br In the present study a large number of GC

In the present study, a large number of GC subjects without preoper-ative chemotherapy was enrolled retrospectively, and mucosal tissues were collected from different stomach microhabitats. We aimed to assess the Lipo3000 and composition of the gastric microbiota across tumoral and peritumoral microhabitats, and compare them to those in the same subjects with normal gastric mucosal morphology. We em-phasized the major shifts in the gastric tumor microbiota relative to that of matched, normal gastric tissue from the same individual, which allow us to survey microbial communities in stomach microhab-itats with an intrinsic control for the effects of environment and host ge-netics. This study will provide a better understanding of the microbial transition in tumors and tumor-free tissues, and of the association be-tween bacterial colonisation and GC development. Ultimately, this will help to identify novel microbiome-related diagnostic tools and thera-peutic interventions.
2. Materials and methods
In total, 276 GC patients without preoperative chemotherapy were enrolled from March 2009 to August 2013 from the First Affiliated Hospital, School of Medicine, Zhejiang University (Zhejiang, China). Stomach tissues were obtained from patients with primary GC who accepted gastrectomy. Finally, 229 tumoral tissues, 247 peritumoral tis-sues and 230 normal tissues were selected for microbiota analysis, which were based on DNA amount and quality appropriate for 16S rRNA gene amplification and sequence analysis. The tumor and tumor-free tissues were collected and confirmed by pathological diag-nosis. N99% of the GC was moderately/poorly differentiated. According to Lauren, GC can be divided into adenocarcinomas of diffuse and intes-tinal types [19]. 96 intestinal-type patients, 56 diffuse-type patients and 124 mixed-type patients were included. Patients were determined to be HP positive (HP+) by positive result in rapid urease test or histopathol-ogy. The tumor and tumor-free (2-5 cm adjacent to the cancer tissue, Peritumor; N5 cm adjacent to the cancer tissue, Normal) tissues were collected, which were confirmed by pathological diagnosis. The clinical and pathological staging were based on the 7th edition American Joint Committee on Cancer (AJCC) cancer staging manual of GC TNM Staging [20]. Tumor stage was categorized as follows: stage I, invades mucosa or submucosa; stage II, invades mucularis mucosa; stage III, invades ad-ventitia; stage IV, invades adjacent structures. 142 cases of early-stage GC (AJCC pathologic stage I and II) and 134 cases of late-stage GC (AJCC pathologic stage III and IV) were included [21]. The following criteria were used to exclude subjects: body mass index (BMI = weight in kilograms divided by the height in meters squared) N 30; use of anti-biotics, probiotics, prebiotics, or synbiotics in the previous month; pre-operative chemotherapy, radiotherapy, or other biological treatment before gastrectomy. Basic demographic data, clinical data and informa-tion about possible confounders of microbiota analysis were collected at the time of inclusion for all GC patients (Table 1). All research was ap-proved by the Ethics Committee of the First Affiliated Hospital, School
Table 1
was determined by Qubit (Invitrogen). Negative DNA extraction con-
Summary of the study subjects' characteristics.
trols (lysis buffer and kit reagents only) were amplified and sequenced
Characteristics Patients (n = 276) as contamination controls. Sequencing was performed on a homologous structures MiSeq in-
strument (Illumina) using a 300 × 2 V3 kit together with PhiX Control
2.3. Bioinformatic analysis
Complications, no
The 16S rRNA gene sequence data set generated from the MiSeq run
Hypertension 74
were first merged and demultiplexed into per samples using the QIIME
Diabetes mellitus 17
Tumor localization, no
version 1.9.0 with default parameters [24]. Chimera sequences were de-
Proximal stomach 35
tected and removed using the USEARCH software based on the UCHIME
Body/Fundus 109
algorithm [25]. Open-reference operational taxonomic unit (OTU) pick
was then performed with USEARCH V7 referenced against Greengenes
Tumor differentiation, no
High differentiated 2
Moderately/poor differentiated 274
number of sequences b 0.005% of the total number of sequences were
Lauren typing, no
discarded as recommended [28]. The result was an OTU table, which
Intestinal type 96
was used for downstream analysis.
Diffuse type 56
For taxonomic assignment, the most abundant sequences were