Reproducibility and validity of microbiomial markers in colorectal cancer

描述(申请人提供)：尽管结直肠癌的发病率和死亡率都有所下降，部分原因是内窥镜检查的使用迅速增加，但结直肠癌(CRC)仍然是美国第四大常见癌症和第二大最常见的癌症死亡原因。因此，制定新的预防战略至关重要。人类的结肠直肠是组成微生物区系的数十亿细菌的宿主。微生物区系在大肠健康的几个功能中是必不可少的，包括免疫、营养代谢、生长和能量收集。然而，直到最近，对微生物区系在健康中的作用的评估一直是有限的，因为大多数细菌是不可培养的。独立于培养的下一代16S核糖体RNA测序使对微生物区系在健康中的作用进行了评估。然而，以往关于大肠微生物群的流行病学研究很少，而且样本量很小。此外，最近的研究发现，肠道中的氧气梯度是身体中最陡峭的，从粘膜到管腔中央接近缺氧。因此，与粘膜细菌相比，腔内微生物更有可能是厌氧的。因此，在进行大规模流行病学研究之前，有必要解决两个关键的方法学或实际问题。第一，样本(S)的来源(S)是最适合分析人类结直肠微生物组的；第二，现场样本是否足够可靠地代表与结直肠癌发生有关的结直肠微生物组。这项拟议的研究将通过使用储存的粪便样本和来自100名有结直肠腺瘤病史的参与者(每人4份样本)的2个时间点的直肠拭子来解决这两个关键问题。在母研究中，在与粪便和直肠拭子样本相同的两个时间点，检测正常直肠组织中的结直肠癌生物标志物(细胞凋亡、炎症、增殖和Wnt途径)。在这项拟议的初步研究中，将使用下一代16S rRNA测序对样本进行分析。我们将比较1)拭子和粪便，以及2)拭子和粪便的组合与单一样本类型与免疫组织化学致癌生物标志物的关系。我们还将检查直肠拭子和粪便样本中的微生物生物标记物是否随时间保持稳定；以及进一步2)比较在一个时间点(现场采集)收集的粪便和直肠拭子中的微生物生物标记物与两个时间点(相隔3个月)的微生物生物标记物与免疫组织化学致癌生物标记物的相关性。这项拟议的研究不仅将调查微生物生物标记物在不同类型或组合的样本中的有效性，还将检查来自一个现场样本的这些生物标记物与从两个时间点收集的与直肠组织中的致癌生物标记物有关的可靠性。拟议的研究是独一无二的，样本量大，样本类型多，跨时间点收集多个样本。拟议研究的结果将为未来与CRC相关的微生物区系大规模流行病学研究奠定坚实的基础。

DESCRIPTION (provided by applicant): Despite a reduction in both incidence and mortality of colorectal cancer, partially due to rapidly increased use of endoscopy, colorectal cancer (CRC) still remains the 4th most common incident cancer and the 2nd most common cause of cancer death in the US. Thus, it is critical to develop new prevention strategies. The human colorectum is host to billions of bacteria which comprise the microbiota. The microbiota is essential in several functions of colorectal health including immunity, nutrient metabolism, growth, and energy harvesting. However, until recently, evaluation of the role of microbiota in health has been limited because the majority of bacteria are uncultivable. Cultivation-independent next-generation sequencing of 16S ribosomal RNA has permitted the evaluation of the role of microbiota in health. However, previous epidemiological studies of the colorectal microbiome are few and with small sample sizes. Furthermore, recent studies found oxygen gradients in the intestine are the steepest in the body, which reduce from mucosa to near anoxia at the middle of the lumen. Thus, microbes in luminal communities are more likely to be anaerobic compared to mucosal bacteria. Thus, before conducting large-scale epidemiological studies, it is necessary to address two key methodological or practical questions. First, what source(s) of sample(s) is/are most appropriate for analysis of the human colorectal microbiome, and, second, whether a spot sample is adequately reliable for representing the colorectal microbiome in relation to colorectal carcinogenesis. This proposed study will address these two key issues by using banked fecal samples and paired rectal swabs from 2 time points among 100 participants with a history of colorectal adenoma (4 samples from each person). In the parent study, colorectal carcinogenesis biomarkers (apoptosis, inflammation, proliferation and Wnt pathway) are measured in normal rectal tissue at the same 2 time points as the fecal and rectal swab samples. In this proposed pilot study, the samples will be analyzed using next-generation sequencing of 16S rRNA. We will compare 1) swabs to stool and 2) the combination of swab and stool to the single sample type in association with immunohistochemistry carcinogenesis biomarkers. We will also examine whether the microbial biomarkers in rectal swabs and stool samples remain stable over time; and further 2) to compare microbial biomarkers in stool and rectal swabs collected at one time point (spot collection) with microbial biomarkers from two time points (3 months apart) in their correlation with immunohistochemistry carcinogenesis biomarkers. The proposed study will not only investigate the validity of microbial biomarkers in different types or combinations of samples, but also examine the reliability of these biomarkers from one spot sample vs. collections from two time points in relation to carcinogenesis biomarkers in rectal tissue. The proposed study is unique with a large sample size, multiple sample types, and multiple collections across time points. The results from the proposed study will lay a solid foundation for future large-scale epidemiologic studies of microbiota in association with CRC.