Introduction

The human gut microbiome plays a crucial role in human health, and disruption of its balance has been linked to various intestinal and extra-intestinal conditions. Therefore, there is a strong demand for an in vitro platform to study the human gut microbiome mechanistically, ideally without the need for animal experiments.

Aims & Methods

The main goal of this study was to optimize stool sampling and culture conditions, including the possible time between sample collection and the process strategy, to resemble physiological conditions in the gut in an in vitro fermentation model.
Stool samples were collected using an anaerobic microbiome collection kit and stored in GutAlive^® (MicroViable therapeutics, Spain) containers for up to 48 hours. Fecal slurries were cultured in a DASbox^® mini bioreactor system (Eppendorf, Germany) for five days using three different process strategies: no-feeding, semi-continuous (batch) feeding, and continuous feeding with a nutrient flow of ~3 ml/hour and the retention time of 28.3 hours, which is comparable to the human colon transit time. Cell viability, pH, temperature, cell count (optical density), and dissolved oxygen were continuously monitored. 16S rRNA gene sequencing was used to analyze the changes in the structure of the microbial community.

Results

Sample storage in GutAlive^® containers did not significantly alter the microbial community structure for up to two days. Analysis of the 16S rRNA gene sequencing data revealed a significant decline in alpha diversity metrics throughout the experiment (all p < 0.050), independent of the duration of stool preservation in the collection container before culturing. Throughout the course of the experiment, notable differences in beta diversity similarity metrics occurred, which were found to be dependent upon the sampling time (p = 0.001) and showed no significant associations with the length of stool preservation in the collection container. The donors’ microbiome had distinct beta diversity values, but they assimilated throughout time. MaAsLin2 analysis revealed that the five most considerably diminished genera were the Eubacterium eligens group, Eubacterium hallii group, Roseburia, Akkermansia, and Anaerostipes. Alpha diversity analysis in the feeding strategy experiments showed the tendency (p = 0.072) that the no-feeding strategy resulted in a higher richness of the microbiome over time, compared to semi-continuous and continuous feeding. Based on the beta diversity parameters of the feeding strategy analysis, it was observed that there were significant differences based on the feeding strategy and the time (both p < 0.050), with the least difference in beta diversity similarity metrics in the no-feeding strategy group throughout the experimental time.

Conclusion

In conclusion, this study provides the evidence that stool samples from patients can be stored for up to two days in GutAlive^® containers without significant changes in the microbiome structure. The choice of feeding strategy showed differences in microbiome diversity metrics; thus it suggests that feeding strategy has an immense impact on the microbiome structure.

Introduction

Clostridioides difficile infection (CDI) is the most common cause of healthcare associated infectious diarrhea in hospitalized patients, affecting primarily elderly patients with comorbidities after antibiotic exposure. Despite introduction of novel antibiotic options, recurrent CDI (rCDI) remain a major clinical challenge. Bezlotoxumab, an anti-Toxin B monoclonal antibody used in conjunction with standard of care antibiotic therapy, has been shown to reduce the frequency of rCDI in registration trials. To date, there is limited post-marketing data on this compound concerning its clinical efficacy and safety outside trials. The aim of the BODEGA study was to assess the clinical efficacy, safety and logistics of administration of bezlotoxumab in a real-life setting.

Aims & Methods

In this observational, prospective and retrospective multicenter study, we collected data from 100 patients receiving bezlotoxumab in combination with standard of care for the prevention of rCDI. Follow-up with respect to response and safety was performed at days 10, 45 and 90. For each patient, variables with respect to demography, fitness, previous CDI treatments and CDI associated hospitalizations, diagnostic and outcome were documented. Documentation was performed by use of the online platform ClinicalSurveys.Net. For statistical analysis, SPSS version 29.0.0.0 (241) was used.

Results

At the time of this analysis, data from 88/100 unique patients with documented CDI was evaluable, as 12 had not yet completed follow-up. The evaluable study population consists of 62,5% (n=55) females with an average age of 59 years (range: 20-91). Overall, 94,3% (n=83) of patients presented with additional comorbidities. Gastrointestinal (n=33, 37,5,4%) and cardiovascular diseases (n=38, 43,2%) were most frequently documented. Patients experienced a median of 3 episodes (range=0-10) of CDI prior to the episode in which bezlotoxumab was administered. The most recent CDI episode was non-complicated in 90,9% (n=80) and community acquired in 52,9% (n=46). Patients received fidaxomicin standard (n=36, 40,9%), fidaxomicin extended (n=16, 18,2%), vancomycin standard (n=13, 14,8%), vancomycin tapered (n=13, 14,8%) and others (n=4, 4,5%) as antibiotic treatment for the episode during which bezlotoxumab was administered. In their CDI patient history, most patients received the following treatments sequentially: vancomycin and fidaxomicin (n=34, 38,6%) fidaxomicin only (n=25, 28,4%, multiple administrations possible) and vancomycin only (n=16, 18,2%, multiple administrations possible). In 76/88 patients (86,4%) and 67/83 (76,1%) there was no further recurrence on day 45 and 90, respectively.

Conclusion

Preliminary data from the BODEGA study demonstrates comparatively low recurrence rates at days 45 and 90 in a real-life setting. Our study population was characterized by cases of multiply recurrent CDI with an uncomplicated course of CDI, that was often acquired in an ambulatory care setting. In these aspects, the study population differs significantly from patients included in registration trials. Although follow-up is not yet complete for all study patients, we conclude that bezlotoxumab is a promising option for treating patients with multiply recurrent CDI.

References

N/A

Disclosure

Funded with an unrestricted grant from Merck/MSD

Introduction

Quantitative fluorescence molecular endoscopy (qFME) enables real-time in vivo visualization and quantification of fluorescence and can thereby highlight tumor tissue. qFME has proven its potential in detection of lesions and residual disease but is limited to the mucosal side only^1,2. Ultrasound guided needle biopsy (USNB/SFF) single fiber fluorescence spectroscopy may enable assessment of lymph nodes and all tissue layers of the esophageal wall. This pilot study evaluated the clinical impact of USNB/SFF spectroscopy using the fluorescent tracer bevacizumab-800CW for the identification of residual tumor in patients with esophageal cancer.

Aims & Methods

Patients diagnosed with locally advanced esophageal adenocarcinoma scheduled for neoadjuvant chemoradiotherapy followed by surgery were included. Patients were intravenously injected with either 4.5, 10 or 25 mg bevacizumab-800CW 2-3 days prior to the qFME to establish the optimal dose. FME collected real-time white light and fluorescence images of the primary tumor site and normal esophageal tissue. Quantitative fluorescent measurements were acquired superficially using multi-diameter single fiber reflectance / single fiber fluorescence (MDSFR/SFF) and inside the esophageal wall and within lymph nodes using USNB-SFF spectroscopy. Additionally, the reduced scattering coefficient and absorption coefficient were determined from the MDSFR/SFF data.

Results

Fifteen patients were included in the dose-escalation, where 25 mg showed the best differentiation between primary tumor site and normal tissue and was extended with 3 additional patients (TBR: 2.63±0.60, p=0.0002). VEGF immunohistochemistry confirmed the higher uptake of VEGF at the primary tumor site. USNB was evaluated in 6/8 patients from the 25mg cohort. The primary tumor site (p=0.071) and lymph nodes (p=0.090) showed higher fluorescence intensity compared to normal tissue. Fluorescence measurements of normal tissue were comparable between the luminal MDSFR/SFF and USNB/SFF measurements. In 33% of all patients residual tumor was found only below a tumor-free mucosa signifying the need to analyze the deeper tissue layers. Last, the optical properties, the reduced scattering coefficient and absorption coefficient. The combination of the intrinsic fluorescence, absorption and scattering coefficient was converted into the new variable omega and improved the differentiation between tumor and normal tissue even further.

Conclusion

We showed the potential of bevacizumab-800CW combined with qFME and USNB/SFF for response monitoring in esophageal cancer. This combination allows for both in vivo superficial and deeper tissue measurements to ensure that partial responders with a tumor-free mucosa are also adequately selected. A phase II study should establish its value for stratification of patients for active surveillance or surgery.

References

1. Tjalma JJJ, Koller M, Linssen MD, Hartmans E, de Jongh S, Jorritsma-Smit A, et al. Quantitative fluorescence endoscopy: an innovative endoscopy approach to evaluate neoadjuvant treatment response in locally advanced rectal cancer. Gut. 2020;69(3):406-10.
2. Nagengast WB, Hartmans E, Garcia-Allende PB, Peters FTM, Linssen MD, Koch M, et al. Near-infrared fluorescence molecular endoscopy detects dysplastic oesophageal lesions using topical and systemic tracer of vascular endothelial growth factor A. Gut. 2019;68(1):7-10.

Disclosure

Nothing to disclose

Introduction

The ability to achieve endoscopic R0 resection in colorectal T1 cancer depends on the submucosal (SM) invasion distance of the malignancy. In this study, we looked for correlations between the SM invasive distance and achievement of R0 resection.

Aims & Methods

We classified fibrosis of the SM layer as noncancerous (Type B) and cancerous (Type C) and further categorized each into three phases from mild (1) to severe (3). Measurements of SM invasion distances for each tumor were based on the criteria specified by the Japanese Society for Cancer of the Colon and Rectum (JSCCR). For 142 patients (70 male and 72 female, average age of 68.1 years), 143 lesions were diagnosed as cancerous fibrosis in subjects who underwent ESD and 1,813 lesions were scored as colorectal epithelial neoplasms, with T1 cancer being diagnosed by postoperative pathological diagnosis. We evaluated the diagnostic ability of cancerous fibrosis in 308 patients including 165 Type B cases. We also compared the degree of SM invasion in Type C-1, C-2, and C-3 malignancies in which we observed macroscopic typing of the 143 Type C lesions. We then scored the degree of SM invasion in R0 resected Type C lesions. Using an ROC curve and a 95% cutoff index, we calculated the maximal SM invasion distance for which R0 resection could be achieved.

Results

The macroscopic subtypes of Type C lesions were as follows: 0-Is+IIa (LST-G (nodular mixed type)): 55, 0-IIa (LST-NG (flat elevated typ)): 23, 0-IIc+IIa (LST-NG(pseudodepressed typ)): 19, 0-Is: 23, 0-IIa+IIc: 15, 0-IIc: 8. Perioperative endoscopy was able to accurately diagnose 83.9% Type C cases and 93.9% of Type B cases. With regard to diagnostic potential, perioperative endoscopy had 83.9% sensitivity, 93.9%, and specificity. Diagnostic precision was 92.3%, PPV was 92.3%, and NPV was 87.0%. Pathology was used to sub-classify the 143 Type C cases based on severity. Seventy-one patients were C-1, 34 were C-2, and 38 were C-3. The mean SM invasion distances in C-1, C-2 and C-3 cases were 801.2 ± 956.1 mm, 1,745 ± 891.6 mm, and 3063.5 ± 2008.4 mm, respectively; differences between each group were significant (p<0.01). We classified macroscopic types of Type C into Flat and Depressed (FD:0-IIa, 0-IIc+IIa, 0-IIa+IIc, 0-IIc) and Protruded (P: 0-Is+IIa, 0-Is ).Although the corresponding values in the P group were larger (C-1:970.6 mm, C-2:2292.3 mm, and C-3:3483.3 mm, respectively), this difference was not statistically significant. The R0 rate according to the invasive distance was 94.7% when SM was <1,500 mm, and 92.9% when SM was <1,000. For SM between 1,000 mm and 1,500 mm the R0 rate was 19/19 (100%).The percentage of cancer-positive cases in the vertical margin (VM (+)) increased proportionally with the degree of invasion. For SM values between 1,500 and 2,000, VM (+) was 79.2%, whereas for SM values of 2,000 or more, the R0 rate decreased to 45.5%. We identified a statistically significant difference (p=0.037) between less than 1,500 mm and 1,500-2,000 mm. The maximum Youden Index was 0.636 and the R0 rate was 95.4%, when the upper limit was less than 1,625 mm.

Conclusion

An SM of less than 1,625 mm (regardless of the macroscopic type) is predictive of an R0 resection for colorectal T1 cancer, and provided the possibility of curative treatment. Also, we infer that the quantitative and qualitative advantages of ESD can help endoscopists avoid the pitfalls associated with preoperative endoscopic diagnosis, which may be useful in clinical practice.

Introduction

The role of inhaled and subsequently swallowed aeroallergens in treatment outcomes of adults with eosinophilic esophagitis (EoE) is unclear. We hypothesized that the pollen season contributes to the failure of dietary therapy in EoE. In this international two-centre study, we compared six-food elimination diet (SFED) outcomes of EoE patients assessed during versus outside the pollen season.

Aims & Methods

Consecutive adult patients (≥18 years) with EoE who underwent SFED and skin prick test (SPT) for birch and grass pollen were included. Demographics, atopy, SPT results, endoscopy findings (EREFS), and the date of the endoscopy following SFED were retrospectively extracted. Individual sensitization profiles and historical and location specific pollen count data were scrutinized to define whether each patient had been assessed during or outside the pollen season following SFED. For patients without pollen sensitization, Spring was considered as pollen season for comparison purposes. Patients were divided into group A (positive SPT for pollens) or B (negative SPT for pollens). All patients had active EoE before SFED (eosinophils ≥15/high-power field, HPF) and adhered to SFED under the supervision of a dietitian. Response to SFED (eos<15/HPF) was assessed after a minimum of 6 weeks. Data are described as median/interquartile range (Q1-Q3) or as counts/percent. Kruskal-Wallis Rank Sum Test, Pearson’s Chi-Square and Fisher’s exact test were used as appropriate. Significance threshold was p<0.05.

Results

Fifty-eight patients were included, 62.0% had positive SPT for grass and/or birch (group A), while in 37.9% SPT was negative (group B). Significantly more patients from group A also had positive SPT for dust mite, cat and/or dog dander (85.7% vs 31.8%, p<0.001). The two groups were comparable in sex, age, baseline symptoms, atopy, baseline EREFS, and duration of SFED (Table1). The overall SFED response was 56.9%, (95% CI, 44.1%-68.8%) regardless of SPT results and pollen season. When stratifying SFED response according to whether the assessment of the assessment had been performed during or outside the pollen season, patients sensitized to pollens showed significantly lower response to SFED when the assessment was performed during compared to outside the pollen season (21.4% vs 77.3%; p=0.003). Additionally, during the pollen season, patients with positive SPT for pollens had significantly lower response to SFED compared to those with negative SPT (21.4% vs 77.8%; p=0.01). Patients without pollen sensitization did not show seasonal variations in SFED response (66.7% vs 53.8%; p=0.67) (Table 2).

Conclusion

Pollens may have a role in sustaining esophageal eosinophilia in sensitized adults with EoE despite avoidance of trigger foods. SPT for pollens may have a clinical utility in the dietary management of EoE by identifying patients less likely to respond to SFED during pollen season. Further studies are needed to clarify if alternative/combined therapy during pollen season will improve outcomes in sensitized EoE patients undergoing SFED or if elimination diets need to be undertaken outside of the pollen season.

Introduction

Chemoradiotherapy (CRT) is a standard non-surgical treatment for esophageal squamous cell carcinoma (ESCC). One of the major disadvantages of CRT is poor local control, and salvage esophagectomy is generally indicated for local failure; however, it is invasive with a high rate of postoperative complications and mortality so that patients often refused or are intolerant to salvage esophagectomy. Alternatively, photodynamic therapy (PDT) with talaporfin sodium (TS) is less invasive salvage treatment for local failure after CRT/ radiotherapy (RT) and was reported to have relatively high local complete response (L-CR) rate and few adverse events (AEs) in the short term. However, long-term outcomes of PDT with TS are unknown.

Aims & Methods

This retrospective study aimed to clarify the long-term outcomes of PDT with TS for local failure after CRT/RT for ESCC. Patients who underwent PDT with TS for histologically proven local failure of ESCC between December 2012 and December 2021 at the National Cancer Center Hospital East were enrolled. Patients with other active cancers or a prior history of PDT were excluded. Basic indication criteria of PDT were as follows: invasion depth limited to the shallow muscle layer, <1/2 of the esophageal circumference, absence of metastasis, and patient’s refusal or unsuitable physical condition for salvage surgery. We assessed L-CR rate, incidence of AEs, clinical courses after PDT, 3-year overall survival (OS), progression free survival (PFS) and major PFS (mPFS) in which major disease progression after PDT which defined as disease progression requiring more invasive treatment than repeated endoscopic treatment and death from all causes were considered as events. We also assessed pre-PDT factors associated with L-CR. L-CR was defined as no apparent residual tumor, disappearance of post-PDT ulcer and confirmation of the scar formation, and absence of cancer cells in biopsy.

Results

108 patients (92 men; median age 74 years [range, 52-88 years]) were enrolled. Patients with pre-CRT/RT cT1/ T2/ T3/ T4/ unknown were 50/ 22/ 24/ 9/ 3. Those with residual lesion/ local recurrence/ unknown were 63/ 39/ 6. Patients with pre-PDT ycT1a/ T1b/ T2 were 6/ 85/ 17. Pre-PDT median lesion size was 20 mm (range, 4-50 mm). 60 patients achieved L-CR after PDT, 41 had L-nonCR, and 7 were not evaluable. L-CR rate of PDT was 58.8% (60/101). During median follow-up of 23 months (range, 1-90 months), 39 patients (36.1%) finally had no recurrence after PDT. 18 patients, including 8 with L-nonCR after PDT and 10 with local recurrence after achieving L-CR, underwent re-endoscopic treatments alone and remained disease-free afterward. The other 44 patients had major disease progression, and 15 received salvage surgery among them. As for the lesion characteristics associated with L-CR, the lesions with <1/2 circumference and with <20 mm in size were more likely to achieve L-CR than those with ≥1/2 circumference (64.4% vs. 16.7%, P=0.003) and with ≥20 mm (80.0% vs. 45.2%, P=0.001), respectively. The 3-year OS/ PFS/ mPFS of all patients were 78.1%/ 29.8%/ 48.1%. The 3-year OS of L-CR cases was better than that of L-nonCR cases (89.3% vs. 67.7%, P=0.003). There was no grade 3 < AEs. Non-cardiac chest pain (60.2%), skin phototoxicity (4.6%), and esophageal stricture requiring balloon dilatation (4.7%) occurred.

Conclusion

PDT with TS for local failure after CRT/RT for ESCC showed the preferable long-term outcomes. Moreover, achieving local control with PDT could improve prognosis of patients even with local failure after CRT/RT.

Introduction

Colorectal cancer is one of the most common types of cancer worldwide. The majority of patients are operable at the time of diagnosis. One of the most severe surgical complications is anastomotic leakage (AL). The incidence of AL depends on where the anastomosis is located (1-2% for ileocolic anastomoses, increasing to 10-15% of anastomoses to the rectum). AL leads to prolonged hospital stay, decreased quality of life, increased risk of cancer recurrence, and increased mortality.
Experimental studies have shown that certain bacteria can contribute to the development of AL.
In Denmark, people aged 50-74 years are offered free of charge screening for colorectal cancer. They collect a stool sample at home to be analyzed for the presence for blood (a fecal immunochemistry sample). It is possible to extract bacterial DNA from the fecal sample, to characterize the gut microbiome at the time of sampling.

Aims & Methods

The objective of the study was to use third-generation sequencing methods to analyze screening fecal samples in a cohort of colorectal cancer patients, and to characterize and compare the gut microbiome between patients with or without postoperative AL.
Fecal samples sent to the Colorectal Cancer Screening Program that tested positive for blood, from 2016-2018, were place in a biobank and stored at -80 ⁰C. By combining the biobank with data from the Danish Colorectal Cancer Group, we identified 18 patients who had surgery for colonic or rectal cancer in the period and suffered a postoperative AL (AL group). We matched these patients in a 1:2-ratio, with 36 patients without AL (Control group). We analyzed the 16S ribosomal RNA genes in the screening fecal samples with third-generation sequencing (Oxford Nanopore Technologies).

Results

The data are preliminary, as the data analysis has not yet been completed.
The AL-group and Control group were similar on basic demographic parameters (age, gender, type of resection, smoker status and ASA classification). The gut microbiome composition was significant different in the two groups, based on the two main components of a principal coordinate analysis (PCoA) chart (PERMANOVA, p-value = 0.01).
The Bacteroidota phylum was more abundant in the Control group (p < 0.05). On the order level, Campylobacterales and Bacteroidales were more abundant in the Control group (p < 0.001). On family level, Campylobacteraceae were more abundant in the Control group (p < 0.001), whereas Burkholderiaceae were abundant in the AL-group (p < 0.001). The AL-group also had more Ralstonia and Burkholderia at genus level (p < 0.001), whereas the Control group had more Campylobacter and Oridobacter genera (p < 0.001).

Conclusion

The preoperative gut microbiome composition in colorectal patients with subsequent anastomotic leakage were significantly different from the control group without anastomotic leakage. Campylobacter and Oridobacter seem protective against AL, whereas Ralstonia and Burkholderia seem harmful. We are awaiting further analysis for the species level.
Further studies should focus on validating our results, including changes in the gut microbiome prior to surgery - for instance the impact of bowel preparation – and on estimating the predictive value of our findings.