Microbial dynamics with CRC progression: a study of the mucosal microbiota at multiple sites in cancers, adenomatous polyps, and healthy controls.

Accumulating evidence has related the gut microbiota to colorectal cancer (CRC). Fusobacterium nucleatum has repeatedly been linked to colorectal tumorigenesis. The aim of this study was to investigate microbial composition in different sampling sites, in order to profile the microbial dynamics with CRC progression. Further, we characterized the tumor-associated F. nucleatum subspecies. Here, we conducted Illumina Miseq next-generation sequencing of the 16S rRNA V4 region in biopsy samples, to investigate microbiota alterations in cancer patients, patients with adenomatous polyp, and healthy controls in Norway. Further, Fusobacterium positive tumor biopsies were subjected to MinION nanopore sequencing of Fusobacterium-specific amplicons to characterize the Fusobacterium species and subspecies. We found enrichment of oral biofilm-associated bacteria, Fusobacterium, Gemella, Parvimonas, Granulicatella, Leptotrichia, Peptostreptococcus, Campylobacter, Selenomonas, Porphyromonas, and Prevotella in cancer patients compared to adenomatous polyp patients and control patients. Higher abundance of amplicon sequence variants (ASVs) classified as Phascolarctobacterium, Bacteroides vulgatus, Bacteroides plebeius, Bacteroides eggerthii, Tyzzerella, Desulfovibrio, Frisingicoccus, Eubacterium coprostanoligenes group, and Lachnospiraceae were identified in cancer and adenomatous polyp patients compared to healthy controls. F. nucleatum ssp. animalis was the dominating subspecies. F. nucleatum ssp. nucleatum, F. nucleatum ssp. vincentii, Fusobacterium pseudoperiodonticum, Fusobacterium necrophorum, and Fusobacterium gonidiaformans were identified in five samples. Several biofilm-associated bacteria were enriched at multiple sites in cancer patients. Another group of bacteria was enriched in both cancer and polyps, suggesting that they may have a role in polyp development and possibly early stages of CRC.

The CRCbiome study: a large prospective cohort study examining the role of lifestyle and the gut microbiome in colorectal cancer screening participants.

BACKGROUND: Colorectal cancer (CRC) screening reduces CRC incidence and mortality. However, current screening methods are either hampered by invasiveness or suboptimal performance, limiting their effectiveness as primary screening methods. To aid in the development of a non-invasive screening test with improved sensitivity and specificity, we have initiated a prospective biomarker study (CRCbiome), nested within a large randomized CRC screening trial in Norway. We aim to develop a microbiome-based classification algorithm to identify advanced colorectal lesions in screening participants testing positive for an immunochemical fecal occult blood test (FIT). We will also examine interactions with host factors, diet, lifestyle and prescription drugs. The prospective nature of the study also enables the analysis of changes in the gut microbiome following the removal of precancerous lesions. METHODS: The CRCbiome study recruits participants enrolled in the Bowel Cancer Screening in Norway (BCSN) study, a randomized trial initiated in 2012 comparing once-only sigmoidoscopy to repeated biennial FIT, where women and men aged 50-74 years at study entry are invited to participate. Since 2017, participants randomized to FIT screening with a positive test result have been invited to join the CRCbiome study. Self-reported diet, lifestyle and demographic data are collected prior to colonoscopy after the positive FIT-test (baseline). Screening data, including colonoscopy findings are obtained from the BCSN database. Fecal samples for gut microbiome analyses are collected both before and 2 and 12 months after colonoscopy. Samples are analyzed using metagenome sequencing, with taxonomy profiles, and gene and pathway content as primary measures. CRCbiome data will also be linked to national registries to obtain information on prescription histories and cancer relevant outcomes occurring during the 10 year follow-up period. DISCUSSION: The CRCbiome study will increase our understanding of how the gut microbiome, in combination with lifestyle and environmental factors, influences the early stages of colorectal carcinogenesis. This knowledge will be crucial to develop microbiome-based screening tools for CRC. By evaluating biomarker performance in a screening setting, using samples from the target population, the generalizability of the findings to future screening cohorts is likely to be high. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01538550 .

Correction to: Detection of Fusobacterium nucleatum in stool and colonic tissues from Norwegian colorectal cancer patients.

Two affiliations of author John Christopher Noone were not included in the original article and have been added here. Also, Acknowledgments of the originally published article is not complete. Please see the corrected section below.

Detection of Fusobacterium nucleatum in stool and colonic tissues from Norwegian colorectal cancer patients.

Norway has one of the world's highest incidences of colorectal cancer (CRC). Accumulating research suggests that the intestinal microbiota may have an important role in initiation and progression of colorectal cancer. In order to evaluate microbiome-based biomarkers for non-invasive detection of CRC, the levels of Fusobacterium nucleatum and selected Escherichia coli toxin genes in stool and mucosa from a small cohort of Norwegian patients were investigated. The study cohort included 72 patients scheduled for colonoscopy. The patients were divided into three groups upon their examinations: cancer, polyp, and control groups. Levels of F. nucleatum in stool samples were significantly higher in the cancer group compared with the control group and the polyp group. High levels of F. nucleatum in stool reflected detection of F. nucleatum in the tumor tissues of colorectal cancer patients. However, no difference in the levels of E. coli toxin genes in neither stool nor biopsy samples between the patient groups was observed. This study suggests that a quantitative PCR assay targeting F. nucleatum in stool samples has the potential to be included in a larger panel of biomarkers for non-invasive testing for colorectal cancer.

The NEOLETEXE trial: a neoadjuvant cross-over study exploring the lack of cross resistance between aromatase inhibitors.

The aromatase inhibitor letrozole (Femar®/Femara®) and the aromatase inactivator exemestane (Aromasin®) differ in their biochemical effect on the aromatase enzyme. Letrozole is a competitive aromatase inhibitor while exemestane binds irreversibly to the aromatase enzyme. This pharmacological difference is of clinical interest since a lack of cross-resistance has been documented. It has been demonstrated in several clinical trials that exemestane may cause a disease regression following resistance to nonsteroidal aromatase inhibitors. The exact mechanism(s) behind this phenomenon is yet unknown. Here, we present the NEOLETEXE trial with the aim of exploring the individual mechanisms involved behind the observed lack of cross resistance. Clinical trial registration: The trial has been approved by the Regional Ethics Committee of South-East Norway (project number 2015/84).

Somatic EP300-G211S mutations are associated with overall somatic mutational patterns and breast cancer specific survival in triple-negative breast cancer.

PURPOSE: We have compared the mutational profiles of human breast cancer tumor samples belonging to all major subgroups with special emphasis on triple-negative breast cancer (TNBC). Our major goal was to identify specific mutations that could be potentially used for clinical decision making in TNBC patients. PATIENTS AND METHODS: Primary tumor specimens from 149 Norwegian breast cancer patients were available. We analyzed the tissue samples for somatic mutations in 44 relevant breast cancer genes by targeted next-generation sequencing. As a second confirmatory technique, we performed pyrosequencing on selected samples. RESULTS: We observed a distinct subgroup of TNBC patients, characterized by an almost completely lack of pathogenic somatic mutations. A point mutation in the adenoviral E1A binding protein p300 (EP300-G211S) was significantly correlated to this TNBC subgroup. The EP300-G211S mutation was exclusively found in the TNBC patients and its presence reduced the chance for other pathological somatic mutations in typical breast cancer genes investigated in our gene panel by 94.9% (P < 0.005). Interestingly, the EP300-G211S mutation also predicted a lower risk for relapses and decreased breast cancer-specific mortality during long-term follow-up of the patients. CONCLUSION: Next-generation sequencing revealed specific mutations in EP300 to be associated with the mutational patterns in typical breast cancer genes and long-term outcome of triple-negative breast cancer patients.

Decoding immune-related gene-signatures in colorectal neoplasia.

Background: Colorectal cancer (CRC) is a significant health issue, with notable incidence rates in Norway. The immune response plays a dual role in CRC, offering both protective effects and promoting tumor growth. This research aims to provide a detailed screening of immune-related genes and identify specific genes in CRC and adenomatous polyps within the Norwegian population, potentially serving as detection biomarkers. Methods: The study involved 69 patients (228 biopsies) undergoing colonoscopy, divided into CRC, adenomatous polyps, and control groups. We examined the expression of 579 immune genes through nCounter analysis emphasizing differential expression in tumor versus adjacent non-tumorous tissue and performed quantitative reverse transcription polymerase chain reaction (RT-qPCR) across patient categories. Results: Key findings include the elevated expression of CXCL1, CXCL2, IL1B, IL6, CXCL8 (IL8), PTGS2, and SPP1 in CRC tissues. Additionally, CXCL1, CXCL2, IL6, CXCL8, and PTGS2 showed significant expression changes in adenomatous polyps, suggesting their early involvement in carcinogenesis. Conclusions: This study uncovers a distinctive immunological signature in colorectal neoplasia among Norwegians, highlighting CXCL1, CXCL2, IL1B, IL6, CXCL8, PTGS2, and SPP1 as potential CRC biomarkers. These findings warrant further research to confirm their role and explore their utility in non-invasive screening strategies.

Detection of colorectal-cancer-associated bacterial taxa in fecal samples using next-generation sequencing and 19 newly established qPCR assays.

We have previously identified increased levels of distinct bacterial taxa within mucosal biopsies from colorectal cancer (CRC) patients. Following prior research, the aim of this study was to investigate the detection of the same CRC-associated bacteria in fecal samples and to evaluate the suitability of fecal samples as a non-invasive material for the detection of CRC-associated bacteria. Next-generation sequencing (NGS) of the 16S ribosomal RNA (rRNA) V4 region was performed to evaluate the detection of the CRC-associated bacteria in the fecal microbiota of cancer patients, patients with adenomatous polyp and healthy controls. Furthermore, 19 novel species-specific quantitative PCR (qPCR) assays were established to detect the CRC-associated bacteria. Approximately, 75% of the bacterial taxa identified in biopsies were reflected in fecal samples. NGS failed to detect low-abundance CRC-associated taxa in fecal samples, whereas qPCR exhibited high sensitivity and specificity in identifying all targeted taxa. Comparison of fecal microbial composition between the different patient groups showed enrichment of Fusobacterium nucleatum, Parvimonas micra, and Gemella morbillorum in cancer patients. Our findings suggest that low-abundance mucosa-associated bacteria can be detected in fecal samples using sensitive qPCR assays.

Species-level verification of Phascolarctobacterium association with colorectal cancer.

We have previously demonstrated an association between increased abundance of Phascolarctobacterium and colorectal cancer (CRC) and adenomas in two independent Norwegian cohorts. Here we seek to verify our previous findings using new cohorts and methods. In addition, we characterize lifestyle and sex specificity, the functional potential of the Phascolarctobacterium species, and their interaction with other microbial species. We analyze Phascolarctobacterium with 16S rRNA sequencing, shotgun metagenome sequencing, and species-specific qPCR, using 2350 samples from three Norwegian cohorts-CRCAhus, NORCCAP, and CRCbiome-and a large publicly available data set, curatedMetagenomicData. Using metagenome-assembled genomes from the CRCbiome study, we explore the genomic characteristics and functional potential of the Phascolarctobacterium pangenome. Three species of Phascolarctobacterium associated with adenoma/CRC were consistently detected by qPCR and sequencing. Positive associations with adenomas/CRC were verified for Phascolarctobacterium succinatutens and negative associations were shown for Phascolarctobacterium faecium and adenoma in curatedMetagenomicData. Men show a higher prevalence of P. succinatutens across cohorts. Co-occurrence among Phascolarctobacterium species was low (<6%). Each of the three species shows distinct microbial composition and forms distinct correlation networks with other bacterial taxa, although Dialister invisus was negatively correlated to all investigated Phascolarctobacterium species. Pangenome analyses showed P. succinatutens to be enriched for genes related to porphyrin metabolism and degradation of complex carbohydrates, whereas glycoside hydrolase enzyme 3 was specific to P. faecium.IMPORTANCEUntil now Phascolarctobacterium has been going under the radar as a CRC-associated genus despite having been noted, but overseen, as such for over a decade. We found not just one, but two species of Phascolarctobacterium to be associated with CRC-Phascolarctobacterium succinatutens was more abundant in adenoma/CRC, while Phascolarctobacterium faecium was less abundant in adenoma. Each of them represents distinct communities, constituted by specific microbial partners and metabolic capacities-and they rarely occur together in the same patients. We have verified that P. succinatutens is increased in adenoma and CRC and this species should be recognized among the most important CRC-associated bacteria.

Exploring the gut DNA virome in fecal immunochemical test stool samples reveals associations with lifestyle in a large population-based study.

Stool samples for fecal immunochemical tests (FIT) are collected in large numbers worldwide as part of colorectal cancer screening programs. Employing FIT samples from 1034 CRCbiome participants, recruited from a Norwegian colorectal cancer screening study, we identify, annotate and characterize more than 18000 DNA viruses, using shotgun metagenome sequencing. Only six percent of them are assigned to a known taxonomic family, with Microviridae being the most prevalent viral family. Linking individual profiles to comprehensive lifestyle and demographic data shows 17/25 of the variables to be associated with the gut virome. Physical activity, smoking, and dietary fiber consumption exhibit strong and consistent associations with both diversity and relative abundance of individual viruses, as well as with enrichment for auxiliary metabolic genes. We demonstrate the suitability of FIT samples for virome analysis, opening an opportunity for large-scale studies of this enigmatic part of the gut microbiome. The diverse viral populations and their connections to the individual lifestyle uncovered herein paves the way for further exploration of the role of the gut virome in health and disease.

Long-term follow-up of colorectal cancer screening attendees identifies differences in Phascolarctobacterium spp. using 16S rRNA and metagenome sequencing.

Background: The microbiome has been implicated in the initiation and progression of colorectal cancer (CRC) in cross-sectional studies. However, there is a lack of studies using prospectively collected samples. Methods: From the Norwegian Colorectal Cancer Prevention (NORCCAP) trial, we analyzed 144 archived fecal samples from participants who were diagnosed with CRC or high-risk adenoma (HRA) at screening and from participants who remained cancer-free during 17 years of follow-up. We performed 16S rRNA sequencing of all the samples and metagenome sequencing on a subset of 47 samples. Differences in taxonomy and gene content between outcome groups were assessed for alpha and beta diversity and differential abundance. Results: Diversity and composition analyses showed no significant differences between CRC, HRA, and healthy controls. Phascolarctobacterium succinatutens was more abundant in CRC compared with healthy controls in both the 16S and metagenome data. The abundance of Bifidobacterium and Lachnospiraceae spp. was associated with time to CRC diagnosis. Conclusion: Using a longitudinal study design, we identified three taxa as being potentially associated with CRC. These should be the focus of further studies of microbial changes occurring prior to CRC diagnosis.

The epidermal growth factor receptor (EGFR / HER-1) gatekeeper mutation T790M is present in European patients with early breast cancer.

The epidermal growth factor receptor (EGFR) is one of the major oncogenes identified in a variety of human malignancies including breast cancer (BC). EGFR-mutations have been studied in lung cancer for some years and are established as important markers in guiding therapy with tyrosine kinase inhibitors (TKIs). In contrast, EGFR-mutations have been reported to be rare if not absent in human BC, although recent evidence has suggested a significant worldwide variation in somatic EGFR-mutations. Therefore, we investigated the presence of EGFR-mutations in 131 norwegian patients diagnosed with early breast cancer using real-time PCR methods. In the present study we identified three patients with an EGFR-T790M-mutation. The PCR-findings were confirmed by direct Sanger sequencing. Two patients had triple-negative BC (TNBC) while the third was classified as luminal-A subtype. The difference in incidence of T790M mutations comparing the TNBC subgroup with the other BC subgroups was statistical significant (P = 0.023). No other EGFR mutations were identified in the entire cohort. Interestingly, none of the patients had received any previous cancer treatment. To our best knowledge, the EGFR-T790M-TKI-resistance mutation has not been previously detected in breast cancer patients. Our findings contrast with the observations made in lung cancer patients where the EGFR-T790M-mutation is classified as a typical "second mutation"causing resistance to TKI-therapy during ongoing anticancer therapy. In conclusion, we have demonstrated for the first time that the EGFR-T790M-mutation occurs in primary human breast cancer patients. In the present study the EGFR-T790M mutation was not accompanied by any simultaneous EGFR-activating mutation.

The aryl hydrocarbon receptor-mediated disruption of vitellogenin synthesis in the fish liver: Cross-talk between AHR- and ERalpha-signalling pathways.

BACKGROUND: In the fish liver, the synthesis of egg yolk protein precursor vitellogenin (VTG) is under control of the estrogen receptor alpha (ERalpha). Environmental contaminants such as 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) are suspected to have antiestrogenic effects. The aryl hydrocarbon receptor (AHR) is the initial cellular target for TCDD and related compounds. The AHR is a ligand-activated transcription factor that stimulates the expression of the genes encoding xenobiotic metabolizing enzymes, such as cytochrome P450 1A (CYP1A). In this study, the effects of activation of AHR on the hepatic expression of VTG and ERalpha genes, in primary cultured salmon hepatocytes, have been investigated. RESULTS: The expression of the genes encoding VTG and ERalpha were strongly induced by 17beta-estradiol (E2). However, the expression of VTG was disrupted by exposure of the cells to TCDD while CYP1A expression was enhanced. The effect of TCDD on VTG and CYP1A expression was annulled by the AHR-inhibitor alpha-naphthoflavone. Furthermore, exposure of the cells to TCDD abolished E2-induced accumulation of ERalpha mRNA. The AHR-mediated inhibitory effects on the expression of the VTG and ERalpha genes may occur at transcriptional and/or post-transcriptional levels. Nuclear run-off experiments revealed that simultaneous exposure of the cells to E2 and TCDD strongly inhibited the initiation of transcription of the VTG and ERalpha genes. In addition, inhibition of RNA synthesis by actinomycin D treatment showed that post-transcriptional levels of VTG and ERalpha mRNAs were not significantly altered upon treatment of the cells with TCDD. These results suggested that activation of AHR may inhibit the transactivation capacity of the ERalpha. Further, electrophoretic mobility shift assays using nuclear extracts prepared from cells treated for one or two hours with E2, alone or in mixture with TCDD, showed a strong reduction in the DNA binding activities upon TCDD treatment. These results also suggested that activation of the AHR signalling pathway caused a marked decrease in the number of the nuclear ERalpha or that activated AHR blocked the ability of ERalpha to bind to its target DNA sequence. Finally, our results from Northern hybridizations indicated that E2 treatment of the cells did not cause any significant effect on the TCDD-induced levels of CYP1A mRNA. CONCLUSION: In fish hepatocytes E2 induces ERalpha and VTG gene expression. The presence of dioxin (TCDD) abolishes this induction, probably through the action of AHR in complex with AHR nuclear translocator, and possibly by direct interference with the auto-regulatory transcriptional loop of ERalpha. Furthermore, E2 does not interfere with TCDD induced CYP1A gene expression, suggesting that cross-talk between the ERalpha- and AHR-signalling pathways is unidirectional.

A rapid, high-throughput screening method for carriage of methicillin-resistant Staphylococcus aureus.

Rapid screening of methicillin-resistant Staphylococcus aureus (MRSA) colonization prior to hospital admittance is important to reduce nosocomial infections and health care costs. Molecular detection of mecA and S. aureus specific target genes has become widely established for this purpose. However, there are still limitations in potential for high-throughput screening in the methods described. We have compared the time aspects and workload of four different DNA preparation platforms, resulting in an automated and simple MRSA screening method which combines two liquid handling systems and a simple lysis buffer. We have further transferred our in-house dual real-time PCR to a fast-PCR protocol, reducing the time and labour spent on these samples to a minimum.