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.

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.

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.

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.

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.

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.

Paradoxical coexpression of proinflammatory and down-regulatory cytokines in intestinal T cells in childhood celiac disease.

BACKGROUND & AIMS: Specific T-lymphocyte reactions are central in the pathogenesis of celiac disease, an inflammatory small-bowel enteropathy caused by a permanent intolerance to gluten. To delineate local T-lymphocyte responses to gluten, the cytokine expression in jejunal T lymphocytes of pediatric celiac patients with active disease, i.e., untreated and gluten-challenged celiac patients, was determined and compared with that of treated, symptom-free celiac patients and controls. METHODS: Biopsy samples were collected from celiac patients and controls. Intraepithelial and lamina propria T lymphocytes were isolated separately, and the cytokine messenger RNA levels were determined by using quantitative real-time reverse-transcription polymerase chain reaction. Interferon (IFN)-gamma and interleukin (IL)-10 were determined at the protein level by immunohistochemistry. RESULTS: Active celiac disease was characterized by distortions in cytokine expression by T lymphocytes, with highly significant increases of IFN-gamma and IL-10 but no concomitant increases in tumor necrosis factor alpha, transforming growth factor beta1, or IL-2 and no induction of IL-4. A marked shift of IFN-gamma and IL-10 production from the lamina propria to the epithelium was characteristic of active celiac disease, and as many as one fourth of the intraepithelial lymphocytes expressed IFN-gamma. Intraepithelial T lymphocytes in treated, symptom-free celiac patients still had increased IFN-gamma levels compared with controls. CONCLUSIONS: In celiac patients, gluten intake seems to cause an overreaction in intraepithelial T lymphocytes, with uncontrolled production of IFN-gamma and IL-10. This may cause both recruitment of intraepithelial lymphocytes and a leaky epithelium, leading to a vicious circle with amplified immune activity and establishment of intestinal lesions.

Detection of occult tumour cells in lymph nodes of colorectal cancer patients using real-time quantitative RT-PCR for CEA and CK20 mRNAS.

The purpose of our study was to develop specific, sensitive, objective assays for early detection of disseminated tumour cells in patients with colorectal cancer (CRC). Carcinoembryonic antigen (CEA) and cytokeratin 20 (CK20) were chosen as markers because they are selectively expressed in epithelial cells with maintained expression in CRC. Real-time quantitative RT-PCR assays with RNA copy standards were constructed. Regional lymph nodes were collected from patients with CRC (n = 51) and benign intestinal disease (n = 10). Results were compared to routine histopathology and anti-CEA immunohistochemistry. Lymph node levels of CEA and CK20 mRNA correlated strongly (p < 0.0001, r = 0.8). Lymph nodes from non-CRC patients had <0.01 CEA and <0.001 CK20 mRNA copies/18S rRNA unit. Lymph nodes from 3/6 Dukes' A, 17/26 Dukes' B, 10/10 Dukes' C and 7/9 Dukes' D patients had CEA mRNA levels above cut-off. Corresponding figures for CK20 mRNA were 3/6, 10/26, 9/10 and 5/9, respectively. CEA mRNA levels varied from 0.001 to 100 copies/18S rRNA unit in Dukes' A and B, and 50% of the Dukes' B patients had CEA mRNA levels within the range of Dukes' C patients. Three Dukes' B patients have died from CRC or developed distant metastases. All 3 had high CEA and CK20 mRNA levels. Determination of mRNA was superior to immunohistochemistry in showing CEA expression in lymph nodes. The present qRT-PCR assay for CEA mRNA seems to be a superior tool to identify individuals with disseminated tumour cells. Future extended studies will establish the clinically most relevant cut-off level.