Differences in circulating lymphocyte subpopulations among patients with inflammatory polyps, colorectal adenomas and colorectal cancer.

BACKGROUND AND STUDY AIMS: Colorectal cancer (CRC) may develop from focal changes within benign or precancerous polyps. The immune system's failure to detect and eradicate tumor cells due to immune surveillance evasion, allows cancer to develop and spread. This study aims to analyze the differences in circulating lymphocyte subpopulations in patients with colorectal inflammatory polyps, colorectal adenomas and CRC. PATIENTS AND METHODS: We retrospectively reviewed patients from September 2016 to December 2019 at the Shaoxing Second Hospital. Using flow cytometry, the subset distribution and immunophenotype of T cells, CD4+ T cells, CD8+ T cells, B cells and NK cells were investigated in peripheral blood mononuclear cell samples. The counts of lymphocytes were determined by white blood cell counts. RESULTS: In total, 518 patients were included in this study. The counts of lymphocytes, T cells and NK cells in patients with inflammatory polyps, colorectal adenomas and CRC were lower than controls. The counts and percentages of CD8+ T cells in patients with inflammatory polyps, colorectal adenomas and CRC were lower than controls. The counts of CD4+ T cells were lower in patients with CRC than inflammatory polyps. The percentages of CD4+ T cells in patients with inflammatory polyps, colorectal adenomas and CRC were higher than controls, but lower in the CRC than inflammatory polyps, colorectal adenomas. The counts and percentages of B cells were lower in CRC patients than colorectal adenomas patients. In addition, the percentages of B cells were higher in patients with inflammatory polyps and colorectal adenomas than in controls. CONCLUSIONS: The decrease in counts of lymphocyte, T cells, CD8+ T cells and NK cells in patients may be related to the dysplasia of epithelial cells. Furthermore, the B cells and CD4+ T cells may be related to the malignant growth of the dysplastic epithelial cells.

Investigation of miRNA dysregulation and association with immune cell profile during malignant transformation of colorectal cells.

BACKGROUND: Colorectal cancer (CRC) is one of the most prevalent and life-threatening cancer among the world. Accumulated somatic mutations during malignant transformation process endow cancer cells with increased growth, invasiveness and immunogenicity. These highly immunogenic cancer cells develop multiple strategies to evade immune attack. Through post-transcriptional regulation, microRNAs (miRNAs) not only participate in cancer development and progression but also manipulate anti-cancer immune response. This study aims to identify miRNAs associated with the colorectal cell malignant transformation process and their association with immune cell population using synchronous adjacent normal, polyp and CRC specimens. METHODS: We conducted a Low Density Array to compare the miRNA expression profile of synchronous colorectal adenoma, adenocarcinoma and adjacent normal colon mucosa collected from 8 patients, in order to identify candidate miRNAs involved in CRC progression. These findings were further validated in 14 additional patients and GEO dataset GSE41655. The relative abundance of dendritic cells, natural killer cells, neutrophil and macrophage was determined and correlated with dysregulated miRNA levels. RESULTS: MicroRNA microarray identified 39 miRNAs aberrantly expressed during the colorectal cell transformation process. Seven novel miRNAs were shortlisted, and dysregulation of miR-149-3p, miR-192-3p, miR-335-5p and miR-425 were further validated by the qPCR validation experiment and data retrieved from the GEO dataset. Furthermore, these miRNAs demonstrated certain associations with level of dendritic cells, natural killer cells, neutrophil and macrophage within the polyp or CRC specimens. CONCLUSION: This study revealed miRNA dysregulated during stepwise malignant transformation of colorectal mucosal cells and their association with immune cell population.

Differential pre-malignant programs and microenvironment chart distinct paths to malignancy in human colorectal polyps.

Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.

Genomic and functional characterization of a mucosal symbiont involved in early-stage colorectal cancer.

Colorectal cancer is a major health concern worldwide. Growing evidence for the role of the gut microbiota in the initiation of CRC has sparked interest in approaches that target these microorganisms. However, little is known about the composition and role of the microbiota associated with precancerous polyps. Here, we found distinct microbial signatures between patients with and without polyps and between polyp subtypes using sequencing and culturing techniques. We found a correlation between Bacteroides fragilis recovered and the level of inflammatory cytokines in the mucosa adjacent to the polyp. Additional analysis revealed that B. fragilis from patients with polyps are bft-negative, activate NF-κB through Toll-like receptor 4, induce a pro-inflammatory response, and are enriched in genes associated with LPS biosynthesis. This study provides fundamental insight into the microbial microenvironment of the pre-neoplastic polyp by highlighting strain-specific genomic and proteomic differences, as well as more broad compositional differences in the microbiome.

CD163+ macrophages suppress T cell response by producing TGF-ß in pediatric colorectal polyps.

The local immune response plays an important role in the pathogenesis of colorectal carcinoma. Patients with colorectal polyps are at increased risk of colorectal cancer. However, the immunoregulation of early-stage colorectal polyps remain unknown. In the study, 202 biopsy samples from 80 pediatric patients with colorectal polyps and from 42 normal controls were collected. We found that the number of CD4+, CD8+T cells and CD19+B cells were reduced, whereas CD68+macrophages (Mϕ) were increased in colorectal polyps compared to the distal normal tissue from the same patients and the tissue from healthy donors. The frequency of Mϕwas negatively correlated with the number of CD4+ and CD8+T cells but not CD19+B cells in colorectal polyps. We further identified that CD163 was highly expressed on Mϕϕ from colorectal polyps compared to those from normal controls. Furthermore, real-time PCR revealed that TGF-ß, but not IL-10 and IL-4, was increased in colorectal polyps. Immunofluorescence and flow cytometry showed that TGF-ß was predominantly produced by CD163+Mϕ. In vitro experiments demonstrated that the supernatant from cultured polyps induced CD163 expression and TGF-ß production in blood-derived Mϕ. A co-culture experiment revealed that purified Mϕ from colorectal polyps suppressed T cell proliferation. Based on these results, we hypothesized that abundant CD163+Mϕ may promote the progression of colorectal polyps by inhibiting the local T cell response through TGF-ß production.

Serrated Lesions in Inflammatory Bowel Disease: Genotype-Phenotype Correlation.

BACKGROUND: Patients with inflammatory bowel disease (IBD) and hyperplastic/serrated polyposis have an increased risk of colorectal cancer. The aim of our study was to elucidate the nature of serrated lesions in IBD patients. MATERIALS AND METHODS: Sixty-five lesions with serrated morphology were analyzed in 39 adult IBD patients. Lesions were classified according to the WHO 2019 criteria or regarded as reactive, and molecular analysis was performed. RESULTS: 82.1% of patients had ulcerative colitis, 17.9% had Crohn's disease; 51.3% were female, and the mean age was 54.5 years. The duration of IBD varied significantly (16.7 ± 11.4 years). Endoscopy showed polypoid lesions in 80.3%; the size ranged from 2 to 20 mm. A total of 21.6% of the lesions were located in the right colon. Five lesions were classified as inflammatory pseudopolyps, 28 as hyperplastic polyp, 21 and 2 as sessile serrated lesion without and with dysplasia, respectively, and 9 as traditional serrated adenoma with low-grade dysplasia. Analysis of all true serrated lesions revealed 31 mutations in KRAS and 32 in BRAF gene. No mutations were identified in inflammatory pseudopolyps. In the right colon BRAF mutations were more frequent than KRAS (16 vs 3), while KRAS mutations prevailed on the left side (28 vs 16, P < .001). One patient with traditional serrated adenomas progressed to an adenocarcinoma after 61 months. CONCLUSION: The molecular analysis could help discriminate true serrated lesions (IBD-associated or not) from reactive pseudopolyps with serrated/hyperplastic epithelial change. These should help in more accurate classification of serrated lesions.

Increased Th17-Related Cytokine Serum Levels in Patients With Multiple Polyps of Unexplained Origin.

OBJECTIVES: Most patients with multiple colonic polyps do not have a known genetic or hereditary origin. Our aim was to analyze the presence of inflammatory cytokines and levels of glucose, insulin, and C-reactive protein (CRP) in patients with multiple colonic polyps. METHODS: Eighty-three patients with 10 or more adenomatous or serrated polyps and 53 control people with normal colonoscopy were included. Smoking habits were registered, and glucose, CRP, and basal insulin in the serum/blood were measured. Quantification of IL-2, IL-4, IL-6, IL-10, IL-11, IL-17A, and IL-23 cytokine levels in the serum was performed by a high-sensitivity enzyme-linked immunosorbent assay. RESULTS: Smoking and diabetes were more prevalent in those with colonic polyps than in the control people (67% vs 16%, P = 0.001; 11% vs 2%, P = 0.048). In addition, the cytokine serum levels were higher, i.e., IL-2 (P = 0.001), IL-4 (P = 0.001), IL-6 (P = 0.001), IL-17A (P = 0.001), IL-23 (P = 0.014), and CRP (P = 0.003). Adjusting for sex, smoking, and diabetes in a multivariate analysis, IL-2, IL-4, IL-6, IL-17A, and IL-23 remained independently elevated in cases with multiple polyps. DISCUSSION: These results indicate that immune responses mediated by Th17 cells may be involved in the pathogenesis of multiple colonic polyps.

Distribution of regulatory T cells in inflammatory colorectal polyps of miniature dachshunds.

Inflammatory colorectal polyp (ICRP) is an emerging disease in Miniature Dachshunds (MDs). Animals with this disease exhibit multiple polyps with severe neutrophil infiltration that respond to immunosuppressive therapy. Macrophages in polypoid lesions have been described to play an important role in neutrophil infiltration in the lesion by producing IL-8. In contrast, IL-10, an anti-inflammatory cytokine, was also reported to be upregulated in polypoid lesions, but its significance in the pathogenesis of ICRP has not been clarified. Regulatory T cells (Tregs) are the main source of IL-10 production and contribute to the maintenance of intestinal homeostasis. Therefore, the objective of this research was to compare the distribution of Tregs in polypoid lesions of ICRPs and the association between the distribution and expression of pro- or anti-inflammatory cytokines. Tissue biopsy specimens of polypoid lesions were collected from 28 MDs with ICRP. Those of macroscopically non-polypoid colonic mucosa from 24 MDs with ICRPs and 21 control dogs were further included as controls. Real-time quantitative polymerase chain reaction was used to quantify gene expression of IL-1ß, IL-4, IL-6, IL-8, IL-10, IL-17, IL-22, IFN-γ, TNF-α, TGF-ß, and forkhead box protein P3 (Foxp3) in each tissue sample. The numbers of Foxp3-positive cells (Tregs) and ionized calcium binding adapter molecule 1 (Iba-1)-positive cells (macrophages) were determined by immunohistochemistry. The gene expression of IL-1ß, IL-6, IL-8, TNF-α, IFN-γ, IL-17, IL-10, TGF-ß, and Foxp3 was significantly upregulated in polypoid lesions relative to control levels. The numbers of Foxp3-positive Tregs and Iba-1-positive macrophages were significantly increased in polypoid lesions compared to those in the non-polypoid colonic mucosa of MDs with ICRPs and control dogs. The upregulation of IL-10 was moderately correlated with the distribution of Tregs in polypoid lesions from MDs with ICRPs. In addition, the relative upregulation of IL-1ß, IL-6, and IL-8 in polypoid lesions, compared to expression in non-polypoid colonic mucosa of MDs with ICRPs, was significantly greater than that of IL-10. These results indicate that increases in Treg numbers and anti-inflammatory cytokines in polypoid lesions comprise reactive changes in response to the inflammation, which warrants further investigation.

Interleukin 1 beta and Matrix Metallopeptidase 3 Contribute to Development of Epidermal Growth Factor Receptor-Dependent Serrated Polyps in Mouse Cecum.

BACKGROUND & AIMS: Transgenic mice (HBUS) that express the epidermal growth factor receptor (EGFR) ligand HBEGF (heparin-binding epidermal growth factor-like growth factor) and a constitutively active G protein-coupled receptor (US28) in intestinal epithelial cells develop serrated polyps in the cecum. Development of serrated polyps depends on the composition of the gut microbiota and is associated with bacterial invasion of the lamina propria, accompanied by induction of inflammation and up-regulation of interleukin 1 beta (IL1B) and matrix metalloproteinase (MMP) 3 in the cecum. We investigated the mechanisms by which these changes contribute to development of serrated polyps. METHODS: We performed studies with C57BL/6 (control) and HBUS mice. To accelerate polyp development, we increased the exposure of the bacteria to the lamina propria by injecting HBUS mice with diphtheria toxin, which binds transgenic HBEGF expressed by the epithelial cells and causes apoptosis. Mice were given injections of IL1B-neutralizing antibody and the MMP inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. Intestinal tissues were collected from mice and analyzed by histology, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry. We examined fibroblast subsets in polyps using single-cell RNA sequencing. RESULTS: Administration of diphtheria toxin to HBUS mice accelerated development of serrated polyps (95% of treated mice developed polyps before 100 days of age, compared with 53% given vehicle). IL1B stimulated subsets of platelet-derived growth factor receptor alpha+ (PDGRFA+) fibroblasts isolated from cecum, resulting in increased expression of MMP3. Neutralizing antibodies against IL1B or administration of the MMP inhibitor reduced the number of serrated polyps that formed in the HBUS mice. Single-cell RNA sequencing analysis showed subsets of fibroblasts in serrated polyps that express genes that regulate matrix fibroblasts and inflammation. CONCLUSIONS: In studies of mice, we found that barrier breakdown and expression of inflammatory factors contribute to development of serrated polyps. Subsets of cecal PDGFRA+ fibroblasts are activated by release of IL1B from myeloid cells during the early stages of serrated polyp development. MMP3 produced by PDGFRA+ fibroblasts is important for serrated polyp development. Our findings confirm the functions of previously identified serrated polyp-associated molecules and indicate roles for immune and stromal cells in serrated polyp development.

Can colonic inflammatory polyp with numerous immunoglobulin G4-positive plasma cells represent a colonic manifestation of immunoglobulin G4-related disease? A case report.

We present an asymptomatic case of a 79-year-old Japanese man who had a 6 mm colonic inflammatory polyp with numerous immunoglobulin G4 (IgG4)-positive plasma cells. No symptoms or abnormal laboratory data, such as changes in serum IgG4 levels, were found at the time of diagnosis or during the 1 year of follow-up thereafter. Additionally, no diffuse/localized swelling or masses were found in organs, except for colonic polyps, by abdominal computed tomography 1 year prior to the polypectomy. Inflammatory myofibroblastic tumor was unlikely from the lack of spindle cell proliferation and ALK immunoreactivity. This is the first case of this colonic polyp in an asymptomatic person. This polyp could be probable for single organ manifestation of IgG4-related disease (IgG4-RD), according to the comprehensive diagnostic criteria for IgG4-RD published in 2012; however, colonic manifestation of IgG4-RD has not been clarified owing to its rarity, and colon-specific criteria for IgG4-RD have not been proposed. Thus, we could not definitively establish the colonic polyp as IgG4-RD. Therefore, careful clinicopathological evaluation is needed to reveal whether this colonic polyp represents a nonspecific inflammatory response or an early manifestation of IgG4-RD.

NK-Cell Enteropathy and Similar Indolent Lymphoproliferative Disorders: A Case Series With Literature Review.

Objectives: We report four new cases of natural killer-cell enteropathy (NKCE) and similar lymphoproliferative disorders (LPDs), as well as review the literature concerning indolent natural killer (NK)-cell LPDs of the gastrointestinal tract. Methods: Pathologic and clinical data were obtained from institutional/referral records. Results: Patient 1 (45-year-old man) had anemia; a small intestinal lesion was endoscopically biopsied. Patient 2 (65-year-old woman) had biliary colic, treated with cholecystectomy. Patient 3 (62-year-old man) had a small colonic polyp, biopsied on routine colonoscopy. Patient 4 (68-year-old man) had presumed Crohn disease; multiple biopsies were performed over more than 10 years. Diagnostic specimens showed atypical infiltrates of Epstein-Barr virus-negative lymphocytes with immunophenotypes suggestive of NK cells. In all cases, there was distortion of glandular architecture but no marked intraepithelial lymphocytosis or necrosis. The patients did not receive therapy for lymphoma and were well on follow-up. Conclusions: These cases support the indolent nature of NKCE and similar LPDs, and they indicate that involvement outside the alimentary canal may occur.

Immune Profiling of Premalignant Lesions in Patients With Lynch Syndrome.

Importance: Colorectal carcinomas in patients with Lynch syndrome (LS) arise in a background of mismatch repair (MMR) deficiency, display a unique immune profile with upregulation of immune checkpoints, and response to immunotherapy. However, there is still a gap in understanding the pathogenesis of MMR-deficient colorectal premalignant lesions, which is essential for the development of novel preventive strategies for LS. Objective: To characterize the immune profile of premalignant lesions from a cohort of patients with LS. Design, Setting, and Participants: Whole-genome transcriptomic analysis using next-generation sequencing was performed in colorectal polyps and carcinomas of patients with LS. As comparator and model of MMR-proficient colorectal carcinogenesis, we used samples from patients with familial adenomatous polyposis (FAP). In addition, a total of 47 colorectal carcinomas (6 hypermutants and 41 nonhypermutants) were obtained from The Cancer Genome Atlas (TCGA) for comparisons. Samples were obtained from the University of Texas MD Anderson Cancer Center and "Regina Elena" National Cancer Institute, Rome, Italy. All diagnoses were confirmed by genetic testing. Polyps were collected at the time of endoscopic surveillance and tumors were collected at the time of surgical resection. The data were analyzed from October 2016 to November 2017. Main Outcomes and Measures: Assessment of the immune profile, mutational signature, mutational and neoantigen rate, and pathway enrichment analysis of neoantigens in LS premalignant lesions and their comparison with FAP premalignant lesions, LS carcinoma, and sporadic colorectal cancers from TCGA. Results: The analysis was performed in a total of 28 polyps (26 tubular adenomas and 2 hyperplastic polyps) and 3 early-stage LS colorectal tumors from 24 patients (15 [62%] female; mean [SD] age, 48.12 [15.38] years) diagnosed with FAP (n = 10) and LS (n = 14). Overall, LS polyps presented with low mutational and neoantigen rates but displayed a striking immune activation profile characterized by CD4 T cells, proinflammatory (tumor necrosis factor, interleukin 12) and checkpoint molecules (LAG3 [lymphocyte activation gene 3] and PD-L1 [programmed cell death 1 ligand 1]). This immune profile was independent of mutational rate, neoantigen formation, and MMR status. In addition, we identified a small subset of LS polyps with high mutational and neoantigen rates that were comparable to hypermutant tumors and displayed additional checkpoint (CTLA4 [cytotoxic T-lymphocyte-associated protein 4]) and neoantigens involved in DNA damage response (ATM and BRCA1 signaling). Conclusions and Relevance: These findings challenge the canonical model, based on the observations made in carcinomas, that emphasizes a dependency of immune activation on the acquisition of high levels of mutations and neoantigens, thus opening the door to the implementation of immune checkpoint inhibitors and vaccines for cancer prevention in LS.

The tumour microenvironment creates a niche for the self-renewal of tumour-promoting macrophages in colon adenoma.

Circulating CCR2+ monocytes are crucial for maintaining the adult tissue-resident F4/80hiMHCIIhi macrophage pool in the intestinal lamina propria. Here we show that a subpopulation of CCR2-independent F4/80hiMHCIIlow macrophages, which are the most abundant F4/80hi cells in neonates, gradually decline in number in adulthood; these macrophages likely represent the fetal contribution to F4/80hi cells. In colon adenomas of ApcMin/+ mice, F4/80hiMHCIIlow macrophages are not only preserved, but become the dominant subpopulation among tumour-resident macrophages during tumour progression. Furthermore, these pro-tumoural F4/80hiMHCIIlow and F4/80hiMHCIIhi macrophages can self-renew in the tumour and maintain their numbers mostly independent from bone marrow contribution. Analyses of colon adenomas indicate that CSF1 may be a key facilitator of macrophage self-renewal. In summary, the tumour microenvironment creates an isolated niche for tissue-resident macrophages that favours macrophage survival and self-renewal.

Macrophage depletion using clodronate liposomes decreases tumorigenesis and alters gut microbiota in the AOM/DSS mouse model of colon cancer.

We examined the role of macrophages in inflammation associated with colorectal cancer (CRC). Given the emerging evidence on immune-microbiota interactions in CRC, we also sought to examine the interaction between macrophages and gut microbiota. To induce CRC, male C57BL/6 mice ( n = 32) received a single injection of azoxymethane (AOM), followed by three cycles of dextran sodium sulfate (DSS)-supplemented water in weeks 1, 4, and 7. Prior to the final DSS cycle ( week 7) and twice weekly until euthanasia, mice ( n = 16/group) received either 200 µl ip of clodronate-filled liposomes (CLD) or phosphate-buffered saline (PBS) encapsulated liposomes to deplete macrophages. Colon tissue was analyzed for polyp burden, macrophage markers, transcription factors, and inflammatory mediators. Stool samples were collected, and DNA was isolated and subsequently sequenced for 16S rRNA. Clodronate liposomes decreased tumor number by ∼36% and specifically large (≥1 mm) tumors by ∼36% ( P < 0.05). This was consistent with a decrease in gene expression of EMR1 in the colon tissue and polyp tissue as well as expression of select markers associated with M1 (IL-6) and M2 macrophages (IL-13, IL-10, TGFß, CCL17) in the colon tissue ( P < 0.05). Similarly, there was a decrease in STAT3 and p38 MAPK and ERK signaling in colon tissue. Clodronate liposomes increased the relative abundance of the Firmicutes phylum ( P < 0.05) and specifically Lactobacillaceae and Clostridiaceae families, which have been associated with reduced CRC risk. Overall, these data support the development of therapeutic strategies to target macrophages in CRC and provide support for further evaluation of immune-microbiota interactions in CRC. NEW & NOTEWORTHY We found that macrophage depletion during late-stage tumorigenesis is effective at reducing tumor growth. This was associated with a decrease in macrophage markers and chemokines in the colon tissue and a decrease in transcription factors that are linked to colorectal cancer. The macrophage-depleted group was found to have an increased abundance of Firmicutes, a phylum with documented anti-tumorigenic effects. Overall, these data support the development of therapeutic strategies to target macrophages in colorectal cancer.

Human Secretory IgM Emerges from Plasma Cells Clonally Related to Gut Memory B Cells and Targets Highly Diverse Commensals.

Secretory immunoglobulin A (SIgA) enhances host-microbiota symbiosis, whereas SIgM remains poorly understood. We found that gut IgM+ plasma cells (PCs) were more abundant in humans than mice and clonally related to a large repertoire of memory IgM+ B cells disseminated throughout the intestine but rare in systemic lymphoid organs. In addition to sharing a gut-specific gene signature with memory IgA+ B cells, memory IgM+ B cells were related to some IgA+ clonotypes and switched to IgA in response to T cell-independent or T cell-dependent signals. These signals induced abundant IgM which, together with SIgM from clonally affiliated PCs, recognized mucus-embedded commensals. Bacteria recognized by human SIgM were dually coated by SIgA and showed increased richness and diversity compared to IgA-only-coated or uncoated bacteria. Thus, SIgM may emerge from pre-existing memory rather than newly activated naive IgM+ B cells and could help SIgA to anchor highly diverse commensal communities to mucus.

Association Between Natural Killer Cell Activity and Colorectal Cancer in High-Risk Subjects Undergoing Colonoscopy.

BACKGROUND & AIMS: Low activity of natural killer (NK) cells has been associated with increased risk of cancer and has been reported in patients with colorectal cancer (CRC). Activity of NK cells can be measured in a small volume of whole blood by a commercially available test. We investigated whether this test could be used to identify patients with CRC, using findings from colonoscopy as a reference standard. METHODS: We performed an open-label, prospective, cross-sectional study of 872 high-risk subjects (more than 40 years old) screened for CRC by colonoscopy at a university hospital in Montreal, Canada from October 2014 through January 2016. Blood samples were collected on the day of colonoscopy, prior to the procedure. The test involves stimulation of whole blood with cytokine that induces NK cells to secrete interferon gamma (IFNG), which is quantified by an ELISA. Tissue samples were taken from lesions during the colonoscopy and analyzed histologically; subjects were classified as having no evidence of disease, adenomatous polyps of less than 10 mm, of 10 mm or more, or CRC. We used the non-parametric Mann-Whitney test to compare NK cell activity between subjects with no evidence of CRC and subjects found to have CRC. Receiver operating characteristic curve analysis was used to assess the ability of the test to identify individuals with CRC. The primary objective was to determine the difference in NK cell activity between subjects with vs without CRC. The secondary objective was the test performance, based on receiver operating characteristic analysis, and cut-off value that most accurately identified individuals with CRC. RESULTS: We found a significant difference in NK cell activity between the 23 subjects with CRC (based on pathology analysis) and the 849 subjects without CRC: subjects found to have CRC by colonoscopy had a median level of 86.0 pg IFNG/mL (inter-quartile range, 43.3-151.0 pg IFNG/mL), whereas subjects without CRC had a median level of 298.1 pg IFNG/mL (inter-quartile range, 100.4-920.2 pg IFNG/mL) (P = .0002). The cut-off value that most accurately identified subjects with CRC was 181 pg/mL. The NK cell activity test identified subjects with CRC with 87.0% sensitivity, 60.8% specificity, a positive predictive value of 5.7%, and a negative predictive value of 99.4%. The odds ratio for detection of CRC in subjects with low NK cell activity vs subjects with higher NK cell activity was 10.3 (95% CI, 3.03-34.9). CONCLUSIONS: Using colonoscopy as the reference standard, a test for NK cell activity in whole blood samples identified patients with CRC with 87.0% sensitivity and a negative predictive value of 99.4%. Subjects with low NK cell activity had a 10-fold higher risk of CRC compared with subjects with high NK cell activity. This test might be used in clinical practice to assess patients for risk of CRC. Clinicaltrials.gov number: NCT02291198.

Stimulation of colorectal biopsies from miniature dachshunds with inflammatory colorectal polyps with toll-like receptor ligands: A pilot study.

Inflammatory colorectal polyps (ICRPs) in miniature dachshunds (MDs) are a possible novel form of breed-specific canine inflammatory bowel disease (IBD). In this pilot study, we investigated the effects of different Toll like receptor (TLR2, TLR4, TLR5 and TLR9) ligands on pro-inflammatory cytokines (IL-1ß, IL-6, IL-8 and TNF-α) gene expression in ex vivo-cultured colorectal samples from four MDs with ICRPs and four healthy MDs. At baseline, no significant difference in the mRNA expression levels of TLRs and pro-inflammatory cytokines was observed between cases and control MDs. After 4-h incubation, the relative ratios of TNF-α mRNA expression in the TLR2- or TLR4-stimulated colorectal samples, and IL-1ß mRNA expression in the TLR9-stimulated colorectal samples form cases showed higher tendency compared with healthy MDs (P<0.05), although statistically not significant. The results of this pilot study using small number of cases indicated that reactivity against TLR2, TLR4 or TLR9 ligand in the production of pro-inflammatory cytokines might be enhanced in the colorectal mucosa of ICRPs. Further research is needed to perform the functional analysis of TLRs in the sole cell population using intestinal epithelial primary culture and the mononuclear cells isolated from colonic mucosa.