Benno Mann

Quantitative real‐time RT‐PCR for detection of disseminated tumor cells in peripheral blood of patients with colorectal cancer using different mRNA markers

The detection of disseminated tumor cells in peripheral blood from colorectal cancer patients by RT‐PCR could be an attractive method for selecting patients for adjuvant therapy. We here report on real‐time RT‐PCR assays (LightCycler) to quantitate potential mRNA markers. We investigated specimens from colon carcinoma and normal colon mucosa tissues, cell lines, blood samples from 129 patients with colorectal cancer (all stages) and 58 reference blood samples (healthy donors, persons suffering from inflammatory bowel or infectious diseases). The expression profile in tissues showed high values for CEA and CK20, whereas in cell lines ProtM was predominant. All markers were detected in reference and patient blood samples (ProtM, 22, 17%; CEA, 84, 86%; CK20, 85, 88%). After quantitative analysis, the definition of cutoff values for each marker and the combination of markers, 13% of patients were judged to have elevated marker concentrations in their blood, from which only 6 had values significantly differing from cutoff value. There were no differences between stages of disease. In the case of 19 patients, investigated prior to and 1 week after surgery, 2 samples revealed a significant postoperative increase in CEA or CK20 mRNA concentration. In spite of high expression levels in tissues and cell lines, we were not able to differentiate satisfyingly mRNA markers originating from tumor cells and those from illegitimate transcription in hematopoetic cells in blood. We conclude that either copy numbers of analyzed markers in circulating tumor cells are not sufficient for detection or, more probably, peripheral blood is not a suitable compartment for detection of tumor cells in colorectal cancer.

Differential gene expression in colon carcinoma cells and tissues detected with a cDNA array.

Expression of selected genes coding for proteins with defined cellular functions was analysed in human cell lines derived from normal colonic mucosa, non‐mucinous colonic carcinomas and mucinous colonic carcinomas. Altered expression of 10 genes in colon carcinoma cells was found by using a cDNA array; 6 of these alterations (60%) were confirmed by Northern blotting or semi‐quantitative reverse transcription‐polymerase chain reaction (RT‐PCR). Among these 6 genes, 3 transcription factors as well as the topoisomerase II α and the mitosis inhibitor WEE1Hu gene were significantly suppressed in the tumour cell lines. In addition, the gene coding for the cell cycle inhibitor p21 was overexpressed only in cell lines derived from mucinous carcinomas. The significant suppression of the kinase WEE1Hu gene in carcinoma cells of both phenotypes and the tendency of the mucinous phenotype to overexpress p21 protein were confirmed in human colon carcinoma tissues. Our data show that the cDNA array method permits a correct identification of changes in gene expression with a relatively high accuracy. The different expression of the p21 gene in the non‐mucinous and mucinous carcinoma cells supports the hypothesis that these phenotypes may develop along different genetic pathways. The detection of WEE1Hu gene suppression in colon carcinoma cells and tissues suggests its potential role in tumourigenesis. Int. J. Cancer 82:868–874, 1999.

Differential expression of genes encoding tight junction proteins in colorectal cancer: frequent dysregulation of claudin-1, -8 and -12

Background and aimsAs integral membrane proteins, claudins form tight junctions together with occludin. Several claudins were shown to be up-regulated in various cancer types. We performed an expression analysis of genes encoding tight junction proteins to display differential gene expression on RNA and protein level and to identify and validate potential targets for colorectal cancer (CRC) therapy.Patients and methodsAmplified and biotinylated cRNA from 30 microdissected CRC specimen and corresponding normal tissues was hybridized to Affymetrix U133set GeneChips. Quantification of differential protein expression of claudin-1, -8 and -12 between normal and corresponding tumour tissues was performed by Western blot analyses. Paraffin-embedded CRC tissue samples, colon cancer cell lines and normal tissue microarray were analysed for protein expression of claudin-1 by immunohistochemistry (IHC).ResultsClaudin-1 (CLDN1) and -12 (CLDN12) are frequently overexpressed in CRC, whereas claudin-8 (CLDN8) shows down-regulation in tumour tissue on RNA level. Quantification of proteins confirmed the overexpression of claudin-1 in tumour tissues, whereas changes of claudin-8 and -12 were not significantly detectable on protein level. IHC confirmed the markedly elevated expression level of claudin-1 in the majority of CRC, showing membranous and intracellular vesicular staining.ConclusionsDifferential expression of genes encoding claudins in CRC suggests that these tight junction proteins may be associated to and involved in tumorigenesis. CLDN1 is frequently up-regulated in large proportion of CRC and may represent potential target molecule for blocking studies in CRC.

Low O-acetylation of sialyl-LEx contributes to its overexpression in colon carcinoma metastases

Two factors potentially determining the consistent over‐expression of sialyl‐Lex antigen in colon carcinoma and metastases were investigated: (i) the expression of the mucins MUC1 and MUC2, known to carry sialyl‐Lex, by Northern blotting; (ii) the extent of sialic acid O‐acetylation, by Western blotting and HPLC. RNA and sialyl‐Lex‐positive mucins were purified from normal colonic mucosa (N), primary carcinomas (T) and their liver metastases (M). Northern blots showed that mRNA expression both of MUC1 and of MUC2 decreases during the progression of the disease, and is lowest in metastatic tissue. The expression of mucin‐bound sialyl‐Lex increased strongly from N to T and, to a lesser extent, to M. After alkali treatment of the mucins these differences disappeared, indicating that the total amount of mucin‐bound sialyl‐Lex is the same in the 3 types of tissues. The O‐acetylation of mucin‐bound sialyl‐Lex gradually decreased from N to M. HPLC analysis showed that in N about 70%, in T 45% and in M only 20% of mucin‐bound sialic acids are O‐acetylated. Thus, the increase of sialyl‐Lex detectable during colon‐carcinoma progression is due to diminished O‐acetylation and not to increased expression of mucin protein cores. The decrease of O‐acetylation is therefore the primary chemical alteration contributing to colon carcinoma‐associated overexpression of sialyl‐Lex. Int. J. Cancer 72:258–264, 1997.

An expression module of WIPF1-coexpressed genes identifies patients with favorable prognosis in three tumor types.

Wiskott–Aldrich syndrome (WAS) predisposes patients to leukemia and lymphoma. WAS is caused by mutations in the protein WASP which impair its interaction with the WIPF1 protein. Here, we aim to identify a module of WIPF1-coexpressed genes and to assess its use as a prognostic signature for colorectal cancer, glioma, and breast cancer patients. Two public colorectal cancer microarray data sets were used for discovery and validation of the WIPF1 co-expression module. Based on expression of the WIPF1 signature, we classified more than 400 additional tumors with microarray data from our own experiments or from publicly available data sets according to their WIPF1 signature expression. This allowed us to separate patient populations for colorectal cancers, breast cancers, and gliomas for which clinical characteristics like survival times and times to relapse were analyzed. Groups of colorectal cancer, breast cancer, and glioma patients with low expression of the WIPF1 co-expression module generally had a favorable prognosis. In addition, the majority of WIPF1 signature genes are individually correlated with disease outcome in different studies. Literature gene network analysis revealed that among WIPF1 co-expressed genes known direct transcriptional targets of c-myc, ESR1 and p53 are enriched. The mean expression profile of WIPF1 signature genes is correlated with the profile of a proliferation signature. The WIPF1 signature is the first microarray-based prognostic expression signature primarily developed for colorectal cancer that is instrumental in other tumor types: low expression of the WIPF1 module is associated with better prognosis.

A genome-wide map of aberrantly expressed chromosomal islands in colorectal cancer

BackgroundCancer development is accompanied by genetic phenomena like deletion and amplification of chromosome parts or alterations of chromatin structure. It is expected that these mechanisms have a strong effect on regional gene expression.ResultsWe investigated genome-wide gene expression in colorectal carcinoma (CRC) and normal epithelial tissues from 25 patients using oligonucleotide arrays. This allowed us to identify 81 distinct chromosomal islands with aberrant gene expression. Of these, 38 islands show a gain in expression and 43 a loss of expression. In total, 7.892 genes (25.3% of all human genes) are located in aberrantly expressed islands. Many chromosomal regions that are linked to hereditary colorectal cancer show deregulated expression. Also, many known tumor genes localize to chromosomal islands of misregulated expression in CRC.ConclusionAn extensive comparison with published CGH data suggests that chromosomal regions known for frequent deletions in colon cancer tend to show reduced expression. In contrast, regions that are often amplified in colorectal tumors exhibit heterogeneous expression patterns: even show a decrease of mRNA expression. Because for several islands of deregulated expression chromosomal aberrations have never been observed, we speculate that additional mechanisms (like abnormal states of regional chromatin) also have a substantial impact on the formation of co-expression islands in colorectal carcinoma.

Expression of SIALYL-Lex antigen defined by MAb AM-3 is an independent prognostic marker in colorectal carcinoma patients

Expression of mucin‐bound sialyl‐Lex antigen during the progression of colorectal carcinoma and its potential prognostic value were analysed in sections of tumours from 182 patients with a documented follow‐up by immunohistochemistry using the monoclonal antibody (MAb) AM‐3. Two groups of colonic carcinomas with weak (n = 79) and strong (n = 103) sialyl‐Lex expression were discerned. The percentage of strongly expressing tumours increased with the progression of the disease (UICC stage I = 10%, stage II = 46%, stage III = 63%, stage IV = 68%, p < 0.0001). Seventy‐four percent of patients with carcinomas exhibiting a strong sialyl‐Lex expression but only 34% of patients with weak sialyl‐Lex expression died of the disease (p = 0.0026). In multivariate analysis, strong sialyl‐Lex expression increased the relative risk of cancer‐related death 3.8‐fold (95% CI = 1.8–7.9, p = 0.00034). The separate analyses of patients in UICC stage II (n = 56), III (n =5 9) and IV (n = 57) revealed that strong sialyl‐Lex expression was associated with a reduction of the 5‐year overall survival rate in UICC stage II (84% vs. 54%, p = 0.0013) and in stage III patients (86% vs. 35%, p = 0.0008) after curative resection but was not relevant in patients with distant metastases. In conclusion, the strong expression of sialyl‐Lex antigen defined by the MAb AM‐3 in colorectal carcinomas is an independent unfavourable prognostic factor after curative resection in stage II and III patients. The predictive power of the sialyl‐Lex expression may be helpful to define subgroups of patients at high risk for whom preventive adjuvant therapy can be selectively applied before the occurrence of detectable metastases. Int. J. Cancer 88:281–286, 2000.

Transcriptional census of 36 microdissected colorectal cancers yields a gene signature to distinguish UICC II and III

UICC stage II and III colorectal cancers (CRC) differ fundamentally in prognosis and therapeutic concepts. To analyze differential gene expression between both stages and to establish a relationship between molecular background and clinical presentation, tumor material from 36 unselected consecutive patients presenting with sporadic CRC, 18 UICC stage II and 18 UICC stage III, were laser microdissected to separate epithelial tumor cells. Gene expression levels were measured using U133A Affymetrix gene arrays. Twelve CRC associated signal transduction pathways as well as all 22,000 probe sets were screened for differential gene expression. We identified a signature consisting of 45 probe sets that allowed discrimination between UICC stage II and stage III with a rate of correct classification of about 80%. The most distinctive elements in this signature were the gene GSTP‐binding elongation factor (GSPT2) and the transcription factor HOXA9. Differential expression of these genes was confirmed by quantitative real‐time polymerase chain reaction (p(HOXA9) = 0.04, p(GSTP2) = 0.02). Despite the reliability of the presented data, there was no substantial differential expression of genes in cancer‐related pathways. However, the comparison with recently published data corroborates the 45 gene signature showing structural agreement in the direction of fold changes of gene expression levels for our set of genes chosen to discriminate between both stages.

Factors influencing lymph node harvest in colorectal surgery

Background and aimsLymphadenectomy in colorectal cancer is a critical component concerning prognosis and survival of patients. Several variables influence the number of harvested lymph nodes (LN). However, results of studies are contradictory, and influencing factors remain to be identified. The aim of the present study was to identify factors that have a significant influence on the number of assessed LN in oncologic colorectal cancer resection.Materials and methodsThree hundred and forty-one patients (190 men and 151 women), who underwent a colorectal cancer resection in a curative intention in the years 2000–2005, were analysed retrospectively. All specimens were histologically examined by two pathologists.ResultsIn a median, 15.1 LN per operation were resected. Early tumour stage (p < 0.01), length of resected bowel segment (p < 0.05) and right-sided location (p < 0.001) had a significant influence on the number of resected LN. Age, gender, surgeon volume, differentiation of the tumour, LN metastases, lymphatic invasion and depth of tumour invasion had no significant association with harvested LN number. Furthermore, the presence or absence of the vermiform appendix and the length of the resected ileum segment in right-sided resections did not significantly affect the assessed LN.ConclusionThe question arises whether for colorectal cancers of all locations the same amount of resected and analysed LNs should be lasting to fulfill oncologic criteria, as the number of harvested LNs depends on several parameters.

Regulation of the intestinal mucin MUC2 gene expression in vivo: evidence for the role of promoter methylation

In the present work we investigated the in vivo regulation of the mucin gene MUC2, which is overexpressed in all mucinous colorectal carcinomas. The inhibition of methylation by 5-azadeoxycytidine induces de novo expression of MUC2 in the colon carcinoma cell line COLO 205. The expression is retained in xenograft tissue and the cells give rise to MUC2-expressing tumours in nude mice. The strong expression of MUC2 in the normal human goblet cells and in the tissue of human mucinous colorectal carcinomas is associated with the average methylation of about 50% at every investigated CpG site of the MUC2 promoter. In contrast, MUC2 promoter in the non-expressing normal columnar cells and in the non-mucinous carcinoma tissue is methylated to nearly 100%. These data show that (i) low methylation of MUC2 promoter is associated with MUC2 expression in vivo and (ii) the pattern of MUC2 promoter methylation in the normal goblet or columnar cells most closely resembles that in mucinous or non-mucinous colorectal carcinomas, respectively. They indicate that MUC2 expression in vivo is regulated by promoter methylation and support the hypothesis that cells with goblet-like differentiation give rise to mucinous colonic carcinomas.