胃癌新辅助化疗不同应答肿瘤微生物组学的改变及其临床意义

Changes in tumor microbiome and underlying value according to response to neoadjuvan chemotherapy for in patients with gastric cancer

目的:探究胃癌新辅助化疗不同应答肿瘤微生物组学的改变。方法:采用回顾性观察性研究的方法,收集2017年1月至2023年1月期间,青岛大学附属医院胃肠外科肿瘤样本库中病理确诊为胃腺癌、无转移及术前仅接受新辅助化疗并成功接受胃癌根治术的31例胃癌患者术后新鲜冷冻标本;排除出现转移或其他原发肿瘤病灶和术前1个月内接受过其他转化治疗包括免疫治疗、靶向治疗和益生菌的患者。采用第8版美国癌症联合委员会(AJCC)分期系统和美国病理学家协会肿瘤消退分级系统(TRG)对患者进行分级和分组,TRG 0~1级、ypT0~1和ypN0被归类为新辅助化疗有反应组(12例),而TRG 2~3级和ypT2~4或ypN+则被归类为新辅助化疗无反应组(19例)。采用16S rRNA测序技术对新鲜冷冻样本进行处理和分析。使用QIIME2的Q2-diversity插件进行Alpha多样性和Beta多样性分析,使用STAMP找出默认参数和两组之间的差异富集细菌类群。通过效应大小线性判别分析进行高维类比较,使用PICRUST2(v2.3.0-b)软件预测微生物组的潜在功能分布。结果:新辅助化疗有反应组与无反应组胃癌患者在性别、年龄、体质指数、吸烟史、肿瘤位置、新辅助化疗前的肿瘤cTNM分期和新辅助化疗方案方面比较,差异没有统计学意义(均 P>0.05),但两组的肿瘤长径和新辅助化疗后的肿瘤ypTNM分期差异均具有统计学意义(均 P=0.001)。胃部微生态的Alpha和Beta多样性结果显示,两组Alpha多样性差异没有统计学意义( P>0.05),而两组Beta多样性差异却有统计学意义( P=0.004)。红螺菌科( Coriobacteriaceae)、瘤胃球菌科( Ruminococcaceae)、韦荣球菌科( Veillonellaceae)和毛螺菌科( Lachnospiraceae)等4种科水平细菌分类群在新辅助化疗有反应组患者中富集,而以 Proteobacteria(变形菌门)为主的4种细菌分类群在新辅助化疗无反应组患者中富集。柠檬酸循环和丙氨酸等在内的各种氨基酸的代谢通路被发现具有潜在预测意义。 结论:不同新辅助化疗反应结果的胃癌患者在胃部微生态的丰度和组成方面存在差异。

Objective To investigate the relationship between efficacy of neoadjuvant chemotherapy(NACT)for gastric cancer and gastric microecology.Methods This was a retrospective observational study using fresh frozen operative specimens.The specimens had been stored in the tumor sample bank of the Department of Gastrointestinal Surgery of the Affiliated Hospital of Qingdao University from January 2017 to January 2023 after having been collected from 31 patients with pathologically diagnosed gastric cancer who had no metastases and had received only neoadjuvant chemotherapy preoperatively.The study patients had all successfully undergone radical gastric cancer surgery.Patients with metastases or other primary tumor foci and/or had received other therapies within 1 month prior to surgery,including immunotherapy,targeted therapies and probiotics,were excluded.The tumors were graded and grouped in accordance with the 8th edition of the American Joint Committee on Cancer staging system and the Tumor Regression Grading System(TRG)of the College of American Pathologists.Those with TRG Grades 0-1,ypT0-1 and ypN0 were classified as responsive(Group R,12 cases),whereas those with TRG Grades 2-3 and ypT2-4 or ypN+were classified as non-responsive(Group NR,19 cases).The fresh frozen samples were processed and analyzed using 16S rRNA sequencing.Alpha and beta diversity analyses were performed using the Q2-diversity plug-in for QIIME2 and STAMP was used to determine the default parameters and differentially enriched bacterial taxa in the two groups.High-dimensional class comparisons were performed by effect size linear discriminant analysis,and potential functional distributions of microbiomes were predicted using PICRUST2(v2.3.0-b)software.Results Groups R and NR did not differ significantly in sex,age,body mass index,smoking history,tumor location,cTNM stage before NACT,and neoadjuvant chemotherapy(all P>0.05),whereas tumor size and ypTNM stage after NACT differed significantly between the two groups(both P=0.001).Alpha and beta diversity analysis of the gastric microbiota did not reveal a statistically significant difference in alpha diversity between the two groups(P>0.05),whereas there was a statistically significant difference in beta diversity between the two groups(P=0.004).Four family-level bacterial taxa,namely Coriobacteriaceae,Ruminococcaceae,Veillonellaceae,and Lachnospiraceae,were enriched in the R group,whereas four bacterial taxa dominated by phylum Proteobacteria were enriched in the NR group.Metabolic pathways of various amino acids,including citric acid cycle and alanine,were found to be potentially predictive.Conclusions There are significant differences in the abundance and composition of gastric microecology in gastric cancer patients with different responses to NACT.