We read with interest the elegant study published by Flemer et al and commend the authors on their work. The authors provide convincing data to illustrate that faecal microbiota does not reflect accurately the underlying mucosal microbiota in patients with colorectal cancer (CRC). The authors also provide supporting evidence to illustrate that microbiota from sampled mucosa both on and off the cancer site is not significantly different. As previously demonstrated, they also provide corroboratory evidence to further conclude that microbiota is different in patients with CRC. We are however concerned about the conclusions relating to the variability of mucosal microbiota based on tumour site. They present data to suggest that Faecalibacterium, Blautia and Clostridium were significantly more abundant in patients with proximal cancer. It is clear from established work that many environmental factors, including body mass index and diet (red meat), influence both mucosal and faecal microbiota. There are also clear relationships between changes in the microbiome and specific pharmacological agents such as antibiotics. Indeed, the authors do their best to try to correct for any confounding variability when assessing the effect of tumour position, by recording body mass index and dietary data (supplementary data). However, it is not clear either from the main manuscript or their supplementary data whether the authors addressed other confounding differences which may exist between patients with tumours in the proximal compared with the distal colon. It is important to highlight that proximal CRCs are more commonly associated with anaemia due to iron deficiency, which has also been shown to effect microbiota populations. Many of these patients may also have received enteral iron supplementation, as part of clinical care, or through over-the-counter supplements, and this is not assessed by the authors. It is known that iron supplementation can have a potential effect on promoting toxic bacterial metabolites. Indeed, in vitro studies and animal models receiving iron supplementation have been found to have increased pathogenic mucosal and faecal microbiota species including Roseburia, Prevotella, Salmonella Clostridium difficile, Clostridium perfringens, and pathogenic Escherichia coli, and decreased the abundance of gut beneficial species including Bifidobacteriaceae and Lactobacillaceae in the proximal colon populations. 4 Moreover, dietary iron also induced an inflammatory cytokine profile in animal models including increased levels of interleukin-6 and Stat-3. Given the potential for iron supplementation to have contributed to the microbiota changes seen in the proximal cancer cohort in this study, are the authors able to clarify whether patients receiving iron supplementation were excluded at screening? If this were not the case, are the authors able to provide the data to illustrate that patients receiving iron supplementation were equally distributed between the proximal and distal cancer groups? If this is not possible, we would certainly encourage caution about this specific conclusion as there may indeed be additional contributory causes which could explain the difference in microbiota. We feel that further work is needed to clarify the microbiota differences between tumour site in CRC.

中文翻译：

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结直肠癌中的肿瘤相关和非肿瘤相关微生物群

我们饶有兴趣地阅读了 Flemer 等人发表的优雅研究，并赞扬了作者的工作。作者提供了令人信服的数据来说明粪便微生物群不能准确反映结直肠癌 (CRC) 患者的潜在粘膜微生物群。作者还提供了支持证据，以说明来自癌症部位内外取样粘膜的微生物群没有显着差异。如前所述，他们还提供了确凿的证据，以进一步得出结论，CRC 患者的微生物群是不同的。然而，我们担心与基于肿瘤部位的粘膜微生物群的变异性有关的结论。他们提供的数据表明，Faecalibacterium、Blautia 和 Clostridium 在近端癌症患者中的含量明显更高。从既定工作中可以清楚地看出，许多环境因素，包括体重指数和饮食（红肉），都会影响粘膜和粪便微生物群。微生物组的变化与抗生素等特定药物之间也存在明显的关系。事实上，作者通过记录体重指数和饮食数据（补充数据），在评估肿瘤位置的影响时，尽最大努力纠正任何混杂的变异性。然而，无论是主要手稿还是他们的补充数据，都不清楚作者是否解决了近端与远端结肠肿瘤患者之间可能存在的其他混杂差异。重要的是要强调近端 CRC 更常与缺铁引起的贫血有关，这也被证明会影响微生物群。其中许多患者可能还接受了肠内补铁，作为临床护理的一部分，或通过非处方补充剂，作者没有对此进行评估。众所周知，补铁可能对促进有毒细菌代谢物产生潜在影响。事实上，已经发现接受铁补充剂的体外研究和动物模型增加了致病性粘膜和粪便微生物群，包括罗氏杆菌、普氏菌、艰难梭菌、产气荚膜梭菌和致病性大肠杆菌，并减少了包括双歧杆菌在内的肠道有益物种的丰度和近端结肠群体中的乳酸杆菌科。4 此外，膳食铁还在动物模型中诱导炎性细胞因子谱，包括增加白细胞介素 6 和 Stat-3 的水平。鉴于补铁可能导致本研究中近端癌症队列中微生物群的变化，作者能否澄清是否在筛选时排除了接受补铁的患者？如果不是这种情况，作者是否能够提供数据来说明接受铁补充剂的患者在近端和远端癌症组之间平均分布？如果这是不可能的，我们当然会鼓励对这一特定结论持谨慎态度，因为可能确实存在其他促成因素可以解释微生物群的差异。