminant analysis effect size (LEfSe: ( http://huttenhower.sph.harvard.edu/galaxy/ )) with LDA score > 2 and P value < 0.05[ 20 ]. Survival analysis was performed using the Kaplan–Meier method, and differences between groups were assessed by log-rank test. Results Patient background During the study period, a total of 54 patients received the combination therapy of Atezolizumab and Bevacizumab at our hospital. Out of these, 37 patients agreed to participate in the study and provided pre-treatment fecal samples. Of these 37 patients, 28 were categorized as responders (11 patients with PR and 17 patients with SD) and 9 were categorized as non-responders (Fig. 1 ). Patient background information for both groups is presented in Table 1 . The median age of patients in the responder and non-responder groups was 74 and 75 years, respectively ( P = 0.184). The sex ratio (male:female) was 23:5 in the responder group and 5:4 in the non-responder group (p = 0.178). The etiology of HCC was as follows: HBV (5 patients), HCV (7 patients), alcohol (10 patients), and non-B non-C (6 patients) in the responder group and HBV (1 patient), HCV (6 patients), alcohol (2 patients), and non-B non-C (0 patients) in the non-responder group ( P = 0.128). The distribution of BCLC stage was as follows: A (3 patients), B (6 patients), and C (19 patients) in the responder group and A (0 patients), B (3 patients), and C (6 patients) in the non-responder group (p = 0.707). The distribution of Child–Pugh score was as follows: 5 (22 patients), 6 (6 patients), and 7 (0 patients) in the responder group and 5 (3 patients), 6 (3 patients), and 7 (3 patients) in the non-responder group ( P = 0.007), indicating significantly better liver function in the responder group. Neutrophil-to-lymphocyte ratio (NLR), which has been reported to be associated with the efficacy of ICIs, did not differ between the two groups [ 21 ]. Other blood test data and the use of PPIs and antibiotics were similar between the two groups. Microbiome profiling and comparison of alpha and beta diversities We examined the gut microbiome profile of the responder and non-responder groups at the phylum level, as shown in Fig. 2 . Both groups were dominated by the major phyla found in human gut microbiota: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. The proportions of each phylum were as follows: Firmicutes (78.0% vs 68.9%), Bacteroidetes (19.7% vs 14.1%), Actinobacteria (5.3% vs 8.3%), and Proteobacteria (2.5% vs 3.1%) in the responder and non-responder groups, respectively. We also compared the alpha and beta diversities of the two groups and found no significant differences (Supplementary Fig. 1). Comparison of relative abundance of bacterial communities between responder and non-responder groups with LEfSe We used LEfSe to compare the relative abundance of bacterial communities between responder and non-responder to identify bacterial genera associated with treatment response. Figure 3 shows the results of the LEfSe analysis, with green and red bars indicating bacterial communities that were significantly increased in the responder and non-responder groups, respectively. One family ( Tannerellaceae ), one genus ( Parabacteroides ), and two species ( Bacteroides stercoris and Parabacteroides merdae ) were found to be more abundant in the responder group. Conversely, the microbiome related to Phylum Synergistota and three families ( Leuconostocaceae, Morganellaceae , and Synergistaceae ), two genera ( Weisella and Lachnospiraceae UCG 008 ), and some species were enriched in the non-responder group. Comparison of progression-free survival (PFS) and overall survival (OS) To investigate the bacteria associated with the effect of atezolizumab and bevacizumab, we focused on Bacteroides stercoris and Parabacteroides merdae , which were enriched in the responder group and have been reported to be associated with a better effect of ICIs [ 22 , 23 ]. The relative abundance of these bacteria in each sample is shown in Supplementary Fig. 2. PFS and OS were calculated for the two groups based on the presence or absence of each bacterium (Supplementary Fig. 3). Both bacteria showed a trend towards better PFS and OS in the group with the bacterium, and particularly the group with Bacteroides stercoris showed significantly better PFS than the group without the bacteria. On the basis of these results, we divided the patients into four groups based on the presence or absence of the bacteria (1: P. merdae (-) + B. stercoris (-), 2: P. merdae (+) + B. stercoris (-), 3: P. merdae (-) + B. stercoris (+), 4: P. merdae (+) + B. stercoris (+)). PFS and OS were analyzed separately. The results showed that the group that was negative for both bacteria had significantly worse PFS and OS than the other groups (Supplementary Fig. 4). Comparison of patient background of each group showed that the group without both bacteria had significantly higher alanine aminotransferase (ALT) and α-fetoprotein (AFP) (Supplementary Table). Because the absence of both bacteria may be related to the poor efficacy of atezolizumab and bevacizumab treatment, we analyzed OS in the group negative for both bacteria (n = 4) and in the group positive for at least one of either bacteria (n = 33). We found that the group without the bacteria had a significantly worse prognosis (Fig. 4 ). Comparison of patient background showed that the group with the absence of these two bacteria were significantly older, with lower serum albumin and higher AFP levels (Table 2 ). Discussion In this study, we analyzed pre-treatment fecal samples from patients undergoing atezolizumab and bevacizumab treatment and identified bacteria enriched in the responder group that were associated with better prognosis. The recent introduction of several new agents has led to advancements in the treatment of HCC. Atezolizumab, an ICI, is commonly used in combination with bevacizumab, an angiogenesis inhibitor, as a first-line treatment for unresectable HCC. However, the efficacy of this treatment and potential adverse events remain unclear. In recent years, numerous studies have reported on the relationship between ICIs and the intestinal microbiome, with intestinal bacteria-mediated mechanisms thought to be involved in their efficacy and in adverse events [ 7 ]. In relation to HCC, a study from Taiwan revealed that Lachnoclostridium and Prevotella 9 are associated with the favorable effects of ICI administration in HCC [ 9 ]. Meanwhile, another study from Japan showed that the efficacy of atezolizumab and bevacizumab was related to the use of antibiotics, suggesting that intestinal bacteria play a role in the effectiveness of these drugs [ 24 ]. In our study, Parabacteroides merdae and Bacteroides stercoris were found to be enriched in the responder group. Both of these bacteria have been reported to have favorable effects on ICIs[ 22 , 23 ]. Bacteroides stercoris was enriched in the responder group of PD-1 and CTLA4-based therapy for patients with malignant melanoma [ 22 ]. Parabacteroides merdae was enriched in responders to anti-PD-1 and anti-PD-L1 inhibitor treatment for non-small cell lung cancer patients [ 23 ]. These bacteria and the contributions they make to the intestinal environment are hypothesized to be involved in host immunity and related to therapeutic effects of ICIs. The absence of both Bacteroides stercoris and Parabacteroides merdae was significantly associated with poor prognosis. Comparing baseline characteristics of these patients, the group without either bacterium was significantly older (83 vs 73 years) and had higher AFP. Even among the patients aged 75 or older, AFP was significantly higher in the group without both bacteria (data not shown). AFP levels at the time of HCC diagnosis have been reported as an independent risk predictor related to pathological grade, progression, and survival [ 25 ]. This suggests that the presence or absence of these bacteria may select patients with higher risk of malignancy. Our study had some limitations. Owing to the small number of cases in this single-center, retrospective study, differences between the microbiome and treatment effects by etiology or liver function were difficult to examine. Further studies with a larger patient cohort or a validation group are needed to address the strong inter-individual variability associated with the intestinal microbiome. Moreover, metabolite measurements were not taken, and thus the mechanism by which the microbiome may be involved in the effects of ICIs is unknown. Conclusion Our study suggests that specific gut bacteria may play a crucial role in determining the treatment response of HCC patients receiving atezolizumab and bevacizumab therapy. These findings may pave the way for the development of novel microbiome-based therapeutic strategies for enhancing the efficacy of immunotherapy in HCC patients. Declarations Consent to participate The study was approved by the ethics committees of hospital (approval number: 2015-0420). Informed consent was obtained from all patients for inclusion in the study. Funding This work was supported by the 39th Grant from The Nitto Foundation for Kenta Yamamoto and by JST [Moonshot R&D][Grant Number JPMJMS2214-11]. Conflict of interest The authors (Yosuke Inukai, Kenta Yamamoto, Takashi Honda, Shinya Yokoyama, Norihiro Imai, Yoji Ishizu, Masanao Nakamura, Hiroki Kawashima) declare that they have no conflict of interest. Takanori Ito received personal fees from Chugai Pharmaceutical Co., Ltd. outside the submitted work. Masatoshi Ishigami received grants from Chugai Pharmaceutical Co., Ltd. outside the submitted work. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. Authorship contributions YI and KY conceived the idea of the study. TH developed the statistical analysis plan and conducted statistical analyses. 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Tables Table 1: Patient characteristics. Responder n=28 Non-responder n=9 P -value Age † 74(63-79) 75(71-85) 0.184 Gender (male/female) 23/5 5/4 0.178 Body mass index† 23.3(20.9-26.8) 24.0(20.9-25.3) 0.804 Etiology (HBV/HCV/Alcohol/NBNC) 5/7/10/6 1/6/2/0 0.128 BCLC stage (A/B/C) 3/6/19 0/3/6 0.707 Child-Pugh grade (A/B) 28/0 6/3 0.011 Child-Pugh score (5/6/7) 22/6/0 3/3/3 0.007 Treatment history (1/2/3/4) 26/2/0/0 4/3/0/2 0.005 AST (IU/L) † 36(27-51) 37(20-46) 0.710 ALT(IU/L) † 29(21-38) 25(15-30) 0.279 γ- GTP(IU/L) † 77(55-136) 33(31-186) 0.583 Total bilirubin (mg/dL) † 0.8(0.71.1) 0.7(0.5-1.0) 0.246 Albumin (g/dL) † 3.8(3.6-4.0) 3.4(2.6-3.8) 0.051 HbA1c(%) † 6.1(5.6-6.8) 6.2(5.7-6.7) 0.657 AFP (ng/mL) † 9.5(5.0-410.8) 564.0(13.0-845.0) 0.123 PPI (yes/no) 18/10 5/4 0.705 Antibiotics(yes/no) 3/25 1/8 1.000 C-reactive protein (mg/dL) † 0.20(0.09-0.50) 0.38(0.04-1.46) 0.645 NLR † 1.96(1.29-3.32) 2.71(1.64-3.62) 0.357 BCLC, the Barcelona Clinic Liver Cancer Classification ; AST, aspartate aminotransferase; ALT, alanine aminotransferase; γ-GTP, γ-glutamyl transpeptidase; AFP, α-fetprotein; PPI, protom pump inhibitor; NLR, Neutrophil-Lymphocyte Ratio †Values are expressed as median (interquartile range). Table 2: Patient background with and without P.merdae and B.stercoris. P.merdae(+)or B.stercoris(+) n=33 P.merdae(-) and B.stercoris(-) n=4 P -value Age* 73(63-79) 83(79-85) 0.047 Gender (male/female) 26/7 2/2 0.244 Body mass index* 24.0(20.9-27.0) 21.7(20.1-23.3) 0.240 Etiology (HBV/HCV/Alcohol/NBNC) 6/10/11/6 0/3/1/0 0.460 BCLCstage (A/B/C) 3/8/22 0/1/3 1 Child-Pugh grade (A/B) 31/2 3/1 0.298 Child-Pugh score (5/6/7) 24/7/2 1/2/1 0.104 Treatment history (1/2/3/4) 27/5/1 3/0/1 0.278 AST (IU/L)* 35(25-45) 134(88-174) 0.106 ALT (IU/L)* 27(21-35)) 52(23-107) 0.365 γ- GTP (IU/L)* 72(34-132) 266(147-447) 0.149 Total bilirubin (mg/dL)* 0.8(0.6-1.2) 0.9(0.7-1.0) 0.825 Albumin (g/dL)* 3.8(3.6-4.0) 3.0(2.8-3.3) 0.039 HbA1c (%)* 6.1(5.7-6.8) 6.2(5.9-6.8) 0.980 AFP (ng/mL)* 10.0(5.0-494.0) 4335.5(429.8-36901.0) 0.027 PPI (yes/no) 21/12 2/2 0.625 Antibiotics(yes/no) 4/29 0/4 1.000 C-reactive protein (mg/dL)* 0.20(0.09-0.44) 1.27(0.82-1.47) 0.186 NLR* 1.97(1.22-2.75) 3.65(3.20-4.08) 0.070 BCLC, the Barcelona Clinic Liver Cancer Classification ; AST, aspartate aminotransferase; ALT, alanine aminotransferase; γ-GTP, γ-glutamyl transpeptidase; AFP, α-fetprotein; PPI, protom pump inhibitor; NLR, Neutrophil-Lymphocyte Ratio †Values are expressed as median (interquartile range). Additional Declarations No competing interests reported. Supplementary Files Supplementalfigure.pdf SupplementaryTable.docx Cite Share Download PDF Status: Published Journal Publication published 26 Apr, 2024 Read the published version in Cancers → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3996460","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275887202,"identity":"1129e759-caf4-4580-a6ef-43f8e2dc7552","order_by":0,"name":"Yosuke Inukai","email":"","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yosuke","middleName":"","lastName":"Inukai","suffix":""},{"id":275887203,"identity":"0b5f2577-496a-423b-8e4d-7a1d8a1a7155","order_by":1,"name":"Kenta Yamamoto","email":"","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kenta","middleName":"","lastName":"Yamamoto","suffix":""},{"id":275887204,"identity":"19bc7670-c945-4a93-9a97-ea1e2a52ea4f","order_by":2,"name":"Takashi Honda","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABA0lEQVRIiWNgGAWjYBACAyCWYKgAkgdQJdgIaDlDshbGNkwtuIE5e+/BGz/nbZPjO8B78OOXisPyDBIJjB9+MPDl4dJi2XMu2bJ3221jyQN8ydIyZw4bNkgkMEv2MLAV43TYjRwzCd5ttxM3HOAxkJZsO8y4/0YCgzTQL4kNeLRI/p1zux6oxfg3UIs9yJbfhLRI8zbcTjA4wGMm+bHtcCJQCxt+W86cMbaWOXbbcOZhHjNrhjPpyQ08D9ssewzw+OV4j+HNNzW35fmO9xjf/FFhbdvAnnz4xo+KYzhDDAGYgYiHoRnIYgQ6yeBYAmEtILU/GOpg7BritIyCUTAKRsFIAADYAFimx2fD5QAAAABJRU5ErkJggg==","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Takashi","middleName":"","lastName":"Honda","suffix":""},{"id":275887205,"identity":"9bffcd60-f4de-4dc5-b22f-4d6362af05d2","order_by":3,"name":"Shinya Yokoyama","email":"","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shinya","middleName":"","lastName":"Yokoyama","suffix":""},{"id":275887206,"identity":"3557981d-63f0-4916-a459-536932e08aa0","order_by":4,"name":"Takanori Ito","email":"","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Takanori","middleName":"","lastName":"Ito","suffix":""},{"id":275887207,"identity":"ef834bbb-0f2d-4185-9dd6-7693da6f014b","order_by":5,"name":"Norihiro Imai","email":"","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Norihiro","middleName":"","lastName":"Imai","suffix":""},{"id":275887208,"identity":"42a0073f-af62-42c1-b3a3-885095eef0e4","order_by":6,"name":"Yoji Ishizu","email":"","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yoji","middleName":"","lastName":"Ishizu","suffix":""},{"id":275887209,"identity":"7384f9a0-1bf3-4f8b-a128-f014c9a73a00","order_by":7,"name":"Masanao Nakamura","email":"","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Masanao","middleName":"","lastName":"Nakamura","suffix":""},{"id":275887210,"identity":"84c3b48c-b772-4f1c-8621-b4f08ec0c82d","order_by":8,"name":"Masatoshi Ishigami","email":"","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Masatoshi","middleName":"","lastName":"Ishigami","suffix":""},{"id":275887211,"identity":"54ebbefe-9440-4c58-a698-3bc9f840bd14","order_by":9,"name":"Hiroki Kawashima","email":"","orcid":"","institution":"Nagoya University Graduate School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hiroki","middleName":"","lastName":"Kawashima","suffix":""}],"badges":[],"createdAt":"2024-02-28 11:07:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3996460/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3996460/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.3390/cancers16091675","type":"published","date":"2024-04-26T22:31:13+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52033431,"identity":"c545b8da-8958-434d-bef1-405cb039749b","added_by":"auto","created_at":"2024-03-05 16:41:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":94540,"visible":true,"origin":"","legend":"\u003cp\u003eStudy design.\u003c/p\u003e\n\u003cp\u003eHCC, hepatocellular carcinoma; PR, Partial Response; SD, Stable Disease; PD, Progressive Disease\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3996460/v1/5dc1da7ce853b985932c5ab6.png"},{"id":52033430,"identity":"b6983c43-b1bb-4c61-b9a4-706ef04e0fac","added_by":"auto","created_at":"2024-03-05 16:41:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":114240,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundances at phylum level in Responder and Non-responder groups.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3996460/v1/724859b6019f73f6e0012797.png"},{"id":52033432,"identity":"50c863f4-c7fd-47c7-94d0-3a98747a1488","added_by":"auto","created_at":"2024-03-05 16:41:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1145556,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of relative abundance of bacterial communities between groups performed with LEfSe.\u003c/p\u003e\n\u003cp\u003eThe graph shows a comparison of the relative abundance of bacterial communities between both groups performed with LEfSe. Green bars show bacterial communities with a higher relative abundance in the Responder group, and red bars represent bacterial communities that were more abundant in the Non-responder group.\u003c/p\u003e\n\u003cp\u003eLEfSe, linear discriminant analysis effect size\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3996460/v1/30570acdfb7a0eb966031664.png"},{"id":52033434,"identity":"f79aefc0-0f4a-4400-9d3c-6f6aa0521657","added_by":"auto","created_at":"2024-03-05 16:41:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":117263,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of Progression Free Survival and Overall Survival grouped by presence or absence of Bacteroides stercoris and Parabacteroides merdae.\u003c/p\u003e\n\u003cp\u003eProgression Free Survival and Overall survival were significantly shorter in the group without both Bacteroides stercoris and Parabacteroides merdae.\u003c/p\u003e\n\u003cp\u003ePFS, Progression Free Survival; OS, Overall Survival; B.stercoris, Bacteroides stercoris; P.merdae, Parabacteroides merdae\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3996460/v1/9116affabed4226a60e2414e.png"},{"id":55690249,"identity":"b2b78320-e8de-4a44-ba5a-21d8216319c8","added_by":"auto","created_at":"2024-05-01 22:31:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":995418,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3996460/v1/0d48b2e8-0359-4e29-91db-2c8a0ca26331.pdf"},{"id":52033433,"identity":"796d73a2-56cd-47d5-94a3-53d394b5a816","added_by":"auto","created_at":"2024-03-05 16:41:31","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":588049,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfigure.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3996460/v1/79decbb0e2c5c0fc5fa712aa.pdf"},{"id":52033436,"identity":"f4f18386-806c-4281-8733-9e444a958079","added_by":"auto","created_at":"2024-03-05 16:41:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":17021,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-3996460/v1/6153f415ea9a821cd0355b39.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intestinal Microbiome Associated with Efficacy of Atezolizumab and Bevacizumab Therapy for Hepatocellular Carcinoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHepatocellular carcinoma (HCC) is a leading cause of death worldwide, despite advancements in Hepatitis B virus (HBV) and Hepatitis C virus (HCV) treatments[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Various drugs, including lenvatinib, regorafenib, ramucirumab, and cabozantinib, have been developed for unresectable HCC [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, atezolizumab and bevacizumab, a combination of immune checkpoint inhibitors (ICIs) and angiogenesis inhibitors, have been shown to be more effective than molecular target agents and are widely used as a first-line treatment[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eICIs are thought to have an indirect anti-tumor effect by inducing T cell activation mechanisms[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The administration of ICIs augments T cell-mediated immune responses against tumor cells and has been found to improve overall survival in patients with various cancer types [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the response to these treatments varies, and markers for treatment efficacy have not yet been identified[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Several studies have shown that the gut microbiome influences the antitumor immune response and that modulation of the microbiome may improve therapeutic response [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Thus, the gut microbiome is thought to be closely related to the function of ICIs.\u003c/p\u003e \u003cp\u003eAlthough several reports have been published on the association between ICI-treatment for HCC and the gut microbiome[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], no reports have investigated whether the gut microbiome is directly related to the efficacy of atezolizumab and bevacizumab for HCC. Therefore, this study aimed to investigate the relationship between the efficacy of combination therapy of atezolizumab and bevacizumab in unresectable HCC and the gut microbiome by analyzing the gut microbiome of patients prior to treatment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThis study was designed as a single-center cohort study to investigate the relationship between the gut microbiome and the efficacy of atezolizumab and bevacizumab therapy in patients with unresectable HCC. Patients who received this combination therapy at Nagoya University Hospital between December 2020 and October 2022 and who provided written informed consent were included in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTreatment response and patient groups\u003c/h2\u003e \u003cp\u003eTreatment response was assessed using the Response Evaluation Criteria in Solid Tumors (RECIST) v1.1 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] during the treatment period, with responses classified as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Treatment efficacy was evaluated every 6 to 8 weeks from the start of treatment, and patients were divided into two groups based on the best response achieved during treatment: a responder group with treatment response classified as either SD, PR, or CR and a non-responder group with no treatment response, or PD.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSurvival analysis\u003c/h2\u003e \u003cp\u003eOverall survival (OS) was defined as the time from the first day of treatment to the date of death or last follow-up. Progression-free survival (PFS) was defined as the time from the first day of treatment to the date of PD onset or death.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEthics approval\u003c/h2\u003e \u003cp\u003e This study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of Nagoya University Hospital (approval number: 2015\u0026thinsp;\u0026minus;\u0026thinsp;0420). All patients provided written informed consent before enrollment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePatients\u003c/h2\u003e \u003cp\u003ePatient data, including sex, age, body mass index (BMI), cause of chronic hepatitis, blood test data, previous treatment, use of proton pump inhibitors (PPIs) and antibiotics use in the month prior to administration were collected from medical records. Hepatocellular carcinoma (HCC) stage was classified using the Barcelona Clinic Liver Cancer Classification (BCLC) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and liver function was classified using the Child\u0026ndash;Pugh score [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Diabetes mellitus was diagnosed in accordance with the criteria of the American Diabetes Association, with a random plasma glucose level of \u0026ge;\u0026thinsp;200 mg/dL, fasting plasma glucose level of \u0026ge;\u0026thinsp;126 mg/dL, or use of any anti-hyperglycemic medication [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Alcohol consumption of more than 80 g/day was considered heavy drinking [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBefore treatment, patients were screened for varices by endoscopy to assess the risk of bleeding. Patients with high-risk varices received endoscopic treatment before administration of atezolizumab and bevacizumab. In addition, patients were screened for endocrine, neovascular, and respiratory abnormalities before administration of combination therapy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTreatment protocol\u003c/h2\u003e \u003cp\u003ePatients received a combination therapy of atezolizumab and bevacizumab (Chugai Pharmaceutical Co. LTD, Tokyo, Japan) via intravenous administration. The dosage consisted of 1200 mg of atezolizumab and 15 mg/kg of bevacizumab, given every 3 weeks until either tumor progression or unacceptable adverse events occurred. If clinical benefits were observed, treatment was continued even beyond tumor progression. In the event of adverse events, patients were allowed to receive a monotherapy of either atezolizumab or bevacizumab, depending on the type of adverse event experienced.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eSample collection and DNA isolation\u003c/h2\u003e \u003cp\u003eSample collection and DNA isolation were performed as previously reported [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In brief, fecal samples were collected from patients before treatment and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. DNA was extracted using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further analysis. The V3-4 regions of the bacterial 16S rRNA gene were amplified using universal primers, and the PCR products were purified with AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). Samples were barcoded and pooled for sequencing on an Illumina MiSeq platform using the MiSeq Reagent Kit v3 with 2\u0026times;300 reads and 600 cycles (Illumina, San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e16S rRNA gene sequencing\u003c/h2\u003e \u003cp\u003eThe 16S rRNA sequencing data was processed using QIIME2 (version 2021.2) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. After demultiplexing, the paired-end reads for 37 microbiome samples were imported into QIIME2. Sequence quality control and feature table construction was performed using the Divisive Amplicon Denoising Algorithm 2 (DADA2) QIIME2 plugin. After denoising, a pre-trained naive Bayes classifier based on the SILVA database version 132 was used to explore the taxonomic distribution of the samples [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using EZR, a graphical user interface for R [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The significance level was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Continuous variables were expressed as medians with interquartile ranges and analyzed using the Mann\u0026ndash;Whitney \u003cem\u003eU\u003c/em\u003e test. Categorical variables were analyzed using chi-squared or Fisher's exact tests. Microbiome data were analyzed and visualized using the MicrobiomeAnalyst online platform[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Alpha diversity was calculated using the Chao 1, Shannon index, and observed genuses, and beta diversity was estimated using the Bray\u0026ndash;Curtis index and visualized using principal coordinate analysis (PCoA). The significance of the PCoA plot was determined using PERMANOVA. Differential taxonomy analysis was performed using linear discriminant analysis effect size (LEfSe: (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://huttenhower.sph.harvard.edu/galaxy/\u003c/span\u003e\u003cspan address=\"http://huttenhower.sph.harvard.edu/galaxy/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e))\u003c/span\u003e with LDA score\u0026thinsp;\u0026gt;\u0026thinsp;2 and \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Survival analysis was performed using the Kaplan\u0026ndash;Meier method, and differences between groups were assessed by log-rank test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003ePatient background\u003c/h2\u003e\n \u003cp\u003eDuring the study period, a total of 54 patients received the combination therapy of Atezolizumab and Bevacizumab at our hospital. Out of these, 37 patients agreed to participate in the study and provided pre-treatment fecal samples. Of these 37 patients, 28 were categorized as responders (11 patients with PR and 17 patients with SD) and 9 were categorized as non-responders (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Patient background information for both groups is presented in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The median age of patients in the responder and non-responder groups was 74 and 75 years, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.184). The sex ratio (male:female) was 23:5 in the responder group and 5:4 in the non-responder group (p\u0026thinsp;=\u0026thinsp;0.178). The etiology of HCC was as follows: HBV (5 patients), HCV (7 patients), alcohol (10 patients), and non-B non-C (6 patients) in the responder group and HBV (1 patient), HCV (6 patients), alcohol (2 patients), and non-B non-C (0 patients) in the non-responder group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.128). The distribution of BCLC stage was as follows: A (3 patients), B (6 patients), and C (19 patients) in the responder group and A (0 patients), B (3 patients), and C (6 patients) in the non-responder group (p\u0026thinsp;=\u0026thinsp;0.707). The distribution of Child\u0026ndash;Pugh score was as follows: 5 (22 patients), 6 (6 patients), and 7 (0 patients) in the responder group and 5 (3 patients), 6 (3 patients), and 7 (3 patients) in the non-responder group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007), indicating significantly better liver function in the responder group. Neutrophil-to-lymphocyte ratio (NLR), which has been reported to be associated with the efficacy of ICIs, did not differ between the two groups [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e]. Other blood test data and the use of PPIs and antibiotics were similar between the two groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eMicrobiome profiling and comparison of alpha and beta diversities\u003c/h2\u003e\n \u003cp\u003eWe examined the gut microbiome profile of the responder and non-responder groups at the phylum level, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Both groups were dominated by the major phyla found in human gut microbiota: Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. The proportions of each phylum were as follows: Firmicutes (78.0% vs 68.9%), Bacteroidetes (19.7% vs 14.1%), Actinobacteria (5.3% vs 8.3%), and Proteobacteria (2.5% vs 3.1%) in the responder and non-responder groups, respectively. We also compared the alpha and beta diversities of the two groups and found no significant differences (Supplementary Fig. 1).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eComparison of relative abundance of bacterial communities between responder and non-responder groups with LEfSe\u003c/h2\u003e\n \u003cp\u003eWe used LEfSe to compare the relative abundance of bacterial communities between responder and non-responder to identify bacterial genera associated with treatment response. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e shows the results of the LEfSe analysis, with green and red bars indicating bacterial communities that were significantly increased in the responder and non-responder groups, respectively. One family (\u003cem\u003eTannerellaceae\u003c/em\u003e), one genus (\u003cem\u003eParabacteroides\u003c/em\u003e), and two species (\u003cem\u003eBacteroides stercoris\u003c/em\u003e and \u003cem\u003eParabacteroides merdae\u003c/em\u003e) were found to be more abundant in the responder group. Conversely, the microbiome related to Phylum Synergistota and three families (\u003cem\u003eLeuconostocaceae, Morganellaceae\u003c/em\u003e, and \u003cem\u003eSynergistaceae\u003c/em\u003e), two genera (\u003cem\u003eWeisella\u003c/em\u003e and \u003cem\u003eLachnospiraceae UCG 008\u003c/em\u003e), and some species were enriched in the non-responder group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eComparison of progression-free survival (PFS) and overall survival (OS)\u003c/h2\u003e\n \u003cp\u003eTo investigate the bacteria associated with the effect of atezolizumab and bevacizumab, we focused on \u003cem\u003eBacteroides stercoris\u003c/em\u003e and \u003cem\u003eParabacteroides merdae\u003c/em\u003e, which were enriched in the responder group and have been reported to be associated with a better effect of ICIs [\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e23\u003c/span\u003e]. The relative abundance of these bacteria in each sample is shown in Supplementary Fig. 2. PFS and OS were calculated for the two groups based on the presence or absence of each bacterium (Supplementary Fig. 3). Both bacteria showed a trend towards better PFS and OS in the group with the bacterium, and particularly the group with \u003cem\u003eBacteroides stercoris\u003c/em\u003e showed significantly better PFS than the group without the bacteria.\u003c/p\u003e\n \u003cp\u003eOn the basis of these results, we divided the patients into four groups based on the presence or absence of the bacteria (1: \u003cem\u003eP. merdae\u003c/em\u003e (-)\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. stercoris\u003c/em\u003e (-), 2: \u003cem\u003eP. merdae\u003c/em\u003e (+)\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. stercoris\u003c/em\u003e (-), 3: \u003cem\u003eP. merdae\u003c/em\u003e (-)\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. stercoris\u003c/em\u003e (+), 4: \u003cem\u003eP. merdae\u003c/em\u003e (+)\u0026thinsp;+\u0026thinsp;\u003cem\u003eB. stercoris\u003c/em\u003e (+)). PFS and OS were analyzed separately. The results showed that the group that was negative for both bacteria had significantly worse PFS and OS than the other groups (Supplementary Fig. 4). Comparison of patient background of each group showed that the group without both bacteria had significantly higher alanine aminotransferase (ALT) and \u0026alpha;-fetoprotein (AFP) (Supplementary Table). Because the absence of both bacteria may be related to the poor efficacy of atezolizumab and bevacizumab treatment, we analyzed OS in the group negative for both bacteria (n\u0026thinsp;=\u0026thinsp;4) and in the group positive for at least one of either bacteria (n\u0026thinsp;=\u0026thinsp;33). We found that the group without the bacteria had a significantly worse prognosis (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Comparison of patient background showed that the group with the absence of these two bacteria were significantly older, with lower serum albumin and higher AFP levels (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we analyzed pre-treatment fecal samples from patients undergoing atezolizumab and bevacizumab treatment and identified bacteria enriched in the responder group that were associated with better prognosis.\u003c/p\u003e \u003cp\u003eThe recent introduction of several new agents has led to advancements in the treatment of HCC. Atezolizumab, an ICI, is commonly used in combination with bevacizumab, an angiogenesis inhibitor, as a first-line treatment for unresectable HCC. However, the efficacy of this treatment and potential adverse events remain unclear.\u003c/p\u003e \u003cp\u003eIn recent years, numerous studies have reported on the relationship between ICIs and the intestinal microbiome, with intestinal bacteria-mediated mechanisms thought to be involved in their efficacy and in adverse events [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In relation to HCC, a study from Taiwan revealed that \u003cem\u003eLachnoclostridium\u003c/em\u003e and \u003cem\u003ePrevotella 9\u003c/em\u003e are associated with the favorable effects of ICI administration in HCC [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Meanwhile, another study from Japan showed that the efficacy of atezolizumab and bevacizumab was related to the use of antibiotics, suggesting that intestinal bacteria play a role in the effectiveness of these drugs [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In our study, \u003cem\u003eParabacteroides merdae\u003c/em\u003e and \u003cem\u003eBacteroides stercoris\u003c/em\u003e were found to be enriched in the responder group. Both of these bacteria have been reported to have favorable effects on ICIs[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. \u003cem\u003eBacteroides stercoris\u003c/em\u003e was enriched in the responder group of PD-1 and CTLA4-based therapy for patients with malignant melanoma [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. \u003cem\u003eParabacteroides merdae\u003c/em\u003e was enriched in responders to anti-PD-1 and anti-PD-L1 inhibitor treatment for non-small cell lung cancer patients [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These bacteria and the contributions they make to the intestinal environment are hypothesized to be involved in host immunity and related to therapeutic effects of ICIs.\u003c/p\u003e \u003cp\u003eThe absence of both \u003cem\u003eBacteroides stercoris\u003c/em\u003e and \u003cem\u003eParabacteroides merdae\u003c/em\u003e was significantly associated with poor prognosis. Comparing baseline characteristics of these patients, the group without either bacterium was significantly older (83 vs 73 years) and had higher AFP. Even among the patients aged 75 or older, AFP was significantly higher in the group without both bacteria (data not shown). AFP levels at the time of HCC diagnosis have been reported as an independent risk predictor related to pathological grade, progression, and survival [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This suggests that the presence or absence of these bacteria may select patients with higher risk of malignancy.\u003c/p\u003e \u003cp\u003eOur study had some limitations. Owing to the small number of cases in this single-center, retrospective study, differences between the microbiome and treatment effects by etiology or liver function were difficult to examine. Further studies with a larger patient cohort or a validation group are needed to address the strong inter-individual variability associated with the intestinal microbiome. Moreover, metabolite measurements were not taken, and thus the mechanism by which the microbiome may be involved in the effects of ICIs is unknown.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study suggests that specific gut bacteria may play a crucial role in determining the treatment response of HCC patients receiving atezolizumab and bevacizumab therapy. These findings may pave the way for the development of novel microbiome-based therapeutic strategies for enhancing the efficacy of immunotherapy in HCC patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the ethics committees of hospital (approval number:\u0026nbsp;2015-0420).\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained from all patients for inclusion in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the 39th Grant from The Nitto Foundation for Kenta Yamamoto and\u0026nbsp;by JST [Moonshot R\u0026amp;D][Grant Number JPMJMS2214-11].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors (Yosuke Inukai, Kenta Yamamoto, Takashi Honda, Shinya Yokoyama, Norihiro Imai, Yoji Ishizu, Masanao Nakamura, Hiroki Kawashima) declare that they have no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTakanori Ito received personal fees from Chugai Pharmaceutical Co., Ltd. outside the submitted work. Masatoshi Ishigami received grants from Chugai Pharmaceutical Co., Ltd. outside the submitted work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYI\u003c/strong\u003e and \u003cstrong\u003eKY\u003c/strong\u003e conceived the idea of the study. \u003cstrong\u003eTH\u0026nbsp;\u003c/strong\u003edeveloped the statistical analysis plan and conducted statistical analyses. \u003cstrong\u003eYI, KY, TH, SY, NI, TI, YI, MN, MI and HK\u003c/strong\u003e contributed to the interpretation of the results. \u003cstrong\u003eYI\u003c/strong\u003e drafted the original manuscript. \u003cstrong\u003e\u0026nbsp;All authors\u003c/strong\u003e reviewed the manuscript draft and revised it critically on intellectual content. \u003cstrong\u003eAll authors\u003c/strong\u003e approved the final version of the manuscript to be published.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. 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Transection of the oesophagus for bleeding oesophageal varices Br J Surg. 1973;60:646\u0026ndash;649.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhelton PK, Carey RM, Aronow WSet al.. 2017 ACC/AHA/AAPA/ABC/\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eACPM/AGS/APhA/ASH/ASPC/NMA/PCNA\u003c/span\u003e\u003cspan address=\"http://ACPM/AGS/APhA/ASH/ASPC/NMA/PCNA\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e Guideline for the Prevention, Detection, Evaluation, and Management of High Blood Pressure in Adults: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines J Am Coll Cardiol. 2018;71:e127-e248.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrabb DW, Im GY, Szabo G, Mellinger JL, Lucey MR. Diagnosis and Treatment of Alcohol-Associated Liver Diseases: 2019 Practice Guidance From the American Association for the Study of Liver Diseases Hepatology. 2020;71:306\u0026ndash;333.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamamoto K, Kuzuya T, Honda Tet al.. Relationship Between Adverse Events and Microbiomes in Advanced Hepatocellular Carcinoma Patients Treated With Sorafenib Anticancer Res. 2020;40:665\u0026ndash;676.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolyen E, Rideout JR, Dillon MRet al.. Author Correction: Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2 Nat Biotechnol. 2019;37:1091.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuast C, Pruesse E, Yilmaz Pet al.. The SILVA ribosomal RNA gene database project: improved data processing and web-based tools Nucleic Acids Res. 2013;41:D590-596.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKanda Y. Investigation of the freely available easy-to-use software 'EZR' for medical statistics Bone Marrow Transplant. 2013;48:452\u0026ndash;458.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDhariwal A, Chong J, Habib S, King IL, Agellon LB, Xia J. MicrobiomeAnalyst: a web-based tool for comprehensive statistical, visual and meta-analysis of microbiome data Nucleic Acids Res. 2017;45:W180-W188.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSegata N, Izard J, Waldron Let al.. Metagenomic biomarker discovery and explanation Genome Biol. 2011;12:R60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValero C, Lee M, Hoen Det al.. Pretreatment neutrophil-to-lymphocyte ratio and mutational burden as biomarkers of tumor response to immune checkpoint inhibitors \u003cem\u003eNat Commun\u003c/em\u003e. 2021;12:729.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndrews MC, Duong CPM, Gopalakrishnan Vet al.. Gut microbiota signatures are associated with toxicity to combined CTLA-4 and PD-1 blockade Nat Med. 2021;27:1432\u0026ndash;1441.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong P, Yang D, Wang Het al.. Relationship between intestinal flora structure and metabolite analysis and immunotherapy efficacy in Chinese NSCLC patients \u003cem\u003eThorac Cancer\u003c/em\u003e. 2020;11:1621\u0026ndash;1632.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaesaka K, Sakamori R, Yamada Ret al.. Pretreatment with antibiotics is associated with reduced therapeutic response to atezolizumab plus bevacizumab in patients with hepatocellular carcinoma PLoS One. 2023;18:e0281459.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBai DS, Zhang C, Chen P, Jin SJ, Jiang GQ. The prognostic correlation of AFP level at diagnosis with pathological grade, progression, and survival of patients with hepatocellular carcinoma Sci Rep. 2017;7:12870.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eTable 1: Patient characteristics.\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse:collapse;border:none;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eResponder\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003en=28\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eNon-responder\u003c/span\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003en=9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e-value\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAge\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026dagger;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e74(63-79)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e75(71-85)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.184\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eGender (male/female)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e23/5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e5/4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.178\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eBody mass index\u0026dagger;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e23.3(20.9-26.8)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e24.0(20.9-25.3)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.804\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eEtiology (HBV/HCV/Alcohol/NBNC)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e5/7/10/6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e1/6/2/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.128\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eBCLC stage (A/B/C)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e3/6/19\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0/3/6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.707\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eChild-Pugh grade (A/B)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e28/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e6/3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:red;'\u003e0.011\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eChild-Pugh score (5/6/7)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e22/6/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e3/3/3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:red;'\u003e0.007\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eTreatment history (1/2/3/4)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e26/2/0/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e4/3/0/2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:red;'\u003e0.005\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAST (IU/L)\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;\u0026dagger;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e36(27-51)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e37(20-46)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.710\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eALT(IU/L)\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;\u0026dagger;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e29(21-38)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e25(15-30)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.279\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e\u0026gamma;-\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eGTP(IU/L)\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;\u0026dagger;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e77(55-136)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e33(31-186)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.583\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eTotal bilirubin (mg/dL)\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;\u0026dagger;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.8(0.71.1)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.7(0.5-1.0)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.246\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAlbumin (g/dL)\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;\u0026dagger;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e3.8(3.6-4.0)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e3.4(2.6-3.8)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.051\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eHbA1c(%)\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026dagger;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e6.1(5.6-6.8)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e6.2(5.7-6.7)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.657\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAFP (ng/mL)\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;\u0026dagger;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e9.5(5.0-410.8)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e564.0(13.0-845.0)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.123\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003ePPI (yes/no)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e18/10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e5/4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.705\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eAntibiotics(yes/no)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e3/25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e1/8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e1.000\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eC-reactive protein (mg/dL)\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;\u0026dagger;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.20(0.09-0.50)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.38(0.04-1.46)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.645\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.6pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003eNLR\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026dagger;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:85.05pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e1.96(1.29-3.32)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:92.15pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e2.71(1.64-3.62)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;padding:0in 5.4pt 0in 5.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:black;'\u003e0.357\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Yu Mincho\",serif;margin-bottom:4.0pt;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eBCLC,\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003ethe Barcelona Clinic Liver Cancer Classification\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;; AST, aspartate aminotransferase; ALT, alanine aminotransferase; \u0026gamma;-GTP, \u003cspan style=\"color:#202124;background:white;\"\u003e\u0026gamma;-glutamyl transpeptidase;\u003c/span\u003e AFP, \u0026alpha;-fetprotein; PPI, protom pump inhibitor; NLR, Neutrophil-Lymphocyte Ratio\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Yu Mincho\",serif;margin-bottom:4.0pt;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026dagger;Values are expressed as median (interquartile range).\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cspan style=\"font-size:14px;font-family:\u0026quot;Arial\u0026quot;,sans-serif;\"\u003e\u003c/span\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eTable 2: Patient background with and without P.merdae and B.stercoris.\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border: none;width:474.9pt;border-collapse:collapse;\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:40.05pt;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:40.05pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eP.merdae(+)or B.stercoris(+)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003en=33\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:40.05pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eP.merdae(-) and B.stercoris(-)\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cstrong\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003en=4\u003c/span\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:40.05pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cem\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eP\u003c/span\u003e\u003c/em\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e-value\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eAge*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e73(63-79)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e83(79-85)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;color:red;'\u003e0.047\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eGender (male/female)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e26/7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e2/2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e0.244\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eBody mass index*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e24.0(20.9-27.0)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e21.7(20.1-23.3)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e0.240\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eEtiology (HBV/HCV/Alcohol/NBNC)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e6/10/11/6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e0/3/1/0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e0.460\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eBCLCstage (A/B/C)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e3/8/22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e0/1/3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:21.35pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eChild-Pugh grade (A/B)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:21.35pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e31/2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:21.35pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e3/1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:21.35pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e0.298\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:21.75pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eChild-Pugh score (5/6/7)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:21.75pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e24/7/2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:21.75pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e1/2/1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:21.75pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e0.104\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:18.85pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eTreatment history (1/2/3/4)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:18.85pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e27/5/1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:18.85pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e3/0/1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:18.85pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e0.278\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.4pt .4pt 0in .4pt;height:14.2pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eAST (IU/L)*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:14.2pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e35(25-45)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:14.2pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e134(88-174)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:14.2pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e0.106\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.4pt .4pt 0in .4pt;height:14.2pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eALT (IU/L)*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:14.2pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e27(21-35))\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.6pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:14.2pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e52(23-107)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:14.2pt;\"\u003e\n \u003cp 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style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e0.625\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.4pt .4pt 0in .4pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eAntibiotics(yes/no)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan 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style=\"width:63.8pt;border:none;border-bottom:solid windowtext 1.0pt;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e0.186\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width:148.35pt;border:none;padding:.4pt .4pt 0in .4pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eNLR*\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:135.15pt;border:none;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e1.97(1.22-2.75)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:127.6pt;border:none;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e3.65(3.20-4.08)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width:63.8pt;border:none;padding:.25pt .25pt 0in .25pt;height:17.4pt;\"\u003e\n \u003cp style='margin:0in;text-align:center;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e0.070\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Yu Mincho\",serif;margin-bottom:4.0pt;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003eBCLC,\u0026nbsp;\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003ethe Barcelona Clinic Liver Cancer Classification\u003c/span\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;; AST, aspartate aminotransferase; ALT, alanine aminotransferase; \u0026gamma;-GTP, \u003cspan style=\"color:#202124;background:white;\"\u003e\u0026gamma;-glutamyl transpeptidase;\u003c/span\u003e AFP, \u0026alpha;-fetprotein; PPI, protom pump inhibitor; NLR, Neutrophil-Lymphocyte Ratio\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;text-align:justify;font-size:14px;font-family:\"Yu Mincho\",serif;margin-bottom:4.0pt;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026dagger;Values are expressed as median (interquartile range).\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp style='margin:0in;text-align:left;font-size:14px;font-family:\"Yu Mincho\",serif;'\u003e\u003cspan style='font-family:\"Arial\",sans-serif;'\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3996460/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3996460/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackgrounds:\u003c/h2\u003e \u003cp\u003eThe combination of Atezolizumab and Bevacizumab has become the first-line treatment for patients with unresectable hepatocellular carcinoma (HCC). However, no studies have reported on specific intestinal microbiota associated with the efficacy of Atezolizumab and Bevacizumab. In this study, we analyzed fecal samples collected before treatment to investigate the relationship between the intestinal microbiome and the efficacy of Atezolizumab and Bevacizumab.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eA total of 37 patients with advanced HCC who were treated with Atezolizumab and Bevacizumab were enrolled. Fecal samples were collected from the patients, and they were divided into responder (n\u0026thinsp;=\u0026thinsp;28) and non-responder (n\u0026thinsp;=\u0026thinsp;9) groups. We compared the intestinal microbiota of the two groups and analyzed the intestinal bacteria associated with prognosis using QIIME2.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eThe alpha and beta diversities were not significantly different between both groups, and the proportion of microbiota was similar. The relative abundance of \u003cem\u003eBacteroides stercoris\u003c/em\u003e and \u003cem\u003eParabacteroides merdae\u003c/em\u003e was higher in the responder group than in the non-responder group. When the prognosis was analyzed by the presence or absence of those bacteria, patients without both had a significantly poorer prognosis.\u003c/p\u003e\u003ch2\u003eConclusion:\u003c/h2\u003e \u003cp\u003eDifferences in intestinal microbiome are involved in the therapeutic effect of Atezolizumab and Bevacizumab.\u003c/p\u003e","manuscriptTitle":"Intestinal Microbiome Associated with Efficacy of Atezolizumab and Bevacizumab Therapy for Hepatocellular Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-05 16:41:26","doi":"10.21203/rs.3.rs-3996460/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4f8fc67a-b9f3-4b1d-83c6-b7d0525589d7","owner":[],"postedDate":"March 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-01T22:31:13+00:00","versionOfRecord":{"articleIdentity":"rs-3996460","link":"https://doi.org/10.3390/cancers16091675","journal":{"identity":"cancers","isVorOnly":true,"title":"Cancers"},"publishedOn":"2024-04-26 22:31:13","publishedOnDateReadable":"April 26th, 2024"},"versionCreatedAt":"2024-03-05 16:41:26","video":"","vorDoi":"10.3390/cancers16091675","vorDoiUrl":"https://doi.org/10.3390/cancers16091675","workflowStages":[]},"version":"v1","identity":"rs-3996460","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3996460","identity":"rs-3996460","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}