Multi-disciplinary team meetings for lung cancer in Norway and Denmark: results from national surveys and observations with MDT-MODe.

BACKGROUND AND PURPOSE: Multi-disciplinary Team (MDT) meetings are widely regarded as the 'gold standard' of lung cancer care. MDTs improve adherence to clinical guidelines for lung cancer patients. In this study, we describe and compare lung cancer MDTs in Denmark and Norway by combining national surveys and the MDT-Metric for the Observation of Decision-making (MDT-MODe) instrument. MATERIALS AND METHOD: Identical surveys were sent out to all lung cancer MDT centers in Denmark and Norway by the Danish Lung Cancer Group and the Norwegian Lung Cancer Group. Six MDT centers, three in Denmark and three in Norway, were observed using the MDT-MODe instrument. RESULTS AND INTERPRETATION: We found similar organization of MDT meetings in both countries, with the main difference being more local MDT meetings in Norway. All lung cancer MDTs were chaired by respiratory physicians and attended by a radiologist. Other members included oncologists, pathologists, thoracic surgeons, specialist nurses, nuclear medicine specialists and junior doctors. Overall, members reported that they had sufficient time for preparation and attending MDT meetings. With the MDT-MODe instrument it was found that the MDT chairs, surgeons, oncologists, radiologists all contributed positively to case discussion. Comorbidities were included in the discussion of most patients while the patient's view and psychosocial issues were less often discussed. A treatment decision was reached in 79.7% of cases discussed. In conclusion, we found similar settings and overall good quality concerning lung cancer MDT meetings in Denmark and Norway.

Reproducible chemostat cultures to minimize eukaryotic viruses from fecal transplant material.

Recent studies indicate an important role of bacteriophages for successful fecal microbiota transplantation (FMT). However, wider clinical applications of FMT are hampered by to donor variability and concerns of infection risks by bacteria and human viruses. To overcome these challenges, mouse cecal and human fecal material were propagated in a chemostat fermentation setup supporting multiplication of bacteria, and phages, while propagation of eukaryotic viruses will be prevented in the absence of eukaryotic host cells. The results showed decrease of the median relative abundance of viral contigs of classified eukaryotic viruses below 0.01%. The corresponding virome profiles showed dilution rate dependency, a reproducibility between biological replicates, and maintained high diversity regarding both the human and mouse inocula. This proof-of-concept cultivation approach may constitute the first step of developing novel therapeutic tools with high reproducibility and with low risk of infection from the donor material to target gut-related diseases.

Overcoming donor variability and risks associated with fecal microbiota transplants through bacteriophage-mediated treatments.

BACKGROUND: Fecal microbiota transplantation (FMT) and fecal virome transplantation (FVT, sterile filtrated donor feces) have been effective in treating recurrent Clostridioides difficile infections, possibly through bacteriophage-mediated modulation of the gut microbiome. However, challenges like donor variability, costly screening, coupled with concerns over pathogen transfer (incl. eukaryotic viruses) with FMT or FVT hinder their wider clinical application in treating less acute diseases. METHODS: To overcome these challenges, we developed methods to broaden FVT's clinical application while maintaining efficacy and increasing safety. Specifically, we employed the following approaches: (1) chemostat-fermentation to reproduce the bacteriophage FVT donor component and remove eukaryotic viruses (FVT-ChP), (2) solvent-detergent treatment to inactivate enveloped viruses (FVT-SDT), and (3) pyronin-Y treatment to inhibit RNA virus replication (FVT-PyT). We assessed the efficacy of these processed FVTs in a C. difficile infection mouse model and compared them with untreated FVT (FVT-UnT), FMT, and saline. RESULTS: FVT-SDT, FVT-UnT, and FVT-ChP reduced the incidence of mice reaching the humane endpoint (0/8, 2/7, and 3/8, respectively) compared to FMT, FVT-PyT, and saline (5/8, 7/8, and 5/7, respectively) and significantly reduced the load of colonizing C. difficile cells and associated toxin A/B levels. There was a potential elimination of C. difficile colonization, with seven out of eight mice treated with FVT-SDT testing negative with qPCR. In contrast, all other treatments exhibited the continued presence of C. difficile. Moreover, the results were supported by changes in the gut microbiome profiles, cecal cytokine levels, and histopathological findings. Assessment of viral engraftment following FMT/FVT treatment and host-phage correlations analysis suggested that transfer of phages likely were an important contributing factor associated with treatment efficacy. CONCLUSIONS: This proof-of-concept study shows that specific modifications of FVT hold promise in addressing challenges related to donor variability and infection risks. Two strategies lead to treatments significantly limiting C. difficile colonization in mice, with solvent/detergent treatment and chemostat propagation of donor phages emerging as promising approaches. Video Abstract.

Two weeks of acarbose treatment shows no effect on gut microbiome composition in patients with type 2 diabetes: a randomised, placebo-controlled, double-blind, crossover study.

Aim: The alpha-glucosidase inhibitor acarbose is approved for the treatment of type 2 diabetes (T2D). It acts in the lumen of the gut by reducing intestinal hydrolysis and absorption of ingested carbohydrates. This reduces postprandial blood glucose concentration and increases the content of carbohydrates in the distal parts of the intestine potentially influencing gut microbiome (GM) composition and possibly impacting the gut microbiome (GM) dysbiosis associated with T2D. Here, we investigated the effect of acarbose on GM composition in patients with T2D. Methods: Faecal samples were collected in a previously conducted randomised, placebo-controlled, double-blind, crossover study in which 15 individuals with metformin-treated T2D (age 57-85 years, HbA1c 40-74 mmol/mol, BMI 23.6-34.6 kg/m2) were subjected to two 14-day treatment periods with acarbose and placebo, respectively, separated by a 6-week wash-out period. Faecal samples were collected before and by the end of each treatment period. The GM profiles were evaluated by 16S rRNA gene amplicon sequencing. Results: The GM profiles after the treatment periods with acarbose or placebo remained unaffected (P > 0.7) when compared with the GM profiles before treatment. This applied to the analysis of within-sample diversity (α-diversity) and between-sample bacterial composition diversity (ß-diversity). Additionally, no dominant bacterial species differentiated the treatment groups, and only minor increases in the relative abundances of Klebsiella spp. and Escherichia coli (P < 0.05) were observed after acarbose treatment. Conclusion: In patients with metformin-treated T2D, 14 days of treatment with acarbose showed only minor effects on GM as seen in increased relative abundances of Klebsiella spp. and Escherichia coli.

Routes to Diagnosis in Danish Lung Cancer Patients: Emergency Presentation, Age and Smoking History-A Population-Based Cohort Study.

BACKGROUND: The fast-track cancer pathway aims to expedite diagnosis of lung cancer and treatment and is the preferred route to diagnosis. Diagnosis following an unplanned admission (unplanned route) has been associated with poor outcomes. OBJECTIVE: This study explores factors associated with lung cancer diagnosis following unplanned admissions, focusing on the elderly population. METHODS: A retrospective cohort study using population-based data from Danish registries. Factors such as age, comorbidity, performance status, smoking history, socioeconomic parameters and treatment modality were analyzed in relation to route to diagnosis and prognosis. RESULTS: Among 17,835 patients, 16% were elderly (≥ 80 years). The unplanned route constituted 28% of diagnostic routes, with higher proportion among the elderly (33%). Poor performance status and advanced disease stage were associated with the unplanned route. Married patients were less likely to undergo an unplanned route to diagnosis. Smoking did not significantly influence diagnostic route. The adjusted odds ratio for curative treatment and dying 12 months after diagnosis, following unplanned route to diagnosis were 0.68 (95% CI, 0.61-0.76) and 1.48 (95% CI, 1.36-1.61), respectively. CONCLUSION: Frailty (poor performance status and high burden of comorbidity) in addition to unfavorable socioeconomic factors, which all were more prevalent among elderly patients, were associated with undergoing an unplanned route to diagnosis. However, age itself was not. Diagnosis following unplanned admission correlated with reduced likelihood of curative treatment and poorer prognosis. Expanding screening initiatives to include frail elderly individuals living alone, along with alertness by primary care clinicians, is warranted to improve outcomes for these patients.

Transfer of modified gut viromes improves symptoms associated with metabolic syndrome in obese male mice.

Metabolic syndrome encompasses amongst other conditions like obesity and type-2 diabetes and is associated with gut microbiome (GM) dysbiosis. Fecal microbiota transplantation (FMT) has been explored to treat metabolic syndrome by restoring the GM; however, concerns on accidentally transferring pathogenic microbes remain. As a safer alternative, fecal virome transplantation (FVT, sterile-filtrated feces) has the advantage over FMT in that mainly bacteriophages are transferred. FVT from lean male donors have shown promise in alleviating the metabolic effects of high-fat diet in a preclinical mouse study. However, FVT still carries the risk of eukaryotic viral infections. To address this, recently developed methods are applied for removing or inactivating eukaryotic viruses in the viral component of FVT. Modified FVTs are compared with unmodified FVT and saline in a diet-induced obesity model on male C57BL/6 N mice. Contrasted with obese control, mice administered a modified FVT (nearly depleted for eukaryotic viruses) exhibits enhanced blood glucose clearance but not weight loss. The unmodified FVT improves liver pathology and reduces the proportions of immune cells in the adipose tissue with a non-uniform response. GM analysis suggests that bacteriophage-mediated GM modulation influences outcomes. Optimizing these approaches could lead to the development of safe bacteriophage-based therapies targeting metabolic syndrome through GM restoration.

Progress against lung cancer, Denmark, 2008-2022.

BACKGROUND AND PURPOSE: There has been marked progress against lung cancer in Denmark. To gain further insight into the different aspects of the improvement, we examined the stage-specific incidence rates, stage-specific survival and mortality rates. MATERIALS AND METHODS: We used information from the Danish Lung Cancer Registry on date of diagnosis and clinical stage to calculate age-standardised incidence rates and patient survival by sex, period and stage. Information about age-standardised lung cancer-specific mortality rates by sex and period was extracted from The Danish Health Data Authority. RESULTS: Firstly, the decrease in incidence rates was due to a reduction in the rates of advanced stages. Secondly, there was a gradual increase in survival across all stages, and thirdly, the mortality rates gradually decreased over time. INTERPRETATION: The improvements in survival and mortality from lung cancer were due to decreasing incidence rates of advanced cancer and improvement in survival at all stages of the disease.

Lung cancer among the Elderly in Denmark - A comprehensive population-based cohort study.

OBJECTIVE: Lung cancer primarily occurs in the elderly with a median age at diagnosis in Denmark of 73 years. However, elderly patients are under-represented in clinical trials as well as in screening studies. In this study, we aim to characterize elderly patients with lung cancer and explore the diagnostic intensity, treatment patterns, and survival. METHOD: Patients diagnosed with lung cancer between 2014 and 2017 according to the Danish Cancer Registry, and with clinical information in the Danish Lung Cancer Registry were included. Patient information was linked by the unique social identification number to information from Statistics Denmark. RESULTS: We included n = 17,835 patients in this study, of whom 2,871 (16.1 %) were 80 years or older. Fewer elderly patients had lung biopsies (47 % vs 53 %) or mediastinal procedures (34 % vs 26 %), compared to the younger patients (p < 0.001). Fewer elderly patients had treatment registration (60 % vs 85 %), and fewer received treatment with curative intent (23 % vs 42 %) compared to patients younger than 80 years (p < 0.001). The elderly patients had 2.1 (CI 95 % 1.9 - 2.2) times higher odds of dying within 12 months after diagnosis than younger patients. CONCLUSION: The diagnostic intensity among lung cancer patients aged eighty years or above is lower compared to younger patients. Being elderly is associated with not undergoing surgical treatment or treatment with curative intent. Across all treatment groups, being older than eighty years of age was associated with an adverse prognosis.

Salmonella infections in Denmark from 2013-2022 with focus on serotype distribution, invasiveness, age, sex, and travel exposition.

PURPOSE: To analyze the nationwide incidence of Salmonella infections in Denmark from 2013 to 2022. METHODS: Confirmed cases of Salmonella enterica subsp. enterica were examined using the National Register of Enteric Pathogens during 2013-2022. Proportions, incidence rates (IR), relative risk (RR), and 95% confidence intervals (CI) were calculated to assess differences in serotypes, invasiveness, age, sex, and travel exposure. RESULTS: We identified 9,944 Danish Salmonella enterica subsp. enterica cases, with an average annual incidence rate of 16.9 per 100,000 inhabitants, declining during the COVID-19 pandemic. Typhoidal cases totaled 206, with an average annual IR of 0.35 per 100,000 inhabitants. Enteric fever patients had a median age of 24 years (IQR:17-36). Leading non-typhoid Salmonella (NTS) serotypes were S. Enteritidis (26.4%), monophasic S. Typhimurium (16.5%), and S. Typhimurium (13.5%). Median age for NTS cases was 42 (IQR: 18-62), with even sex distribution, and a third reported travel prior to onset of disease. The overall percentage of invasive NTS (iNTS) infection was 8.1% (CI: 7.6-8.7). Eleven serotypes were associated with higher invasiveness, with S. Dublin and S. Panama having the highest invasiveness with age and sex-adjusted RR of 7.31 (CI: 6.35-8.43) and 5.42 (CI: 3.42-8.60), respectively, compared to all other NTS serotypes. Increased age was associated with higher RR for iNTS infection. CONCLUSION: During the decade, there was a limited number of typhoidal cases. The dominant NTS serotypes were S. Enteritidis and monophasic S. Typhimurium, whereas S. Dublin and S. Panama exhibited the highest invasive potential.

The Danish lung cancer registry: A nationwide validation study.

BACKGROUND: This study evaluates the validity of the information in the Danish Lung Cancer Registry (DLCR). Since 2000, the DLCR has been a tool for monitoring interventions and outcome of all Danish lung cancer patients with the intent to streamline and improve treatment and survival. The DLCR receives information from the Danish Patient Registries in addition to clinical information from the treating physicians. In the year 2022, more than 50 papers have been published using DLCR as a data source. However, the DLCR has not previously been validated. METHODS: A random sample of 1000 patients diagnosed with non-small cell lung cancer from 2014 to 2016 and recorded in the DLCR were included for validation. Medical records were reviewed and were considered as the "gold standard" to which data listed in the DLCR were compared. RESULTS: Information was retrieved from medical charts for all patients. Agreement on stage at diagnosis was 90.1 % (95 % CI 88.0-91.9) and on date of diagnoses was 93.8 (95 % CI 92.1-93.2). Agreement on smoking status in pack years (+/- 10 pack years) was 91.2 % (95 % CI 88.6-93.2). The positive predictive value of treatment intent was 87.4 (95 % CI 85.1-89.6). CONCLUSION: The data in the DLCR are complete, detailed and accurate. The comparison of data from the DLCR with the medical records revealed overall high validity of the data in the registry.

Introduction of the Danish Lung Nodule Registry: A part of the Danish Lung Cancer Registry.

BACKGROUND: The majority of lung cancer cases are diagnosed late, resulting in poor prognosis and high mortality rates. Early detection and management of lung cancer can improve patient outcomes and reduce mortality rates. Pulmonary nodules are key factors in the early detection of lung cancer, they are common in high-risk populations and require correct classification to determine whether they are benign or malignant. Over the last decade a steep increase in the number of thoracic CT scans has been seen in Denmark, resulting in substantial resources allocated to CT follow-up of incidentally detected pulmonary nodules. The implementation of a nationwide Danish prospective pulmonary nodule registry is to methodically record pulmonary nodules and thereby evaluate the scope of pulmonary nodule follow-up, the nature of the nodules, and the clinical progression of patients with pulmonary nodules. METHODS: A prospective pulmonary nodule registry (Danish Lung Nodule Registry) will be a natural appendix to the Danish Lung Cancer Registry. Three new ICD-10 classification codes will be introduced, defining the type of nodule: /DR91.1/ Solid nodule /DR91.2/ Part-solid nodule; /DR91.3/ Non-solid nodule. Furthermore, an additional letter will describe whether the imaging exam is performed on suspicion of lung cancer (A), or the finding is incidental (B). Registration of the nodules will be performed by the departments of respiratory medicine who manage follow-up of pulmonary nodules. It is estimated that around 7000 nodules will be registered annually. DISCUSSION: The registration of patients in the lung nodule registry complies with current Danish legislation. The registry will be seamlessly integrated with other nationwide Danish registries, including the Danish Lung Cancer Registry, to collect additional patient data and improve the quality and scope of the data acquired. The results from these comprehensive epidemiological studies will be of significant interest and offer valuable research opportunities.

Consequences of Losing Incidental Pulmonary Nodules to Follow-Up: Unmonitored Nodules Progressing to Stage IV Lung Cancer.

INTRODUCTION: Lung cancer is the leading cause of cancer-related death globally. Incidental pulmonary nodules represent a golden opportunity for early diagnosis, which is critical for improving survival rates. This study explores the impact of missed pulmonary nodules on the progression of lung cancer. METHODS: A total of 4,066 stage IV lung cancer cases from 2019 to 2021 in Danish hospitals were investigated to determine whether a chest computed tomography (CT) had been performed within 2 years before diagnosis. CT reports and images were reviewed to identify nodules that had been missed by radiologists or were not appropriately monitored, despite being mentioned by the radiologist, and to assess whether these nodules had progressed to stage IV lung cancer. RESULTS: Among stage IV lung cancer patients, 13.6% had undergone a chest CT scan before their diagnosis; of these, 44.4% had nodules mentioned. Radiologists missed a nodule in 7.6% of cases. In total, 45.3% of nodules were not appropriately monitored. An estimated 2.5% of stage IV cases could have been detected earlier with proper surveillance. CONCLUSION: This study underlines the significance of monitoring pulmonary nodules and proposes strategies for enhancing detection and surveillance. These strategies include centralized monitoring and the implementation of automated registries to prevent gaps in follow-up.

Use of [18F]Fluorodeoxyglucose Positron Emission Tomography/Computed Tomography after Curative Treatment of Non-Small-Cell Lung Cancer Patients: A Nationwide Cohort Study.

[¹8F]Fluorodeoxyglucose positron emission tomography/computed tomography ([¹8F]FDG PET/CT) is a valuable imaging tool in the post-treatment management of non-small-cell lung cancer (NSCLC). This study aimed to investigate the trends in utilization and factors associated with the use of [¹8F]FDG PET/CT after curative-intent treatment. Data from 13,758 NSCLC patients diagnosed between 2007 and 2020 identified in the Danish Lung Cancer Registry, who underwent curative-intent treatment, were analyzed using multivariable regression. The results showed a significant increase in the use of [¹8F]FDG PET/CT scans, from 10.4 per 100 patients per year in 2007 to 39.6 in 2013, followed by a period of stability. Higher utilization rates were observed in patients who received radiotherapy (22% increase compared to surgical resection) and in patients with stage II-III disease (14% and 20% increase compared to stage I, respectively). Additionally, utilization was increased when other diagnostic procedures were performed, such as MRI, ultrasound, endoscopy, and biopsy. These findings highlight an increasing reliance on [¹8F]FDG PET/CT in post-treatment NSCLC, especially after radiotherapy and in patients with locally advanced disease, where treatment-induced radiographic changes and an increased risk of recurrence present a significant diagnostic challenge.

Choice of Ultrafilter Affects Recovery Rate of Bacteriophages.

Studies into the viral fraction of complex microbial communities, like in the mammalian gut, have recently garnered much interest. Yet there is still no standardized protocol for extracting viruses from such samples, and the protocols that exist employ procedures that skew the viral community of the sample one way or another. The first step of the extraction pipeline often consists of the basic filtering of macromolecules and bacteria, yet even this affects the viruses in a strain-specific manner. In this study, we investigate a protocol for viral extraction based on ultrafiltration and how the choice of ultrafilter might influence the extracted viral community. Clinical samples (feces, vaginal swabs, and tracheal suction samples) were spiked with a mock community of known phages (T4, c2, Φ6, Φ29, Φx174, and Φ2972), filtered, and quantified using spot and plaque assays to estimate the loss in recovery. The enveloped Φ6 phage is especially severely affected by the choice of filter, but also tailed phages such as T4 and c2 have a reduced infectivity after ultrafiltration. We conclude that the pore size of ultrafilters may affect the recovery of phages in a strain- and sample-dependent manner, suggesting the need for greater thought when selecting filters for virus extraction.

Outcome of Incidental Pulmonary Nodules in a Real-World Setting.

OBJECTIVES: Early diagnosis of lung cancer is imperative to improve survival. Incidental pulmonary nodules (IPN) may represent early stages of lung cancer and appropriate follow-up and management of these nodules is important, but also very resource demanding. We aim to describe the results of the CT-based follow-up on a cohort of patients with IPN in terms of detected malignancies, the proportion undergoing invasive procedures, and the subsequent outcome. MATERIALS AND METHODS: Retrospective cohort study of patients in a CT IPN follow-up program who underwent a needle biopsy of the lung from 2018 to 2021 at Aarhus University Hospital. RESULTS: A total of 4181 patients with IPN were followed with CT control scans. Out of these 249 (6%) were diagnosed with lung cancer of which 224 (90%) were diagnosed as a result of the IPN follow-up. Seventy-five percent of the patients were diagnosed in stages I to II and curable treatment was possible in 77.9% of the patients. In the CT IPN follow-up program 449 patients underwent a CT guided needle biopsy. Out of these 190 patients underwent biopsy without the detection of malignancy, corresponding to 4.5% of the entire IPN population. CONCLUSION: The cumulated incidence of lung cancer in our population in the IPN follow-up program was 6%. The probability of malignancy when undergoing an invasive procedure on an IPN was 55.7% of which lung cancer was vastly predominant. The majority of lung cancers were diagnosed in an early and potentially curable stage.

Decision support to general practice in choice of chest imaging for patients with pulmonary symptoms.

INTRODUCTION: The choice of chest imaging for patients with respiratory problems is based on risk profile and symptoms. In 2018-2020, GPs in the catchment area of Silkeborg Regional Hospital, Denmark, were offered direct referral for either X-ray or low-dose computed tomography (LDCT) of the chest for patients with respiratory symptoms who did not meet the criteria for a contrast-enhanced CT (CECT) of the chest and upper abdomen as part of the lung cancer referral pathway. The aim of this study was 1) to estimate the percentage of patients referred for LDCT or chest X-ray who met CECT criteria based on the clinical information in the referral letters, and 2) to assess the GPs' response to standard questions regarding the active feedback provided. METHODS: The study was conducted from April to October 2019. Radiographers initially assessed all referrals for X-ray or LDCT, and contacted the GPs if they assessed that symptoms and clinical characteristics justified CECT. RESULTS: In the study period, 1,112 referrals for chest imaging from GPs were received; in 97 cases (9%), the referral information warranted CECT as part of a lung cancer referral package. In 71% (69/97) of these cases, the GP accepted the conversion to CECT; 55 of 73 LDCTs and 14 of 24 X-rays. In 15 cases, the GP adhered to the requested imaging owing to clinical assessment or their agreement with the patient, and in the remaining 13 cases no specific reason was given. CONCLUSION: The feedback provided was well received by GPs and the approach adopted may be a step towards structured decision support to facilitate the choice of chest imaging. FUNDING: None. TRIAL REGISTRATION: Not relevant.

Diagnosis and treatment of lung cancer in Denmark during the COVID-19 pandemic.

BACKGROUND: We examined the number of lung cancers diagnosed, the quality of care and the socio-economic and clinical characteristics among patients with lung cancer during the COVID-19 pandemic compared to previous years. METHODS: We included all patients ≥ 18 years old diagnosed with lung cancer from 01 January 2018 to 31 August 2021 as registered in the Danish Lung Cancer Registry. Using a generalised linear model, we estimated prevalence ratios (PR) and 95% confidence intervals (CI) of the associations between the pandemic and socioeconomic and clinical factors, and indicators of quality. RESULTS: We included 18,113 patients with lung cancer (82.0% non-small cell lung cancer (NSCLC)), which was similar to the preceding years, although a decline in NSCLC cases occurred during the first lockdown period in 2020. No difference in distribution of income or educational level was observed. No difference was observed in the quality of treatment - as measured by curative intent, proportion of patients resected or who died within 90 days of diagnosis. CONCLUSION: Using nationwide population-based data, our study reassuringly shows no adverse effects of the COVID-19 pandemic on the diagnosis, socio-economic characteristics nor quality of treatment of lung cancer, as compared to the preceding years.

Fecal virome transfer improves proliferation of commensal gut Akkermansia muciniphila and unexpectedly enhances the fertility rate in laboratory mice.

Probiotics are intended to improve gastrointestinal health when consumed. However, the probiotics marketed today only colonize the densely populated gut to a limited extent. Bacteriophages comprise the majority of viruses in the human gut virome and there are strong indications that they play important roles in shaping the gut microbiome. Here, we investigate the use of fecal virome transplantation (FVT, sterile filtrated feces) as a mean to alter the gut microbiome composition to lead the way for persistent colonization of two types of probiotics: Lacticaseibacillus rhamnosus GG (LGG) representing a well-established probiotic and Akkermansia muciniphila (AKM) representing a putative next-generation probiotic. Male and female C57BL/6NTac mice were cohoused in pairs from 4 weeks of age and received the following treatment by oral gavage at week 5 and 6: AKM+FVT, LGG+FVT, probiotic sham (Pro-sham)+FVT, LGG+Saline, AKM+Saline, and control (Pro-sham+Saline). The FVT donor material originated from mice with high relative abundance of A. muciniphila. All animals were terminated at age 9 weeks. The FVT treatment did not increase the relative abundance of the administered LGG or AKM in the recipient mice. Instead FVT significantly (p < 0.05) increased the abundance of naturally occurring A. muciniphila compared to the control. This highlights the potential of propagating the existing commensal "probiotics" that have already permanently colonized the gut. Being co-housed male and female, a fraction of the female mice became pregnant. Unexpectedly, the FVT treated mice were found to have a significantly (p < 0.05) higher fertility rate independent of probiotic administration. These preliminary observations urge for follow-up studies investigating interactions between the gut microbiome and fertility.

Biomarker expression and survival in patients with non-small cell lung cancer receiving adjuvant chemotherapy in Denmark.

INTRODUCTION: Programmed cell death ligand-1 (PD-L1) expression may help identify patients with non-small cell lung cancer (NSCLC) who would benefit from immunotherapy. We assessed PD-L1 expression, and epidermal growth factor receptor (EGFR) and V-Ki-Ras2 Kirsten rat sarcoma (KRAS) mutations in NSCLC patients receiving adjuvant chemotherapy. METHODS: Data for stage IB/II/IIIA NSCLC patients (diagnosed: 2001-2012) were retrieved from Danish population-based registries. Tumor tissue samples were tested for PD-L1 expression using VENTANA PD-L1 (SP263) Assay in tumor cells (TC) at ≥25% cutoff and immune cells (IC) at ≥1% and ≥25% cutoffs. KRAS and EGFR mutations were tested using PCR-based assays. Follow-up began 120 days after diagnosis until death/emigration/January 1, 2015, whichever came first. Using Cox proportional hazard regression, hazard ratios (HRs) were computed for overall survival (OS) for each biomarker, adjusting for age, sex, histology, comorbidities, and tissue specimen age. RESULTS: Among 391 patients identified, 40.4% had stage IIIA disease, 49.9% stage II, and 8.7% stage IB. PD-L1-TC was observed in 38% of patients, EGFR mutations in 4%, and KRAS mutations in 29%. KRAS mutations were more frequent among patients with PD-L1 TC≥25% versus TC<25% (37% versus 24%). OS was not associated with PD-L1 TC≥25% versus TC<25% (stage II: adjusted HR = 1.15 [95% confidence interval: 0.66-2.01]; stage IIIA: 0.72 [0.44-1.19]). No significant association was observed with OS and PD-L1-IC ≥1% and ≥25%. EGFR and KRAS mutations were not associated with a prognostic impact. CONCLUSION: A prognostic impact for NSCLC patients receiving adjuvant chemotherapy was not associated with PD-L1 expression, or with EGFR and KRAS mutations.

CRISPR-Cas provides limited phage immunity to a prevalent gut bacterium in gnotobiotic mice.

Many bacteria and archaea harbor the adaptive CRISPR-Cas system, which stores small nucleotide fragments from previous invasions of nucleic acids via viruses or plasmids. This molecular archive blocks further invaders carrying identical or similar nucleotide sequences. However, few of these systems have been confirmed experimentally to be active in gut bacteria. Here, we demonstrate experimentally that the type I-C CRISPR-Cas system of the prevalent gut bacterium Eggerthella lenta can specifically target and cleave foreign DNA in vitro by using a plasmid transformation assay. We also show that the CRISPR-Cas system acquires new immunities (spacers) from the genome of a virulent E. lenta phage using traditional phage assays in vitro but also in vivo using gnotobiotic (GB) mice. Both high phage titer and an increased number of spacer acquisition events were observed when E. lenta was exposed to a low multiplicity of infection in vitro, and three phage genes were found to contain protospacer hotspots. Fewer new spacer acquisitions were detected in vivo than in vitro. Longitudinal analysis of phage-bacteria interactions showed sustained coexistence in the gut of GB mice, with phage abundance being approximately one log higher than the bacteria. Our findings show that while the type I-C CRISPR-Cas system is active in vitro and in vivo, a highly virulent phage in vitro was still able to co-exist with its bacterial host in vivo. Taken altogether, our results suggest that the CRISPR-Cas defense system of E. lenta provides only partial immunity in the gut.