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The identification of transcriptomic and protein biomarkers prognosticating recurrence risk after chemoradiation of localized squamous cell carcinoma of the anus (SCCA) has been limited by a lack of available fresh tissue at initial presentation. We analyzed archival FFPE SCCA specimens from pretreatment biopsies prior to chemoradiation for protein and RNA biomarkers from patients with localized SCCA who recurred (N = 23) and who did not recur (N = 25). Tumor cells and the tumor microenvironment (TME) were analyzed separately to identify biomarkers with significantly different expression between the recurrent and non-recurrent groups. Recurrent patients had higher mean protein expression of FoxP3, MAPK-activation markers (BRAF, p38-MAPK) and PI3K/Akt activation (phospho-Akt) within the tumor regions. The TME was characterized by the higher protein expression of immune checkpoint biomarkers such as PD-1, OX40L and LAG3. For patients with recurrent SCCA, the higher mean protein expression of fibronectin was observed in the tumor and TME compartments. No significant differences in RNA expression were observed. The higher baseline expression of immune checkpoint biomarkers, together with markers of MAPK and PI3K/Akt signaling, are associated with recurrence following chemoradiation for patients with localized SCCA. These data provide a rationale towards the application of immune-based therapeutic strategies to improve curative-intent outcomes beyond conventional therapies for patients with SCCA.

Many clinical studies have evaluated the effect of probiotics, but only a few have assessed their dose effects on gut microbiota and host. We conducted a randomized, double-blind, controlled intervention clinical trial to assess the safety (primary endpoint) of and gut microbiota response (secondary endpoint) to the daily ingestion for 4 weeks of two doses (1 or 3 bottles/day) of a fermented milk product (Test) in 96 healthy adults. The Test product is a multi-strain fermented milk product, combining yogurt strains and probiotic candidate strains Lactobacillus paracasei subsp. paracasei CNCM I-1518 and CNCM I-3689 and Lactobacillus rhamnosus CNCM I-3690. We assessed the safety of the Test product on the following parameters: adverse events, vital signs, hematological and metabolic profile, hepatic, kidney or thyroid function, inflammatory markers, bowel habits and digestive symptoms. We explored the longitudinal gut microbiota response to product consumption and dose, by 16S rRNA gene sequencing and functional contribution by shotgun metagenomics. Safety results did not show any significant difference between the Test and Control products whatever the parameters assessed, at the two doses ingested daily over a 4-week-period. Probiotic candidate strains were detected only during consumption period, and at a significantly higher level for the three strains in subjects who consumed 3 products bottles/day. The global structure of the gut microbiota as assessed by alpha and beta-diversity, was not altered by consumption of the product for four weeks. A zero-inflated beta regression model with random effects (ZIBR) identified a few bacterial genera with differential responses to test product consumption dose compared to control. Shotgun metagenomics analysis revealed a functional contribution to the gut microbiome of probiotic candidates.

Objectives: Aim of this study was to assess the effect of a fermented milk product containing Bifidobacterium lactis CNCM I-2494 (FMP) on gastrointestinal (GI) symptoms and exhaled H2 and CH4 during a nutrient and lactulose challenge in patients with irritable bowel syndrome (IBS)., Methods: We included 125 patients with IBS (Rome III). Fasted subjects were served a 400ml liquid test meal containing 25g lactulose. The intensity of eight GI symptoms and the amount of exhaled H2 and CH4 were assessed before and during 4h after meal intake. The challenge was repeated after 14 days consumption of FMP or a control product in a double-blind, randomized, parallel design. The metabolic potential of fecal microbiota was profiled using 16S MiSeq analysis of samples obtained before and after the intervention., Results: 106 patients with IBS were randomized. No difference between FMP or control groups was found on GI symptoms or breath H2 and CH4 in the whole cohort. A post-hoc analysis in patients stratified according to their fasting H2 levels showed that in high H2 producers (fasting H2 level≥10ppm, n = 35), FMP consumption reduced fasting H2 levels (p = 0.003) and H2 production during the challenge (p = 0.002) and tended to decrease GI discomfort (p = 0.05) vs. control product. The Prevotella/Bacteroides metabolic potential at baseline was higher in high H2 producers (p<0.05) vs. low H2 producers and FMP consumption reduced this ratio (p<0.05) vs. control product., Conclusions: The response to a fermented milk product containing Bifidobacterium lactis CNCM I-2494 (FMP) in patients with IBS seems to be associated with the metabolic potential of the gut microbiota., Trial Registration: ClinicalTrial.gov NCT01252550. These results were presented as congress posters at Digestive Disease Week 2016 in San Diego, USA and United European Gastroenterology Week 2016 in Vienna, Austria., Competing Interests: Funding by Danone Nutricia Research. This does not alter our adherence to PLOS ONE policies on sharing data and materials. B Le Nevé, J Tap, M Derrien, R Brazeilles and S Cools- Portier are employees of Danone Nutricia Research. D Guyonnet is employee of Diana Nova and was employee of Danone Nutricia Research when this work was performed. H Törnblom has received an unrestricted research grant from Tillotts Pharma and served as a consultant/advisory board member for Allergan, Almirall Nordic and Shire. M Simrén has received unrestricted research grants from Danone and Ferring and served as a Consultant/Advisory Board member for AstraZeneca, Danone, Novartis, Almirall, Allergan, Glycom, Nestlé and Shire/Movetis. L Ohman and S Störsrud have nothing to disclose.

The adequate storage of fecal samples from clinical trials is crucial if analyses are to be performed later and in long-term studies. However, it is unknown whether the composition of the microbiota is preserved during long-term stool storage (>1 year). We therefore evaluated the influence of long-term storage on the microbiota composition of human stool samples collected in RNAlater and stored for approximately five years at -80 °C. We compared storage effects on stool samples from 24 subjects with the effects of technical variation due to different sequencing runs and biological variation (intra- and inter-subject), in another 101 subjects, based on alpha-diversity, beta-diversity and taxonomic composition. We also evaluated the impact of initial alpha-diversity and fecal microbiota composition on beta-diversity instability upon storage. Overall, long-term stool storage at -80 °C had only limited effects on the microbiota composition of human feces. The magnitude of changes in alpha- and beta- diversity and taxonomic composition after long-term storage was similar to inter-sequencing variation and smaller than biological variation (both intra- and inter-subject). The likelihood of fecal samples being affected by long-term storage correlated with the initial relative abundance of some genera and tend to be affected by initial taxonomic richness.

Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

Background & Aims: We have limited knowledge about the association between the composition of the intestinal microbiota and clinical features of irritable bowel syndrome (IBS). We collected information on the fecal and mucosa-associated microbiota of patients with IBS and evaluated whether these were associated with symptoms., Methods: We collected fecal and mucosal samples from adult patients who met the Rome III criteria for IBS at a secondary/tertiary care outpatient clinics in Sweden, as well as from healthy subjects. The exploratory set comprised 149 subjects (110 with IBS and 39 healthy subjects); 232 fecal samples and 59 mucosal biopsy samples were collected and analyzed by 16S ribosomal RNA targeted pyrosequencing. The validation set comprised 46 subjects (29 with IBS and 17 healthy subjects); 46 fecal samples, but no mucosal samples, were collected and analyzed. For each subject, we measured exhaled H 2 and CH 4 , oro-anal transit time, and the severity of psychological and gastrointestinal symptoms. Fecal methanogens were measured by quantitative polymerase chain reaction. Numerical ecology analyses and a machine learning procedure were used to analyze the data., Results: Fecal microbiota showed covariation with mucosal adherent microbiota. By using classic approaches, we found no differences in fecal microbiota abundance or composition between patients with IBS vs healthy patients. A machine learning procedure, a computational statistical technique, allowed us to reduce the 16S ribosomal RNA data complexity into a microbial signature for severe IBS, consisting of 90 bacterial operational taxonomic units. We confirmed the robustness of the intestinal microbial signature for severe IBS in the validation set. The signature was able to discriminate between patients with severe symptoms, patients with mild/moderate symptoms, and healthy subjects. By using this intestinal microbiota signature, we found IBS symptom severity to be associated negatively with microbial richness, exhaled CH 4 , presence of methanogens, and enterotypes enriched with Clostridiales or Prevotella species. This microbiota signature could not be explained by differences in diet or use of medications., Conclusions: In analyzing fecal and mucosal microbiota from patients with IBS and healthy individuals, we identified an intestinal microbiota profile that is associated with the severity of IBS symptoms., Trial Registration Number: NCT01252550., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Patients with recessive dystrophic epidermolysis bullosa (RDEB) lack type VII collagen and therefore have severely impaired dermal-epidermal stability causing recurrent skin and mucosal blistering. There is currently no specific approved treatment for RDEB. We present preclinical data showing that intradermal injections of genetically corrected patient-derived RDEB fibroblasts using a Good Manufacturing Practices grade self-inactivating COL7A1 retroviral vector reverse the disease phenotype in a xenograft model in nude mice. We obtained 50% transduction efficiency in primary human RDEB fibroblasts with an average low copy number (range = 1-2) of integrated provirus. Transduced fibroblasts showed strong type VII collagen re-expression, improved adhesion properties, normal proliferative capabilities, and viability in vitro. We show that a single intradermal injection of 3 × 10(6) genetically corrected RDEB fibroblasts beneath RDEB skin equivalents grafted onto mice allows type VII collagen deposition, anchoring fibril formation at the dermal-epidermal junction, and improved dermal-epidermal adherence 2 months after treatment, supporting functional correction in vivo. Gene-corrected fibroblasts previously showed no tumorigenicity. These data show the efficacy and safety of gene-corrected fibroblast therapy using a self-inactivating vector that has now been good manufacturing grade-certified and pave the way for clinical translation to treat nonhealing wounds in RDEB patients., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)