Reproducibility and validity of microbiomial markers in colorectal cancer

描述（由申请人提供）：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常见癌症死亡原因。因此，制定新的预防战略至关重要。人类结直肠是数十亿细菌的宿主，这些细菌组成了微生物群。微生物群在结肠直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，对微生物群在健康中的作用的评估仍然有限，因为大多数细菌是不可培养的。培养独立的下一代16S核糖体RNA测序已经允许评估微生物群在健康中的作用。然而，以往对结直肠微生物组的流行病学研究很少，样本量也很小。此外，最近的研究发现肠道的氧梯度在体内是最陡峭的，从粘膜减少到管腔中部的近缺氧。因此，与粘膜细菌相比，肠道菌群中的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或实际问题。首先，哪些样本来源最适合分析人类结直肠微生物组，其次，一个斑点样本是否足以可靠地代表与结直肠癌发生有关的结直肠微生物组。本研究将通过在100名有结直肠腺瘤病史的参与者（每人4份样本）中使用2个时间点的粪便样本和成对的直肠拭子来解决这两个关键问题。在母体研究中，在与粪便和直肠拭子样本相同的2个时间点，在正常直肠组织中测量结直肠癌发生的生物标志物（细胞凋亡、炎症、增殖和Wnt通路）。在这项拟议的试点研究中，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与免疫组织化学致癌生物标志物相关的单一样本类型。我们还将研究直肠拭子和粪便样本中的微生物生物标志物是否随时间保持稳定；2)比较一个时间点（斑点采集）的粪便和直肠拭子中的微生物生物标志物与两个时间点（间隔3个月）的微生物生物标志物与免疫组织化学致癌生物标志物的相关性。拟议的研究不仅将调查微生物生物标志物在不同类型或组合样品中的有效性，而且还将检验这些生物标志物在直肠组织中与致癌生物标志物相关的一个地点样本和两个时间点收集的可靠性。该研究的独特之处在于样本量大，样本类型多，收集时间点多。本研究结果将为今后开展与结直肠癌相关的微生物群的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.