Moreover, the source, type and abundance of specific bacterial DNA in plasma is not well characterised. However, to date, there has been limited use of rigorous experimental controls to account for contamination that invariably affects low-biomass microbiome studies. In addition to nucleic and mitochondrial sources of cell-free DNA (cfDNA) found in blood plasma, accumulating evidence indicates that commensal microbiome-derived DNA may also contribute to the composition of cfDNA. It is currently unclear whether assessment of the human microbiome from other types of biospecimens, may also prove to be important cancer biomarkers. Here, there is often significant non-compliance due to the undesirable nature of this type of collection which can be challenging from a patient’s perspective. However, a major barrier for analysing the gut microbiome as a cancer biomarker is the need for collection of stool samples. These and other findings suggest that characterisation of the host microbiome is critically important in order to understand and potentially manipulate responses to cancer therapies. Several landmark studies in mouse models and melanoma patients have examined changes in the gut flora after treatment with checkpoint blockade and found that treatment responses were highly dependent on the diversity and abundance of specific bacterial species. In cancer, the gut microbiome has emerged as having an important influence on disease progression, treatment responses and toxicity to immunotherapy. The human microbiota is a major factor governing health and disease. We analyse additional plasma samples, highlighting the potential of this framework to identify differences in cfmDNA between healthy and cancer patients. Through the application of an in silico decontamination strategy including the filtering of amplicon sequence variants (ASVs) with batch dependent abundances and those with a higher prevalence in negative controls, we identify known gut commensal bacteria, such as Faecalibacterium, Bacteroides and Ruminococcus, and also other uncharacterised ASVs. The microbial community of plasma is strongly influenced by laboratory and reagent contaminants introduced during the DNA extraction and sequencing processes. Compared to matched stool and saliva samples, the absolute concentration of cfmDNA is low but significantly above the levels detected from negative controls. We apply a combination of 16S-rRNA-gene sequencing and droplet digital PCR to determine if the specific detection of cell-free microbial DNA (cfmDNA) is possible in metastatic melanoma patients. Number of plasma ASVs from the extension cohort meeting each decontamination criterion separately and in combination. P-values resulting from hypothesis tests on alpha and beta diversity estimates obtained from the extension cohort. Clinical characteristics of the melanoma patients from the extension cohort. Plasma ASVs and reads shared with stool and saliva samples per patient. Clinical characteristics of melanoma patients from the initial study cohort.
Sequencing and in silico decontamination results from the extension cohort of healthy individuals and melanoma patients. Batch effects due to sequencing run and DEB based on Aitchison distance analysis. Taxonomic profile of plasma samples and their corresponding DENCs from DEBs C-E. Batch effects due to DNA extraction day for plasma samples based on Bray-Curtis dissimilarity analysis. Taxonomic profiles of patient-matching plasma, stool and saliva samples and respective DENCs. Taxonomic profile of a 20 strain evenly mixed mock community sequenced alongside study samples. Number of 16S rRNA gene quality-filtered reads obtained across sample types. The Creative Commons Public Domain Dedication waiver ( ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.Īdditional file 1: Figure S1. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
0 kommentar(er)
