Anne Israelsson

Identification of three new genes and estimation of the size of the carcinoembryonic antigen family

Using carcinoembryonic antigen (CEA) subgroup-specific degenerate PCR primers, we have identified three new CEA gene family member L/N exons (CGM9, CGM10, and CGM11) and all previously reported L/N exons of the CEA subgroup (CEA, BGP, NCA, CGM1, CGM2, CGM6, CGM7, and CGM8). This suggests that the CEA subgroup contains 11 genes. CGM9, CGM10, and CGM11 seem to be pseudogenes. A deletion of an asparagine in CGM9 results in loss of a glycosylation site, which is conserved throughout the CEA gene family. We have previously suggested the number of genes in the pregnancy-specific glycoprotein (PSG) subgroup to be 11, which together with this study indicates that the CEA gene family contains 22 genes in all. Parsimony analysis of the CEA subgroup interrelationships suggests that CGM7 occupies the most primitive position within the CEA subgroup, being a sister group to the rest. CEA, BGP, NCA, and CGM1 form a fairly well-supported group within the CEA subgroup.

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 : 28T, 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 : 28T and CD3 : 33, between CD3 : 32 and Prevotella histicola CCUG 55407T, and between CD3 : 34 and Prevotella melaninogenica CCUG 4944BT. Strains CD3 : 27, CD3 : 28T 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 β-subunit (rpoB) and 60 kDa chaperonin protein subunit (cpn60) gene sequencing, and phenotypic, chemical and biochemical properties, strains CD3 : 27, CD3 : 28T 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 : 28T ( = CCUG 60371T = DSM 26989T) 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.

Evolution of the carcinoembryonic antigen family. structures of CGM9, CGM11 and pregnancy-specific glycoprotein promoters.

Earlier studies have demonstrated that the genes of the human carcinoembryonic antigen (CEA) family can be divided into three subgroups, the CEA subgroup (n = 12), the pregnancy-specific glycoprotein (PSG) subgroup (n = 11), and a third subgroup (n = 6). To further characterize the CEA gene family, we have determined the genomic structures of CGM9 and CGM11, analyzed the promoter regions of all eleven PSGs, studied the CGM15-PSG13 intergenic region and the evolutionary relationships beween the CEA family genes. CGM9, a typical CEA subgroup member, was a pseudogene with the exon structure [5′UTR-L-L/N-TM-Cyt-3′UTR]. CGM11 contained a mixture of exons derived from CEA and PSG subgroup genes. The formula of the CGM11 pseudogene was [5′UTR-L-L/N-C-3′UTR]. Thus both genes lacked the IgC2-like domains typically found in CEA subfamily members. The upstream promoter regions of all eleven PSGs were characterized. All PSG promoters lacked the classical TATA and CCAAT elements, but had putative PEA3 box(es), CACCC box(es), a RARE box, and poly (dG-dT) repeats of different lengths. Five PSGs also had an SP1 site. The complete 10-kb intergenic region between CGM15 and PSG13 was sequenced. Clusters of different types of repetitive sequences were seen. The time of divergence of the CEA and PSG subfamilies was estimated to be 107.7 ± 17.1 million years, or at about the time of human-rodent divergence. Models for the evolution of CEA and PSG and the third family subgroup genes are proposed.

Lymph node tissue kallikrein-related peptidase 6 mRNA : a progression marker for colorectal cancer.

Background:A most important characteristic feature for poor prognosis in colorectal cancer (CRC) is the presence of lymph node metastasis. Determination of carcinoembryonic antigen (CEA) mRNA levels in lymph nodes has proven powerful for quantification of disseminated tumour cells. Here, we investigate the utility of human tissue kallikrein-related peptidase 6 (KLK6) mRNA as a progression biomarker to complement CEA mRNA, for improved selection of patients in need of adjuvant therapy and intensified follow-up after surgery.Methods:Lymph nodes of pTNM stage I-IV CRC- (166 patients/503 lymph nodes) and control (23/108) patients were collected at surgery and analysed by quantitative RT–PCR.Results:Lymph node KLK6 positivity was an indicator of poor outcome (hazard ratio 3.7). Risk of recurrence and cancer death increased with KLK6 lymph node levels. Patients with KLK6 lymph node levels above the 90th percentile had a hazard ratio of 6.5 and 76 months shorter average survival time compared to patients with KLK6 negative nodes. The KLK6 positivity in lymph nodes with few tumour cells, that is, low CEA mRNA levels, also indicated poor prognosis (hazard ratio 2.8).Conclusion:In CRC patients, lymph node KLK6 positivity indicated presence of aggressive tumour cells associated with poor prognosis and high risk of tumour recurrence.

Detection of Tumor Cells in Lymph Nodes of Colon Cancer Patients Using Real-Time Quantitative Reverse Transcription-Polymerase Chain Reaction

Colorectal cancer is ranked third in worldwide incidence for women and fourth for men representing 9% of the world cancer or approximately 1 million new cases for 2002 (Parkin et al., 2005). Two ...

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 mRNA levels in the highest lymph node with low levels (−) and high levels (+). CXCL17(+) CC patients showed 2.2 fold increased risk for recurrence (P = 0.03) and decreased mean disease-free survival time of 33 months compared to CXCL17(−) CC 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.