Comparison of microbial signatures between paired faecal and rectal biopsy samples from healthy volunteers using next-generation sequencing and culturomics.

BACKGROUND: Faecal samples are frequently used to characterise the gut microbiota in health and disease, yet there is considerable debate about how representative faecal bacterial profiles are of the overall gut community. A particular concern is whether bacterial populations associated with the gut mucosa are properly represented in faecal samples, since these communities are considered critical in the aetiology of gastrointestinal diseases. In this study we compared the profiles of the faecal and mucosal microbiota from ten healthy volunteers using bacterial culturing (culturomics) and next-generation sequencing targeting the 16S ribosomal nucleic acid (rRNA) gene. Paired fresh rectal biopsies and faecal samples were processed under stringent anaerobic conditions to maintain the viability of the bacteria. Four different sample types were analysed: faecal (F), faecal homogenised (FHg), biopsy tissue (B) and biopsy wash (BW) samples.  RESULTS: There were no significant statistical differences in either bacterial richness or diversity between biopsy washes (BW) and faecal (F) or faecal homogenised (FHg) samples. Principal coordinates analysis of a Bray-Curtis distance matrix generated from sequence variant tables did not show distinct clustering between these samples (PERMANOVA; p = 0.972) but showed strong clustering of samples from individual donors. However, the rectal biopsy tissue (B) samples had a significantly altered bacterial signature with greater abundance of Proteobacteria and Acidobacteria compared to faecal (F) and faecal homogenised (FHg) samples. A total of 528 bacteria encompassing 92 distinct bacterial species were isolated and cultured from a subset of six volunteer samples (biopsy washes and faeces). This included isolation of 22 novel bacterial species. There was significant similarity between the bacterial species grown in anaerobic culture and those identified by 16S rRNA gene sequencing (Spearman correlation; rho = 0.548, p = 0.001). CONCLUSION: This study showed that the bacterial profiles of paired faecal and rectal biopsy wash samples were very similar, validating the use of faecal samples as a convenient surrogate for rectal biopsy-associated microbiota. Anaerobic bacterial culture results showed similar taxonomic patterns to the amplicon sequence analysis disproving the dogma that culture-based methods do not reflect findings of molecular assessments of gut bacterial composition. Video abstract.

Mercaptopurine versus placebo to prevent recurrence of Crohn's disease after surgical resection (TOPPIC): a multicentre, double-blind, randomised controlled trial.

BACKGROUND: Up to 60% of patients with Crohn's disease need intestinal resection within the first 10 years of diagnosis, and postoperative recurrence is common. We investigated whether mercaptopurine can prevent or delay postoperative clinical recurrence of Crohn's disease. METHODS: We did a randomised, placebo-controlled, double-blind trial at 29 UK secondary and tertiary hospitals of patients (aged >16 years in Scotland or >18 years in England and Wales) who had a confirmed diagnosis of Crohn's disease and had undergone intestinal resection. Patients were randomly assigned (1:1) by a computer-generated web-based randomisation system to oral daily mercaptopurine at a dose of 1 mg/kg bodyweight rounded to the nearest 25 mg or placebo; patients with low thiopurine methyltransferase activity received half the normal dose. Patients and their carers and physicians were masked to the treatment allocation. Patients were followed up for 3 years. The primary endpoint was clinical recurrence of Crohn's disease (Crohn's Disease Activity Index >150 plus 100-point increase in score) and the need for anti-inflammatory rescue treatment or primary surgical intervention. Primary and safety analyses were by intention to treat. Subgroup analyses by smoking status, previous thiopurines, previous infliximab or methotrexate, previous surgery, duration of disease, or age at diagnosis were also done. This trial is registered with the International Standard Randomised Controlled Trial Register (ISRCTN89489788) and the European Clinical Trials Database (EudraCT number 2006-005800-15). FINDINGS: Between June 6, 2008, and April 23, 2012, 240 patients with Crohn's disease were randomly assigned: 128 to mercaptopurine and 112 to placebo. All patients received at least one dose of study drug, and no randomly assigned patients were excluded from the analysis. 16 (13%) of patients in the mercaptopurine group versus 26 (23%) patients in the placebo group had a clinical recurrence of Crohn's disease and needed anti-inflammatory rescue treatment or primary surgical intervention (adjusted hazard ratio [HR] 0·54, 95% CI 0·27-1·06; p=0·07; unadjusted HR 0·53, 95% CI 0·28-0·99; p=0·046). In a subgroup analysis, three (10%) of 29 smokers in the mercaptopurine group and 12 (46%) of 26 in the placebo group had a clinical recurrence that needed treatment (HR 0·13, 95% CI 0·04-0·46), compared with 13 (13%) of 99 non-smokers in the mercaptopurine group and 14 (16%) of 86 in the placebo group (0·90, 0·42-1·94; pinteraction=0·018). The effect of mercaptopurine did not significantly differ from placebo for any of the other planned subgroup analyses (previous thiopurines, previous infliximab or methotrexate, previous surgery, duration of disease, or age at diagnosis). The incidence and types of adverse events were similar in the mercaptopurine and placebo groups. One patient on placebo died of ischaemic heart disease. Adverse events caused discontinuation of treatment in 39 (30%) of 128 patients in the mercaptopurine group versus 41 (37%) of 112 in the placebo group. INTERPRETATION: Mercaptopurine is effective in preventing postoperative clinical recurrence of Crohn's disease, but only in patients who are smokers. Thus, in smokers, thiopurine treatment seems to be justified in the postoperative period, although smoking cessation should be strongly encouraged given that smoking increases the risk of recurrence. FUNDING: Medical Research Council.

Evidence for an age-related influence of microsatellite instability on colorectal cancer survival.

It is well established that microsatellite instability (MSI), the hallmark of defective DNA mismatch repair (MMR), is associated with prolonged survival in colorectal cancer compared with tumours that are microsatellite stable (MSS). MSI in sporadic colorectal tumours is primarily due to epigenetic silencing of MLH1. However, there are no prospective population-based studies of survival in patients with germline MMR gene mutations who develop cancer. Although MSI is almost universal in tumours from HNPCC family members, there is a potential confounding effect of ascertainment and other biases that could explain the apparent survival benefit in HNPCC families. Resolving whether germline MMR gene mutations impact on survival is important because it potentially undermines the rationale for surveillance of mutation carriers. Here, we report an investigation of the influence of MSI on survival in cohorts of cancer patients (aged < 30 years at diagnosis, n = 118; non-age-selected, n = 181) in the context of clinicopathologic variables. There was a substantial age-related influence of tumour MSI status on survival. In young patients with tumour MSI, 65% of patients with MSI tumours had germline MSH2 or MLH1 mutations. Clinicopathologic variables and tumour MSI of the cohort were studied with respect to survival and compared with control groups. Young patients had excess MSI tumours (p < 0.000001), mucinous tumours (p < 0.01), advanced disease (p approximately 0.001) and poorer 5-year survival compared with older cases. Cox proportional hazard analysis identified Dukes' stage, age at diagnosis and calendar year of treatment as independent predictors of survival. There was no detectable association between tumour MSI and survival in young patients, although we confirmed previous observations that MSI is associated with better prognosis in later onset cohorts. These findings underscore the rationale for surveillance and early identification of tumours in MMR gene carriers as well as refining understanding of the influence of MSI on cancer progression.