LGR6 is a prognostic biomarker for less differentiated tumors in lymph nodes of colon cancer patients.

Introduction: The aim was to investigate whether the stem cell marker LGR6 has prognostic value in colon cancer, alone or in combination with the prognostic biomarkers CEA and CXCL16. Methods: LGR6 mRNA levels were determined in 370 half lymph nodes of 121 colon cancer patients. Ability to predict relapse after curative surgery was estimated by Kaplan-Meier survival model and Cox regression analyses. Results: Patients with high LGR6 levels [LGR6(+)] had a decreased mean survival time of 11 months at 5-year follow-up and 47 months at 12-year follow-up, respectively, with hazard ratios of 3.2 and 2.8. LGR6 mRNA analysis added prognostic value to CEA and CXCL16 mRNA analysis. In the poor prognosis groups CEA(+) and CXCL16(+), further division was achieved by LGR6 analysis. LGR6(+) patients had a very poor prognosis. LGR6 also identified a small number of CEA(-), TNM stage I patients who relapsed suggesting stem cell origin of these tumors. LGR6 and LGR5 levels correlated strongly in lymph nodes of stage I and IV patients but not in stage II patients, suggesting that these stem cell markers are differentially regulated. Conclusion: This study highlights LGR6 as a useful prognostic biomarker independently and in combination with CEA, CXCL16 or LGR5 identifying different risk groups.

qRT-PCR analysis of CEACAM5, KLK6, SLC35D3, MUC2 and POSTN in colon cancer lymph nodes-An improved method for assessment of tumor stage and prognosis.

One fourth of colorectal cancer patients having curative surgery will relapse of which the majority will die. Lymph node (LN) metastasis is the single most important prognostic factor and a key factor when deciding on postoperative treatment. Presently, LN metastases are identified by histopathological examination, a subjective method analyzing only a small LN volume and giving no information on tumor aggressiveness. To better identify patients at risk of relapse we constructed a qRT-PCR test, ColoNode, that determines levels of CEACAM5, KLK6, SLC35D3, MUC2 and POSTN mRNAs. Combined these biomarkers estimate the tumor cell load and aggressiveness allocating patients to risk categories with low (0, -1), medium (1), high (2) and very high (3) risk of recurrence. Here we present result of a prospective, national multicenter study including 196 colon cancer patients from 8 hospitals. On average, 21 LNs/patient, totally 4698 LNs, were examined by both histopathology and ColoNode. At 3-year follow-up, 36 patients had died from colon cancer or lived with recurrence. ColoNode identified all patients that were identified by histopathology and in addition 9 patients who were undetected by histopathology. Thus, 25% of the patients who recurred were identified by ColoNode only. Multivariate Cox regression analysis proved ColoNode (1, 2, 3 vs 0, -1) as a highly significant risk factor with HR 4.24 [95% confidence interval, 1.42-12.69, P = .01], while pTN-stage (III vs I/II) lost its univariate significance. In conclusion, ColoNode surpassed histopathology by identifying a significantly larger number of patients with future relapse and will be a valuable tool for decisions on postoperative treatment.

Detection of lymph node metastasis in colon cancer by ectopically expressed fibroblast markers FOXQ1 and THBS2.

Introduction: Approximately 25% of colon cancer (CC) patients having curative surgery will relapse. Therefore, it is crucial to identify patients with increased recurrence risk to offer them adjuvant chemotherapy. Three markers with prominent expression in fibroblasts: forkhead box Q1 (FOXQ1), matrix metalloproteinase-11 (MMP11), and thrombospondin-2 (THBS2), and the fibroblast expressed chemokine CXCL12 were selected for studies because of the critical role of fibroblasts in the microenvironment of the tumor. Methods: The expression levels of the biomarkers were assessed in primary CC tumors, lymph nodes of CC patients and controls, and CC cell lines at mRNA and protein levels by real-time qRT-PCR and immunohistochemistry, respectively. Results: FOXQ1, MMP11, and THBS2 mRNAs were expressed at significantly higher levels in primary tumors compared to normal colon (P=0.002, P<0.0001, and P<0.0001, respectively). In contrast, CXCL12 mRNA levels were higher in normal colon tissue. FOXQ1, MMP11, and THBS2 levels were also expressed at significantly higher levels in metastasis-positive lymph nodes compared to both metastasis-negative- and control nodes (P<0.0001/P=0.002, P<0.0001/P<0.0001, and P<0.0001/P<0.0001, respectively). Immuno-morphometry revealed that 30-40% of the tumor cells expressed FOXQ1, MMP11, and THBS2. FOXQ1 and THBS2 were barely detected in normal colon epithelium (P<0.0001), while MMP11 was expressed in normal colon epithelium at high levels. Discussion: We conclude that CC tumor cells show ectopic expression of FOXQ1 and THBS2 possibly making these tumor cells independent of fibroblast cell support. The high expression levels of these two biomarkers in metastatic lymph nodes suggest that they are potential indicators of patients at risk for recurrence.

Prognostic Significance of GPR55 mRNA Expression in Colon Cancer.

G protein-coupled receptor 55 (GPR55) probably plays a role in innate immunity and tumor immunosurveillance through its effect on immune cells, such as T cells and NK cells. In this study, the prognostic value of GPR55 in colon cancer (CC) was investigated. mRNA expression levels of GPR55 were determined in 382 regional lymph nodes of 121 CC patients with 12 years observation time after curative surgery. The same clinical material had previously been analyzed for expression levels of CEA, CXCL16, CXCL17, GPR35 V2/3 and LGR5 mRNAs. Clinical cutoffs of 0.1365 copies/18S rRNA unit for GPR55 and 0.1481 for the GPR55/CEA ratio were applied to differentiate between the high- and low-GPR55 expression groups. Kaplan-Meier survival analysis and Cox regression risk analysis were used to determine prognostic value. Improved discrimination between the two groups was achieved by combining GPR55 with CEA, CXCL16 or CXCL17 compared with GPR55 alone. The best result was obtained using the GPR55/CEA ratio, with an increased mean survival time of 14 and 33 months at 5 and 12 years observation time, respectively (p = 0.0003 and p = 0.003) for the high-GPR55/CEA group. The explanation for the observed improvement is most likely that GPR55 is a marker for T cells and B cells in lymph nodes, whereas CEA, CXCL16 and CXCL17, are markers for tumor cells of epithelial origin.

The myeloid cell biomarker EMR1 is ectopically expressed in colon cancer.

OBJECTIVE: The microenvironment of colon cancer (CC) is heterogeneous including cells of myeloid lineage affecting tumor growth and metastasis. Two functional subtypes of myeloid cells have been identified; one (M1) is tumor-inhibitory and the other one (M2) is tumor-promoting. Whether the three myeloid markers EMR1, CD206 and CD86 are expressed only in the infiltrating myeloid cells or also in the tumor cells was investigated. METHODS: Expression of the myeloid markers was investigated in CC at the mRNA and protein levels in primary tumors and lymph nodes. mRNA expression was also determined in 5 CC cell lines. Protein expression was investigated by two-color immunofluorescence and consecutive-sections-immune-staining combined with morphometry using specific antibodies for the myeloid cell markers and the epithelial cell markers CEACAM5 and EpCAM. RESULTS: EMR1 and CD86, but not CD206, mRNA levels were significantly higher in CC primary tumors compared to apparently normal colon tissue (P <  0.0001). EMR1 mRNA levels were significantly higher in both hematoxylin-eosin positive (H&E(+)) and H&E(-) lymph nodes of CC patients compared to control nodes (P = 0.03 and P = 0.01, respectively). EMR1 and CD206 mRNAs were expressed in 4/5 and 5/5 CC cell lines, respectively, while CD86 mRNA was not expressed. Immuno-morphometry revealed that about 20% of the tumor cells expressed EMR1 and CD206. Positive cells were tumor cells as revealed by anti-CEACAM5 and anti-EpCAM staining. The number of EMR1, CD206 and CD86 positive cells were significantly increased in CC primary tumors compared to normal colon tissue (P <  0.0001). However, CD206 was also expressed in normal colonocytes. Only EMR1 showed significantly increased numbers of positive tumor cells in H&E(+) nodes compared to H&E(-) nodes (P = 0.001). EMR1 expression in CC tumor cells correlated with CXCL17 expressing tumor cells. CONCLUSION: EMR1, like the chemokine CXCL17, is ectopically expressed in colon cancer possibly in the same cancer cells.

CEACAM5, KLK6, SLC35D3, POSTN, and MUC2 mRNA Analysis Improves Detection and Allows Characterization of Tumor Cells in Lymph Nodes of Patients Who Have Colon Cancer.

BACKGROUND: Lymph node metastasis is the single most important prognostic risk factor for recurrence in patients with colon cancer who have undergone curative surgery. The routine method for detecting disseminated tumor cells in lymph nodes is microscopic examination of one or a few hematoxylin and eosin-stained tissue sections by a trained pathologist. This method, however, is insensitive mainly because less than 1% of the lymph node volume is examined, leading to misclassification. OBJECTIVE: This study aimed to investigate whether analysis of a selected group of biomarker mRNAs improves detection and characterization of lymph node metastases/micrometastases compared with the routine method. DESIGN: This study is a side-by-side comparison of biomarker mRNA analysis and histopathology of 185 lymph nodes from patients with colon cancer representing stages I to IV, and an investigation of the importance of lymph node tissue volume for tumor cell detection. SETTINGS: This is a collaborative study between a high-volume central hospital and a preclinical university institution. PATIENTS: Fifty-seven patients who had undergone tumor resection for colon cancer were included. MAIN OUTCOME MEASURES: The primary outcomes measured were mRNA copies per 18S rRNA copy of CEACAM5, KLK6, SLC35D3, POSTN, and MUC2 by multiplex assay and metastases/micrometastases detected by histopathology. RESULTS: The number of tumor cell-positive lymph nodes was 1.33-fold higher based on CEACAM5 mRNA levels compared with histopathological examination. Increasing the tissue volume analyzed for CEACAM5 levels from an 80-µm section to half a lymph node increased the number of positive nodes from 34 of 107 to 80 of 107 (p < 0.0001). Similarly, the number of positive nodes for the aggressiveness marker KLK6 increased from 9 of 107 to 24 of 107. LIMITATIONS: Only a limited number of individual lymph nodes per patient was available for analysis. CONCLUSIONS: mRNA analysis of CEACAM5, KLK6, and SLC35D3 improves the detection of tumor cells in lymph nodes from patients surgically treated for colon cancer, and, together with POSTN and MUC2, it further allows characterization of the tumor cells with respect to aggressiveness and the tumor cell environment. See Video Abstract at http://links.lww.com/DCR/B650. EL ANLISIS DE ARNM DE CEACAM, KLK, SLCD, POSTN Y MUC MEJORA LA DETECCIN Y PERMITE LA CARACTERIZACIN DE CLULAS TUMORALES EN LOS GANGLIOS LINFTICOS DE PACIENTES CON CNCER DE COLON: ANTECEDENTES:Las metástasis en los ganglios linfáticos son el factor de riesgo pronóstico más importante de recurrencia en pacientes con cáncer de colon que se han sometido a cirugía curativa. El método de rutina para detectar células tumorales diseminadas en los ganglios linfáticos es el examen microscópico de una o algunas secciones de tejido teñidas con hematoxilina-eosina por un patólogo capacitado. Sin embargo, este método es insensible principalmente porque se examina menos del 1% del volumen de los ganglios linfáticos, lo que conduce a una clasificación errónea.OBJETIVO:Investigar si el análisis de un grupo seleccionado de ARNm de biomarcadores mejora la detección y caracterización de metástasis / micrometástasis en los ganglios linfáticos en comparación con el método de rutina.DISEÑO:Una comparación en paralelo del análisis de ARNm de biomarcadores y la histopatología de 185 ganglios linfáticos de pacientes con cáncer de colon que representan las etapas I-IV, e investigación de la importancia del volumen de tejido de los ganglios linfáticos para la detección de células tumorales.ENTORNO CLINICO:Estudio colaborativo entre un hospital central de alto volumen y una institución universitaria preclínica.PACIENTES:Cincuenta y siete pacientes que han sido sometidos a resección tumoral por cáncer de colon.PRINCIPALES MEDIDAS DE VALORACION:copias de ARNm / copia de ARNr 18S de CEACAM5, KLK6, SLC35D3, POSTN y MUC2 mediante análisis múltiple y metástasis / micrometástasis detectadas por histopatología.RESULTADOS:El número de ganglios linfáticos con células tumorales positivas fue 1,33 veces mayor según los niveles de ARNm de CEACAM5 en comparación con el examen histopatológico. El aumento del volumen de tejido analizado para los niveles de CEACAM5 de una sección de 80 µm a la mitad de un ganglio linfático aumentó el número de ganglios positivos de 34/107 a 80/107 (p <0,0001). De manera similar, el número de nodos positivos para el marcador de agresividad KLK6 aumentó de 9/107 a 24/107.LIMITACIONES:Solo un número limitado de ganglios linfáticos individuales / paciente estuvo disponible para el análisis.CONCLUSIONES:El análisis de ARNm de CEACAM5, KLK6 y SLC35D3 mejora la detección de células tumorales en los ganglios linfáticos de pacientes con cáncer de colon tratados quirúrgicamente y, junto con POSTN y MUC2, permite además la caracterización de las células tumorales con respecto a la agresividad y el entorno celular tumoral. Consulte Video Resumen en http://links.lww.com/DCR/B650.

Clinical Significance of Stem Cell Biomarkers EpCAM, LGR5 and LGR4 mRNA Levels in Lymph Nodes of Colon Cancer Patients.

The significance of cancer stem cells (CSCs) in initiation and progression of colon cancer (CC) has been established. In this study, we investigated the utility of measuring mRNA expression levels of CSC markers EpCAM, LGR5 and LGR4 for predicting survival outcome in surgically treated CC patients. Expression levels were determined in 5 CC cell lines, 66 primary CC tumors and 382 regional lymph nodes of 121 CC patients. Prognostic relevance was determined using Kaplan-Meier survival and Cox regression analyses. CC patients with lymph nodes expressing high levels of EpCAM, LGR5 or LGR4 (higher than a clinical cutoff of 0.07, 0.06 and 2.558 mRNA copies/18S rRNA unit, respectively) had a decreased mean survival time of 32 months for EpCAM and 42 months for both LGR5 and LGR4 at a 12-year follow-up (p = 0.022, p = 0.005 and p = 0.011, respectively). Additional patients at risk for recurrence were detected when LGR5 was combined with the biomarkers CXCL17 or CEA plus CXCL16. In conclusion, the study underscores LGR5 as a particularly useful prognostic biomarker and illustrates the strength of combining biomarkers detecting different subpopulations of cancer cells and/or cells in the tumor microenvironment for predicting recurrence.

Evaluating macrophage migration inhibitory factor 1 expression as a prognostic biomarker in colon cancer.

OBJECTIVE: Several studies indicate that macrophage migration inhibitory factor 1 plays a role for tumor progression in colon cancer. We investigated whether determination of migration inhibitory factor 1 mRNA expression levels in lymph nodes of colon cancer patients could be used as a prognostic marker. METHODS: Expression levels of migration inhibitory factor 1 and carcinoembryonic antigen mRNAs were assessed in primary tumors and regional lymph nodes of 123 colon cancer patients (stages I-IV), and in colon cancer- and immune cell lines using quantitative reverse transcriptase-polymerase chain reaction. Expression of migration inhibitory factor 1 protein was investigated by two-color immunohistochemistry and immunomorphometry. RESULTS: Migration inhibitory factor 1 mRNA was expressed at 60 times higher levels in primary colon cancer tumors compared to normal colonic tissue (medians 8.2 and 0.2 mRNA copies/18S rRNA unit; p < .0001). A highly significant difference in mRNA expression levels was found between hematoxylin-eosin positive lymph nodes and hematoxylin-eosin negative lymph nodes (p < .0001). Migration inhibitory factor 1 and carcinoembryonic antigen proteins were simultaneously expressed in many colon cancer-tumor cells. Kaplan-Meier survival model and hazard ratio analysis, using a cutoff level at 2.19 mRNA copies/18S rRNA unit, revealed that patients with lymph nodes expressing high levels of migration inhibitory factor 1 mRNA had a 3.5-fold (p = .04) higher risk for recurrence, associated with a small, but significant, difference in mean survival time (7 months, p = .03) at 12 years of follow-up. CONCLUSION: Although migration inhibitory factor 1 mRNA expression levels were related to severity of disease and lymph node analysis revealed that colon cancer patients with high levels had a shorter survival time after surgery than those with low levels, the difference was small and probably not useful in clinical practice.

Allocating colorectal cancer patients to different risk categories by using a five-biomarker mRNA combination in lymph node analysis.

BACKGROUND AND AIMS: Curative surgery saves ≈50% of all patients with colorectal cancer (CRC) while remaining patients have synchronous or will develop metachronous metastases. Presently, the single most important prognostic factor is histopathological detection of disseminated tumor cells in regional lymph nodes. However, the routine method has several limitations. The aim was to identify biomarker mRNAs that could be combined in a formula that would allow better prediction of patients' survival after surgery. METHODS: Screening for biomarker mRNAs overexpressed in CRC was performed by genome-wide hybridization bead array, with verification by qRT-PCR. Specific qRT-PCR assays with copy standards were developed for 5 selected genes and mRNA expression levels determined in lymph nodes from 174 CRC patients (517 nodes) and 24 control patients (118 nodes). Prognostic value of biomarker mRNAs was estimated. A cut-off was set using univariate Cox regression analysis and used for calculation of differences between patient groups in disease-free survival 12 years after surgery (Kaplan-Meier survival model) and risk for recurrent disease (Cox's regression analysis). A formula was constructed for evaluation of the prognostic value of the biomarkers in combination. RESULTS: Two new biomarkers, SLC35D3 and POSTN with prognostic value were identified. SLC35D3 was expressed in the epithelium derived tumor cells and POSTN in fibroblasts. Combined with CEACAM5, KLK6 and MUC2 they could be used to identify risk groups. A formula was constructed using CEACAM5 as denominator for KLK6, SLC35D3 and MUC2 and 18S rRNA as denominator for POSTN. The formula yielded 5 categories (-1, 0, 1, 2, 3). Categories (-1 and 0) had good prognosis, categories (1 and 2) relatively poor prognosis and category (3) very poor prognosis. CONCLUSION: Lymph node analysis using 5 selected biomarker mRNAs and 18S rRNA in combination allowed allocation of CRC patients to different risk categories with respect to recurrent disease.

The Chemokine CXCL16 Is a New Biomarker for Lymph Node Analysis of Colon Cancer Outcome.

Chemokines are important in the development and progression of tumors. We investigated the expression of CXCL14 and CXCL16 in colon cancer. Expression of mRNA was assessed in primary tumors and lymph nodes and CXCL16 mRNA levels were correlated to patient's survival. Protein expression was investigated by two-color immunofluorescence and immunomorphometry. CXCL14 and CXCL16 mRNA levels and protein expression were significantly higher in colon cancer primary tumors compared to apparently normal colon tissue. Positive cells were tumor cells, as revealed by anti-CEA and anti-EpCAM staining. CXCL16, but not CXCL14, mRNA levels were significantly higher in hematoxylin and eosin positive (H&E(+)) compared to H&E(-) colon cancer lymph nodes or control nodes (P < 0.0001). CXCL16 mRNA was expressed in 5/5 colon cancer cell lines while CXCL14 was expressed significantly in only one. Kaplan-Meier analysis revealed that colon cancer patients with lymph nodes expressing high or very high levels (7.2 and 11.4 copies/18S rRNA unit, respectively) of CXCL16 mRNA had a decreased mean survival time of 30 and 46 months at the 12-year follow-up (P = 0.04, P = 0.005, respectively). In conclusion, high expression of CXCL16 mRNA in regional lymph nodes of colon cancer patients is a sign of a poor prognosis.

Utility of G protein-coupled receptor 35 expression for predicting outcome in colon cancer.

The utility of mRNA and protein determinations of G protein-coupled receptor 35, that is, GPR35a (GPR35 V1) and GPR35b (GPR35 V2/3), as indicators of outcome for colon cancer patients after curative surgery was investigated. Expression levels of V1 and V2/3 GPR35, carcinoembryonic antigen and CXCL17 mRNAs were assessed in primary tumours and regional lymph nodes of 121 colon cancer patients (stage I-IV), colon cancer cell lines and control colon epithelial cells using real-time quantitative reverse transcriptase-polymerase chain reaction. Expression of G protein-coupled receptor 35 was investigated by two-colour immunohistochemistry and immunomorphometry. GPR35 V2/3 mRNA, but not V1 mRNA, was expressed in colon cancer cell lines, primary colon tumours and control colon epithelial cells. Haematoxylin and eosin positive (H&E(+)), but not H&E(-), lymph nodes expressed high levels of GPR35 V2/3 mRNA (P<0.0001). GPR35b and carcinoembryonic antigen proteins were simultaneously expressed in many colon cancer tumour cells. Kaplan-Meier and hazard ratio analysis revealed that patients with lymph nodes expressing high levels of GPR35 V2/3 mRNA and, in particular, in the group of patients with lymph nodes also expressing carcinoembryonic antigen mRNA, had a short disease-free survival time, 67 months versus 122 months at 12-year follow-up (difference: 55 months, P = 0.001; hazard ratio: 3.6, P = 0.002). In conclusion, high level expression of G protein-coupled receptor 35 V2/3 mRNA in regional lymph nodes of colon cancer patients is a sign of poor prognosis.

Lymph node CXCL17 messenger RNA: A new prognostic biomarker for colon cancer.

Lymph node metastasis is the most important prognostic characteristic of colorectal cancer. Carcinoembryonic antigen messenger RNA was shown to detect tumor cells that have disseminated to lymph nodes of colorectal cancer patients and to be at least as good as the hematoxylin and eosin method to predict survival in colorectal cancer patients. CXCL17 was recently shown to be ectopically expressed in colon cancer tumors. Therefore, CXCL17 may serve as prognostic marker alone or in combination with carcinoembryonic antigen. CXCL17 and carcinoembryonic antigen messenger RNA levels were determined using quantitative reverse transcription polymerase chain reaction with RNA copy standard in 389 lymph nodes of 120 colon cancer patients (stages I-IV) and 67 lymph nodes of 12 control patients with inflammatory bowel disease as well as in 68 primary tumors and 30 normal colon tissue samples. Lymph nodes of colon cancer patients were analyzed for CXCL17 and carcinoembryonic antigen protein expression by immunohistochemistry. CXCL17 messenger RNA was expressed in primary tumors at high levels, while it was barely detected in normal colon tissue ( p < 0.0001). Similarly, CXCL17 messenger RNA levels were significantly higher in hematoxylin- and eosin-positive (hematoxylin and eosin (+)) lymph nodes compared to hematoxylin- and eosin-negative nodes ( p < 0.0001). CXCL17 messenger RNA levels were investigated in lymph nodes grouped according to carcinoembryonic antigen messenger RNA levels: low (-), intermediate (int), and high (+). CXCL17 messenger RNA levels were higher in the carcinoembryonic antigen (int) and carcinoembryonic antigen (+) groups compared to the carcinoembryonic antigen (-) group ( p = 0.03 and p < 0.0001, respectively). In lymph nodes of stage III and IV patients, CXCL17 messenger RNA levels correlated with carcinoembryonic antigen messenger RNA levels ( p < 0.0001, r = 0.56 and p = 0.0002, r = 0.66, respectively). Staining of consecutive lymph node sections for CXCL17 and carcinoembryonic antigen demonstrated that the same cells expressed both proteins. Altogether, these results indicate that CXCL17 in lymph nodes is expressed by tumor cells. Patients were grouped according to the CXCL17 messenger RNA levels in the highest lymph node with low levels (-) and high levels (+). CXCL17(+) colon cancer patients showed 2.8-3.6 fold increased risk for recurrence ( p = 0.03) and decreased mean disease-free survival time of 8 months compared to CXCL17(-) colon cancer patients ( p = 0.03). CXCL17(+) carcinoembryonic antigen (int) colon cancer patients showed increased risk for recurrence by 8.3 fold ( p = 0.04) and decreased mean disease-free survival time of 46 months compared to CXCL17(-) carcinoembryonic antigen (int) colon cancer patient at follow-up after 12 years ( p = 0.02). The presence of tumor cells expressing CXCL17 in regional lymph nodes is a sign of poor prognosis. Analysis of CXCL17 messenger RNA is particularly useful to detect less differentiated colon cancer tumors expressing relatively low carcinoembryonic antigen messenger RNA levels. Thus, CXCL17 messenger RNA in combination with carcinoembryonic antigen messenger RNA may be used as a complementary tool to the hematoxylin and eosin method for detection of poorly differentiated, aggressive tumors.

Immunopathology of childhood celiac disease-Key role of intestinal epithelial cells.

BACKGROUND & AIMS: Celiac disease is a chronic inflammatory disease of the small intestine mucosa due to permanent intolerance to dietary gluten. The aim was to elucidate the role of small intestinal epithelial cells in the immunopathology of celiac disease in particular the influence of celiac disease-associated bacteria. METHODS: Duodenal biopsies were collected from children with active celiac disease, treated celiac disease, and clinical controls. Intestinal epithelial cells were purified and analyzed for gene expression changes at the mRNA and protein levels. Two in vitro models for human intestinal epithelium, small intestinal enteroids and polarized tight monolayers, were utilized to assess how interferon-γ, interleukin-17A, celiac disease-associated bacteria and gluten influence intestinal epithelial cells. RESULTS: More than 25 defense-related genes, including IRF1, SPINK4, ITLN1, OAS2, CIITA, HLA-DMB, HLA-DOB, PSMB9, TAP1, BTN3A1, and CX3CL1, were significantly upregulated in intestinal epithelial cells at active celiac disease. Of these genes, 70% were upregulated by interferon-γ via the IRF1 pathway. Most interestingly, IRF1 was also upregulated by celiac disease-associated bacteria. The NLRP6/8 inflammasome yielding CASP1 and biologically active interleukin-18, which induces interferon-γ in intraepithelial lymphocytes, was expressed in intestinal epithelial cells. CONCLUSION: A key factor in the epithelial reaction in celiac disease appears to be over-expression of IRF1 that could be inherent and/or due to presence of undesirable microbes that act directly on IRF1. Dual activation of IRF1 and IRF1-regulated genes, both directly and via the interleukin-18 dependent inflammasome would drastically enhance the inflammatory response and lead to the pathological situation seen in active celiac disease.

Ectopic expression of the chemokine CXCL17 in colon cancer cells.

BACKGROUND: The novel chemokine CXCL17 acts as chemoattractant for monocytes, macrophages and dendritic cells. CXCL17 also has a role in angiogenesis of importance for tumour development. METHODS: Expression of CXCL17, CXCL10, CXCL9 and CCL2 was assessed in primary colon cancer tumours, colon carcinoma cell lines and normal colon tissue at mRNA and protein levels by real-time qRT-PCR, immunohistochemistry, two-colour immunofluorescence and immunomorphometry. RESULTS: CXCL17 mRNA was expressed at 8000 times higher levels in primary tumours than in normal colon (P < 0.0001). CXCL17 protein was seen in 17.2% of cells in tumours as compared with 0.07% in normal colon (P = 0.0002). CXCL10, CXCL9 and CCL2 mRNAs were elevated in tumours but did not reach the levels of CXCL17. CXCL17 and CCL2 mRNA levels were significantly correlated in tumours. Concordant with the mRNA results, CXCL10- and CXCL9-positive cells were detected in tumour tissue, but at significantly lower numbers than CXCL17. Two-colour immunofluorescence and single-colour staining of consecutive sections for CXCL17 and the epithelial cell markers carcinoembryonic antigen and BerEP4 demonstrated that colon carcinoma tumour cells indeed expressed CXCL17. CONCLUSIONS: CXCL17 is ectopically expressed in primary colon cancer tumours. As CXCL17 enhances angiogenesis and attracts immune cells, its expression could be informative for prognosis in colon cancer patients.

Noncontaminated dietary oats may hamper normalization of the intestinal immune status in childhood celiac disease.

OBJECTIVES: Life-long, strict gluten-free diet (GFD) is the only treatment for celiac disease (CD). Because there is still uncertainty regarding the safety of oats for CD patients, the aim was to investigate whether dietary oats influence the immune status of their intestinal mucosa. METHODS: Paired small intestinal biopsies, before and after >11 months on a GFD, were collected from children with CD who were enrolled in a randomized, double-blind intervention trial to either of two diets: standard GFD (GFD-std; n=13) and noncontaminated oat-containing GFD (GFD-oats; n=15). Expression levels of mRNAs for 22 different immune effector molecules and tight junction proteins were determined by quantitative reverse transcriptase (RT)-PCR. RESULTS: The number of mRNAs that remained elevated was higher in the GFD-oats group (P=0.05). In particular, mRNAs for the regulatory T cell (Treg) signature molecules interleukin-10 (IL-10) and transforming growth factor-ß1 (TGF-ß1), the cytotoxicity-activating natural killer (NK) receptors KLRC2/NKG2C and KLRC3/NKG2E, and the tight junction protein claudin-4 remained elevated. Between the two groups, most significant differences were seen for claudin-4 (P=0.003) and KLRC3/NKG2E (P=0.04). CONCLUSIONS: A substantial fraction of pediatric CD patients seem to not tolerate oats. In these patients, dietary oats influence the immune status of the intestinal mucosa with an mRNA profile suggesting presence of activated cytotoxic lymphocytes and Tregs and a stressed epithelium with affected tight junctions. Assessment of changes in levels of mRNA for claudin-4 and KLC3/NKG2E from onset to after a year on oats containing GFD shows promise to identify these CD patients.

Prevotella jejuni sp. nov., isolated from the small intestine of a child with coeliac disease.

Five obligately anaerobic, Gram-stain-negative, saccharolytic and proteolytic, non-spore-forming bacilli (strains CD3 : 27, CD3 : 28(T), CD3 : 33, CD3 : 32 and CD3 : 34) are described. All five strains were isolated from the small intestine of a female child with coeliac disease. Cells of the five strains were short rods or coccoid cells with longer filamentous forms seen sporadically. The organisms produced acetic acid and succinic acid as major metabolic end products. Phylogenetic analysis based on comparative 16S rRNA gene sequence analysis revealed close relationships between CD3 : 27, CD3 : 28(T) and CD3 : 33, between CD3 : 32 and Prevotella histicola CCUG 55407(T), and between CD3 : 34 and Prevotella melaninogenica CCUG 4944B(T). Strains CD3 : 27, CD3 : 28(T) and CD3 : 33 were clearly different from all recognized species within the genus Prevotella and related most closely to but distinct from P. melaninogenica. Based on 16S rRNA, RNA polymerase ß-subunit (rpoB) and 60 kDa chaperonin protein subunit (cpn60) gene sequencing, and phenotypic, chemical and biochemical properties, strains CD3 : 27, CD3 : 28(T) and CD3 : 33 are considered to represent a novel species within the genus Prevotella, for which the name Prevotella jejuni sp. nov. is proposed. Strain CD3 : 28(T) ( = CCUG 60371(T) = DSM 26989(T)) is the type strain of the proposed novel species. All five strains were able to form homologous aggregates, in which tube-like structures were connecting individual bacteria cells. The five strains were able to bind to human intestinal carcinoma cell lines at 37 °C.

Intestinal T-cell responses in celiac disease - impact of celiac disease associated bacteria.

A hallmark of active celiac disease (CD), an inflammatory small-bowel enteropathy caused by permanent intolerance to gluten, is cytokine production by intestinal T lymphocytes. Prerequisites for contracting CD are that the individual carries the MHC class II alleles HLA-DQ2 and/or HLA-DQ8 and is exposed to gluten in the diet. Dysbiosis in the resident microbiota has been suggested to be another risk factor for CD. In fact, rod shaped bacteria adhering to the small intestinal mucosa were frequently seen in patients with CD during the "Swedish CD epidemic" and bacterial candidates could later be isolated from patients born during the epidemic suggesting long-lasting changes in the gut microbiota. Interleukin-17A (IL-17A) plays a role in both inflammation and anti-bacterial responses. In active CD IL-17A was produced by both CD8(+) T cells (Tc17) and CD4(+) T cells (Th17), with intraepithelial Tc17 cells being the dominant producers. Gluten peptides as well as CD associated bacteria induced IL-17A responses in ex vivo challenged biopsies from patients with inactive CD. The IL-17A response was suppressed in patients born during the epidemic when a mixture of CD associated bacteria was added to gluten, while the reverse was the case in patients born after the epidemic. Under these conditions Th17 cells were the dominant producers. Thus Tc17 and Th17 responses to gluten and bacteria seem to pave the way for the chronic disease with interferon-γ-production by intraepithelial Tc1 cells and lamina propria Th1 cells. The CD associated bacteria and the dysbiosis they might cause in the resident microbiota may be a risk factor for CD either by directly influencing the immune responses in the mucosa or by enhancing inflammatory responses to gluten.

Lachnoanaerobaculum gen. nov., a new genus in the Lachnospiraceae: characterization of Lachnoanaerobaculum umeaense gen. nov., sp. nov., isolated from the human small intestine, and Lachnoanaerobaculum orale sp. nov., isolated from saliva, and reclassification of Eubacterium saburreum (Prevot 1966) Holdeman and Moore 1970 as Lachnoanaerobaculum saburreum comb. nov.

Two novel obligately anaerobic, Gram-stain-positive, saccharolytic and non-proteolytic spore-forming bacilli (strains CD3:22(T) and N1(T)) are described. Strain CD3:22(T) was isolated from a biopsy of the small intestine of a child with coeliac disease, and strain N1(T) from the saliva of a healthy young man. The cells of both strains were observed to be filamentous, approximately 5 to >20 µm long, some of them curving and with swellings. The novel organisms produced H(2)S, NH(3), butyric acid and acetic acid as major metabolic end products. Phylogenetic analyses, based on comparative 16S rRNA gene sequencing, revealed close relationships (98% sequence similarity) between the two isolates, as well as the type strain of Eubacterium saburreum and four other Lachnospiraceae bacterium-/E. saburreum-like organisms. This group of bacteria were clearly different from any of the 19 known genera in the family Lachnospiraceae. While Eubacterium species are reported to be non-spore-forming, reanalysis of E. saburreum CCUG 28089(T) confirmed that the bacterium is indeed able to form spores. Based on 16S rRNA gene sequencing, phenotypic and biochemical properties, strains CD3:22(T) and N1(T) represent novel species of a new and distinct genus, named Lachnoanaerobaculum gen. nov., in the family Lachnospiraceae [within the order Clostridiales, class Clostridia, phylum Firmicutes]. Strain CD3:22(T) (=CCUG 58757(T) =DSM 23576(T)) is the type strain of the type species, Lachnoanaerobaculum umeaense gen. nov., sp. nov., of the proposed new genus. Strain N1(T) (=CCUG 60305(T)=DSM 24553(T)) is the type strain of Lachnoanaerobaculum orale sp. nov. Moreover, Eubacterium saburreum is reclassified as Lachnoanaerobaculum saburreum comb. nov. (type strain CCUG 28089(T) =ATCC 33271(T) =CIP 105341(T) =DSM 3986(T) =JCM 11021(T) =VPI 11763(T)).

Lymph node CEA and MUC2 mRNA as useful predictors of outcome in colorectal cancer.

The aim was to explore the utility for staging and prognostic impact of carcinoembryonic antigen (CEA), cytokeratin 20 (CK20), guanylyl cyclase C (GCC), CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor, and bone morphogenic protein 1) containing domain protein 1 (CDCP1) and mucin 2 (MUC2) mRNA levels in mesenteric lymph nodes of colorectal cancer (CRC) patients. Lymph nodes were collected at surgery and bisected; one half was subjected to biomarker mRNA analysis using real-time quantitative RT-PCR and the other half to routine histopathology. Lymph nodes from 174 CRC patients and 24 controls were analyzed. The median follow-up time was 59 (range 17-131) months. Cut-off levels were defined by analyzing quintiles by Cox regression model. CEA mRNA showed the best discriminating power between patients with recurrence in CRC after surgery and patients who were apparently disease-free (p = 0.015). The risk of recurrence for the CEA(+) patients was 4.6 times greater than for the CEA(-) patients (p < 0.0001). The other biomarkers gave lower hazard ratios. Cumulative survival analysis demonstrated that the average survival time was 99 months for CEA(-) patients compared to 39 months for CEA(+) patients, a difference of 60 months (p < 0.0001). Six to nine percent of the Stage I and Stage II patients [H&E(-)] had CEA(+), CK20(+), GCC(+) and/or MUC2(+) lymph nodes. Two of these patients died from recurrent CRC. Low lymph node MUC2/CEA mRNA ratio identified patients with high risk for recurrence (p = 0.011). Thus, quantitative reverse transcriptase-polymerase chain reaction of CEA mRNA is a sensitive method to identify tumor cells in lymph nodes of CRC patients and, in combination with MUC2 mRNA, allows improved prediction of clinical outcome.