Gut microbiota ORIGINAL ARTICLE Gut microbiome analysis as a tool towards Gut: first published as 10.1136/gutjnl-2017-315084 on 25 July 2018. Downloaded from targeted non-invasive biomarkers for early hepatocellular carcinoma Zhigang Ren,1,2,3 Ang Li,2,3,4 Jianwen Jiang,1,4,5 Lin Zhou,1,4 Zujiang Yu,2,3 Haifeng Lu,4 Haiyang Xie,1,4 Xiaolong Chen,2,3 Li Shao,4 Ruiqing Zhang,6,7 Shaoyan Xu,1 Hua Zhang,4 Guangying Cui,2,3 Xinhua Chen,1,4 Ranran Sun,2,3 Hao Wen,7 Jan P Lerut,8 Quancheng Kan,9 Lanjuan Li,4 Shusen Zheng1,4,10 ► Additional material is ABSTRact published online only. To view Objective To characterise gut microbiome in patients Significance of this study please visit the journal online with hepatocellular carcinoma (HCC) and evaluate the (http:// dx. doi. org/ 10. 1136/ What is already known on this subject? gutjnl- 2017- 315084). potential of microbiome as non-invasive biomarkers for HCC. ► Hepatocellular carcinoma (HCC) is the For numbered affiliations see third leading cause of cancer-related death end of article. Design We collected 486 faecal samples from East China, Central China and Northwest China prospectively worldwide due to the poor prognosis, high incidence and postsurgical recurrence. Correspondence to and finally 419 samples completed Miseq sequencing. Professor Shusen Zheng, We characterised gut microbiome, identified microbial ► The gut microbiota promotes HCC development Department of Hepatobiliary markers and constructed HCC classifier in 75 early HCC, by the microbiota-liver axis in HCC animal and Pancreatic Surgery, the First 40 cirrhosis and 75 healthy controls. We validated the models, but microbial characteristics in Affiliated Hospital, School of patients with HCC have not been reported. Medicine, Zhejiang University, results in 56 controls, 30 early HCC and 45 advanced The concept of the gut microbiome serving Hangzhou 310003, China; HCC. We further verified diagnosis potential in 18 HCC ► shusenzheng@ zju. edu. cn, from Xinjiang and 80 HCC from Zhengzhou. as a tool towards for achieving targeted non- Professor Lanjuan Li, State Results Faecal microbial diversity was increased invasive biomarkers for specific diseases or Key Laboratory for Diagnosis from cirrhosis to early HCC with cirrhosis. Phylum cancer, including type 2 diabetes, liver cirrhosis and Treatment of Infectious and colorectal cancer, has been established by Disease, the First Affiliated Actinobacteria was increased in early HCC versus Hospital, School of Medicine, cirrhosis. Correspondingly, 13 genera including compelling studies, but it is unclear whether http://gut.bmj.com/ Zhejiang University, Hangzhou Gemmiger and Parabacteroides were enriched in early gut microbial markers could discriminate HCC. 310003, China; ljli@ zju. edu. HCC versus cirrhosis. Butyrate-producing genera were cn and Professor Quancheng What are the new findings? Kan, Department of Pharmacy, decreased, while genera producing-lipopolysaccharide ► Faecal microbial diversity was decreased the First Affiliated Hospital were increased in early HCC versus controls. The optimal from healthy controls to cirrhosis, but it was of Zhengzhou University, 30 microbial markers were identified through a fivefold increased from cirrhosis to early HCC with Zhengzhou 450052, China; cross-validation on a random forest model and achieved cirrhosis. qckan19632012@ 163. com on October 2, 2021 by guest. Protected copyright. an area under the curve of 80.64% between 75 early ► Butyrate-producing bacterial genera ZR, AL, JJ, LZ and ZY contributed HCC and 105 non-HCC samples. Notably, gut microbial were decreased, while genera producing- equally. markers validated strong diagnosis potential for early lipopolysaccharide were increased in early HCC HCC and even advanced HCC. Importantly, microbial versus healthy controls. Received 17 August 2017 markers successfully achieved a cross-region validation of Revised 5 June 2018 ► The optimal 30 microbial markers were Accepted 7 June 2018 HCC from Northwest China and Central China. identified through a fivefold cross-validation on Conclusions This study is the first to characterise gut a random forest model and achieved an area microbiome in patients with HCC and to report the under the curve of 80.64% between 75 early successful diagnosis model establishment and cross- HCC and 105 non-HCC samples. region validation of microbial markers for HCC. Gut ► Gut microbial markers validated strong microbiota-targeted biomarkers represent potential non- diagnosis potential for early HCC and even invasive tools for early diagnosis of HCC. advanced HCC. Importantly, microbial markers successfully achieved a cross-region validation © Author(s) (or their employer(s)) 2018. Re-use of HCC from Northwest China and Central permitted under CC BY-NC. No China. commercial re-use. See rights INTRODUCTION and permissions. Published Hepatocellular carcinoma (HCC) is the third by BMJ. leading cause of cancer-related death worldwide.1 2 Currently, there are an estimated 29 200 new HCC the prevalence of hepatitis B virus (HBV) persistent To cite: Ren Z, Li A, Jiang J, et al. Gut Epub ahead of cases in males and 11 510 cases in females in the USA infection and HBV-induced cirrhosis. Due to the 3 print: [please include Day in 2017. More seriously, estimated new HCC cases absence of specific symptoms in early stages and the Month Year]. doi:10.1136/ achieved 343 700 in males and 122 300 in females lack of early diagnostic markers, most patients with gutjnl-2017-315084 in China in 2015,4 which is mainly attributed to HCC are often diagnosed in an advanced stage with Ren Z, et al. Gut 2018;0:1–10. doi:10.1136/gutjnl-2017-315084 1 Gut microbiota of the First Affiliated Hospital, School of Medicine, Zhejiang Significance of this study University (2014–334); the First Affiliated Hospital of Zheng- zhou University (2017-XY-002) and the First Affiliated Hospital Gut: first published as 10.1136/gutjnl-2017-315084 on 25 July 2018. Downloaded from How might it impact on clinical practice in the foreseeable of Xinjiang Medical University. All participants signed written future? informed consents on enrolment. ► This is the first report to illustrate gut microbial A total of 486 faecal samples from East China, Central characteristics in patients with early HCC through large- China and Northwest China were prospectively collected, and cohort Miseq sequencing. finally 419 samples were included and subjected to 16S rRNA ► Gut microbial alterations may contribute to the development Miseq sequencing. Participants’ demographics, clinicopatholog- of HCC, which implies that the changed gut microbiota may ical data, CT scan, histopathology images and diet habit were represent a potential target to prevent HCC development by collected from hospital electronic medical records and ques- the gut-microbiota-liver axis. tionnaires (online supplementary table S1). Detailed diagnostic ► This study is the first to report the successful diagnosis criteria and inclusion and exclusion criteria of the participants model establishment and cross-region validation of microbial are described in the online supplementary methods. markers for HCC, notably including data from three different regions of China. Gut microbiota-targeted biomarkers Faecal sample collection and DNA extraction represent potential non-invasive tools for early diagnosis of Each individual provided a fresh tail stool sample at 06:30– HCC. 08:30 hours. Each stool sample completed faecal routine testing. The sample was divided into five aliquots of 200 mg and imme- diately stored at −80°C. DNA extraction was conducted as we poor prognosis (the overall ratio of mortality to incidence is previously described.22 23 The details are described in the online 0.95).5 6 Therefore, novel diagnostic markers for early HCC and supplementary methods. new therapeutic strategies are urgently needed to improve the prognosis of this population. The human gut microbiota have been considered the most Stool moisture measurement important microecosystem living in symbiosis with the body.7–11 Stool consistency was assessed using stool routine testing results. It is identified as a crucial determinant of intestinal inflammation Stool moisture content was determined in duplicate on the and as a key player in chronic inflammatory liver diseases.12 Gut frozen homogenised faecal material (−80°C) as the percentage microbial alterations contribute to the onset and progression of of stool mass loss after lyophilisation (Labconco FreeZone, alcoholic liver disease, non-alcoholic fatty liver disease,13 liver LABCONCO, USA).24 cirrhosis and its complications.14 Recent studies in animal models indicate that the intestinal microbiota promote HCC develop- 15 16 PCR amplification, MiSeq sequencing and Sequence data ment through the gut microbiota-liver axis. However, until process now, gut microbial characteristics in clinical patients with HCC Extracted DNA samples were amplified, DNA libraries were have not yet been reported. constructed and the sequencing was performed on an Illumi- http://gut.bmj.com/ The concept of gut microbiome serving as a non-invasive naMiSeq platform by Shanghai Itechgene Technology, China. diagnosis tool for specific diseases or cancer, including type 2 17 18 Amplified reads were processed. The details are described in diabetes (T2D) and colorectal cancer (CRC), has been estab- the online supplementary methods. Raw Illumina read data lished by compelling studies. We previously established an accu- for all samples were deposited in the European