Neoplastic Colorectal Tissues Research Articles

Expression of TSP50, SERCA2 and IL-8 in Colorectal Adenoma and Carcinoma: Correlation to Clinicopathological Factors.

Background: Colorectal cancer (CRC) is the third most common type of cancer, it is considered a genetically heterogeneous disease with different molecular pathways being involved in its initiation and progression. Testes-specific protease 50 (TSP50) gene is a member of cancer/testis antigens that encodes for threonine protease enzyme. Overexpression of TSP50 was found to enhance the progression and invasion of breast cancer and other malignant tumors. SERCA2 is widely expressed in several body tissues; its aberrant expression has been involved in many cancers. IL-8 is an inflammatory cytokine. Alongside its role in inflammation, its expression was reported to induce the migration of tumor cells. Aim: Study the expression of TSP50, SERCA2 and IL-8 in colorectal adenoma (CRA), CRC and normal colonic tissues to compare the expression of these biomarkers in relation to clinicopathological parameters and prognostic factors. Results: TSP50, SERCA2 and IL-8 expression varied between normal colonic tissues, CRA and CRC. Significant statistical association was detected between the three biomarkers’ overexpression and degree of dysplasia in CRA. Also, significant statistical relation was found between the three biomarkers’ overexpression and presence of lympho-vascular invasion, advanced TNM staging and high intra-tumoral inflammatory infiltrate. Multivariable analysis showed that the overexpression of the three biomarkers is significantly associated with worse prognosis. Conclusion: The expression of TSP50, SERCA2 and IL-8 was different between the normal tissue and neoplastic colorectal tissue on one hand and between CRA and CRC on the other. Increased expression of these biomarkers in neoplastic epithelial cells of colorectal carcinoma is associated with adverse prognostic factors and could be considered as independent prognostic factors.

HFE variants in colorectal cancer and their clinicopathological correlations

The study aimed to screen mutation of human homeostatic iron regulator (HFE) in colorectal carcinoma (CRC) and detect their associations with clinicopathological parameters. Expression of HFE was determined by quantitative polymerase chain reaction in matched CRC and non neoplastic colorectal mucosal tissue of 76 patients. Genomic DNA extracted were subjected to high high-resolution melt curve analysis and Sanger sequencing to detect mutations in HFE. The associations of the identified mutations with a variety of clinical features were determined. Approximately 60% of CRC showed low HFE expression. Of the ten 10 mutations identified in exons 2 and 4, c.187C>G (H63D), c845G>A (C282Y), c.193A>T (S65C), g.3828T>C, g.5795T>C, and g.5728G>A were known mutations. Four novel mutations were discovered; : c.184G>A, c.220T>G, c.322A>C, and c.324T>C. Heterozygous H63D and C282Y mutations were seen in 71% and 49% of cancer tissue, respectively. Tumour site (p=0.048) and gender (p=0.039) were significantly associated with H63D and C282Y mutation status, respectively. Local spread of cancer was significantly associated with C282Y mutations in CRC cancer and adjacent non-neoplastic tissue (p=0.029 & and p=0.004, respectively). There was a statistically significant association between H63D and C282Y negativity in matched non-neoplastic colorectal mucosa tissue and pathological staging of cancer (p=0.047 & and p=0.001, respectively). Patients with H63D and C282Y mutations in cancer tissue tend to have higher survival rates. Hence HFE mutations are common in CRC and are associated with clinicopathological parameters, implying the potential clinical significance of HFE mutations in colorectal carcinogenesis.

Identifying molecular contributors to autofluorescence of neoplastic and normal colon sections using excitation-scanning hyperspectral imaging.

Autofluorescence, the endogenous fluorescence present in cells and tissues, has historically been considered a nuisance in biomedical imaging. Many endogenous fluorophores, specifically, collagen, elastin, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide (FAD), are found throughout the human body. In fluorescence imaging scenarios, these signals can be prohibitive as they can outcompete signals introduced for diagnostic purposes. However, autofluorescence also contains information that has diagnostic value. Recent advances in hyperspectral imaging have allowed the acquisition of significantly more data in a shorter time period by scanning the excitation spectra of fluorophores. The reduced acquisition time and increased signal-to-noise ratio allow for separation of significantly more fluorophores than previously possible. We propose to utilize excitation-scanning hyperspectral imaging of autofluorescence to differentiate neoplastic lesions from surrounding non-neoplastic "normal" tissue. The spectra of isolated autofluorescent molecules are obtained using a custom inverted microscope (TE-2000, Nikon Instruments) with an Xe arc lamp and thin-film tunable filter array (VersaChrome, Semrock, Inc.). Scans utilize excitation wavelengths from 360 to 550nm in 5-nm increments. The resultant molecule-specific spectra are used to analyze hyperspectral image stacks from normal and neoplastic colorectal tissues. Due to a limited number of samples, neoplastic tissues examined here are a pool of both colorectal adenocarcinoma and adenomatous polyps. The hyperspectral images are analyzed with ENVI software and custom MATLAB scripts, including linear spectral unmixing. Initial results indicate the ability to separate signals of endogenous fluorophores and measure the relative concentrations of fluorophores among healthy and diseased states, in this case, normal colon versus neoplastic colon. These results suggest pathology-specific changes to endogenous fluorophores can be detected using excitation-scanning hyperspectral imaging. Future work will focus on expanding the library of pure molecules, exploring histogram distance metrics as a means for identifying deviations in spectral signatures, and examining more defined disease states.

Detection of ctDNA biomarkers post resection and relationship to recurrent disease in colorectal cancer patients.

e15616Background: BCAT1 and IKZF1 are aberrantly methylated in almost all neoplastic colorectal tissue specimens. These biomarkers are detected in blood as circulating tumor DNA (ctDNA) in most pat...

Absence of the Epithelial Glycocalyx As Potential Tumor Marker for the Early Detection of Colorectal Cancer.

Detection of cancer at an early stage is pivotal for successful treatment and long term survival, yet early diagnosis requires sensitive and specific markers that can be easily detected by screening procedures. Differences in the surface structure of tumor and healthy cells, if sufficiently pronounced and discernible, may serve that purpose. We analyzed the luminal surface of healthy and neoplastic human colorectal tissues for the presence and architecture of the glycocalyx—a dense network of highly glycosylated proteins—using transmission electron microscopy. The ultrastructural analyses showed that 93% of healthy mucosae were covered by an intact glycocalyx. Contrarily, on over 90% of the surface of neoplastic cells the glycocalyx was absent. The sensitivity and specificity of our marker “absence of a glycocalyx” are excellent, being 91% (83–96%) and 96% (89–99%) for adenocarcinomas and 94% (73–100%) and 92% (85–97%) for precancerous polyps (means and 95% confidence intervals). Using a cell culture model we could demonstrate that a particulate probe targeting a cell surface receptor usually concealed beneath the glycocalyx can bind selectively to glycocalyx-free areas of a tumor cell layer. We propose that the absence of a glycocalyx may serve as novel type of tumor marker. If the absence of the glycocalyx can be detected e.g. via binding of imaging probes to non-shielded surface receptors of anomalously differentiated cells, this tumor marker could be used to enable early diagnosis of colorectal cancer.

Hyaluronic Acid in Normal and Neoplastic Colorectal Tissue: Electrospray Ionization Mass Spectrometric and Fluor Metric Analysis

Aim: The aim of the study was to analyze the expression of hyaluronic acid (HA) and its type in human colorectal cancer (CRC) and non-neoplastic mucosa tissues. Methods : The study included 64 adult CRC patients, who met all the inclusion and exclusion criteria. Two tissue samples (neoplastic and non-neoplastic mucosa) from each patient's large intestine were collected and were subjected to electrospray ionization mass spectrometry (ESI-MS) and fluorimetric assays. Results: The analysis of colorectal neoplastic tissue and non-neoplastic mucosa by ESI-MS allowed the identification of HA and its oligosaccharide fragments. Low molecular weight (LMW), nonbiotinylated isoform of HA, and its fragments were identified in both neoplastic and non-neoplastic mucosa. The expression of HA was found to be slightly lower in tumor tissue (0.561 mg HA/g tissue) than in colorectal non-neoplastic mucosa (0.579 mg HA/g tissue), although the difference was not statistically significant ( P = 0.87). This result was probably influenced by the nonbiotinylated LMW-HA. For the specific group of patients that did not present lymph node metastasis, the average HA levels were higher in tumor tissue (0.674 mg HA/g tissue) than in non-neoplastic tissue (0.529 mg HA/g tissue), which managed to reach statistical significance ( P = 0.04). For the group of patients with lymph node involvement, no difference between tumor and nontumor tissue was observed. The HA expression among the tumor tissues within the variables of each clinicopathological parameters assessed failed to elicit any significant difference (location, P = 0.62; size, P = 0.13; lymph node invasion, P = 0.57; degree of cellular differentiation, P = 0.46; venous infiltration, P = 0.73; lymphatic infiltration, P = 0.36; neural infiltration, P = 0.28; tumor node metastasis classification, P = 0.15; and presence of synchronous metastases, P = 0.35; initial versus advanced stage, P = 0.37). Conclusions: The expression of HA was found to be slightly lower in tumor tissue than in colorectal non-neoplastic mucosa, although this difference was not statistically significant. This finding probably influenced the lower expression of HA in tumor tissue than in colorectal non-neoplastic mucosa. Compared to normal tissues, HA levels are significantly increased in the tumor tissues unless they exhibit lymph node metastasis. Otherwise, the expression of HA in tumor tissue did not correlated with the other clinicopathological parameters.

Colorectal Neoplasia Differentially Expressed (CRNDE), a Novel Gene with Elevated Expression in Colorectal Adenomas and Adenocarcinomas

An uncharacterized gene locus (Chr16:hCG_1815491), now named colorectal neoplasia differentially expressed (gene symbol CRNDE), is activated early in colorectal neoplasia. The locus is unrelated to any known protein-coding gene. Microarray analysis of 454 tissue specimens (discovery) and 68 previously untested specimens (validation) showed elevated expression of CRNDE in >90% of colorectal adenomas and adenocarcinomas. These findings were confirmed and extended by exon microarray studies and RT-PCR assays. CRNDE transcription start sites were identified in CaCo2 and HCT116 cells by 5'-RACE. The major transcript isoforms in colorectal cancer (CRC) cell lines and colorectal tissue are CRNDE-a, -b, -d, -e, -f, -h, and -j. Except for CRNDE-d, the known CRNDE splice variants are upregulated in neoplastic colorectal tissue; expression levels for CRNDE-h alone demonstrate a sensitivity of 95% and specificity of 96% for adenoma versus normal tissue. A quantitative RT-PCR assay measuring CRNDE-h RNA levels in plasma was (with a threshold of 2(-ΔCt) = 2.8) positive for 13 of 15 CRC patients (87%) but only 1 of 15 healthy individuals (7%). We conclude that individual CRNDE transcripts show promise as tissue and plasma biomarkers, potentially exhibiting high sensitivity and specificity for colorectal adenomas and cancers.

The survivin -31 snp in human colorectal cancer correlates with survivin splice variant expression and improved overall survival

Survivin is involved in the regulation of cell division and survival, two key processes in cancer. The majority of studies on survivin in colorectal cancer (CRC) have focused on protein expression and less is known about the expression of survivin splicing variants or survivin gene polymorphisms in CRC. In the present study, the mRNA levels of the five known isoforms of survivin as well as survivin protein were assessed in matched normal and neoplastic colorectal tissue. Moreover, the 9386 C/T and -31 G/C polymorphisms were investigated. Quantitative RT-PCR was used to assess mRNA levels in fresh/frozen tissue samples. Protein levels were immunohistochemically evaluated on formalin-fixed paraffin-embedded tissue sections. Individuals were genotyped using real time PCR. Expression of all 5 survivin splice variants as well as survivin protein was elevated in colorectal carcinomas compared to normal tissue. Specific splice variant expression differentially correlated with clinicopathological parameters. Furthermore, both snps correlated with splice variant levels or their ratios in colorectal carcinomas while the -31 G/C snp may be related to CRC development and improved overall survival. Our results support a role of survivin in colorectal carcinogenesis while the -31 G/C snp may constitute a marker of survival.

Plasma Testing of Gene Expression Biomarkers for Colorectal Neoplasia Discovered in Neoplastic Colorectal Tissue

Plasma Testing of Gene Expression Biomarkers for Colorectal Neoplasia Discovered in Neoplastic Colorectal Tissue

NM23 protein expression in colorectal carcinoma using TMA (tissue microarray): association with metastases and survival

NM23, a metastasis suppressor gene, may be associated with prognosis in patients with colorectal carcinoma. To analyze NM23 expression and its association with the presence of lymph node and liver metastases and survival in patients operated on for colorectal carcinoma. One hundred thirty patients operated on for colorectal carcinoma were investigated. Tissue microarray blocks containing neoplastic tissue and tumor-adjacent non-neoplastic mucosa were obtained and analyzed by immunohistochemical staining using a monoclonal anti-NM23 antibody. Immunohistochemical expression was assessed using a semiquantitative scoring method, counting the percentage of stained cells. The results were compared regarding morphological and histological characteristics of the colorectal carcinoma, presence of lymph node and liver metastases, tumor staging, and patient survival. Statistical analysis was performed using the Mann-Whitney test, the Kruskal-Wallis test and Fisher's exact test. Survival analysis was performed using the Kaplan-Meier method and the log-rank test. NM23 expression was higher in colorectal carcinoma tissue than in adjacent non-neoplastic mucosa (P<0.0001). NM23 protein expression did not correlate with degree of cell differentiation (P = 0.57), vascular invasion (P = 0.85), lymphatic invasion (P = 0.41), perineural infiltration (P = 0.46), staging (P = 0.19), lymph node metastases (P = 0.08), or liver metastases (P = 0.59). Disease-free survival showed significant association (P = 0.01) with the intensity of NM23 protein immunohistochemical expression in colorectal carcinoma tissue, whereas overall survival showed no association with NM23 protein expression (P = 0.13). NM23 protein expression was higher in neoplastic colorectal carcinoma tissue than in adjacent non-neoplastic mucosa, showing no correlation with morphological aspects, presence of lymph node or liver metastases, colorectal carcinoma staging, or overall survival. Disease-free survival was higher in patients with increased NM23 expression.

Expression of nuclear factor Y, subunit C (NFY-C), and retinoic acid receptor-related orphan receptor alpha (RORA) in colorectal adenocarcinoma.

e14085 Background: NFY-C gene is one of the three subunits of nuclear factor Y, a highly conserved transcription factor, which binds with high specificity to CCAAT motifs in the promoters of various genes. RORA is a member of the NR1 subfamily of nuclear hormone receptors. It can bind as a monomer or a homodimer to hormone response elements upstream of several genes to enhance their expression. Methods: mRNA levels of NFY-C and RORA were evaluated by quantitative RT-PCR in 81 neoplastic colorectal tissue specimens and 51 normal tissue specimens from patients with colorectal adenocarcinoma. All patients had undergone curative resections at the University Hospital of Patras, between 1995 and 2005. mRNA levels were normalised to the Alu-Sq levels and were analysed in relation to clinicopathological parameters. Protein expression of NFY-C was assessed by immunochemistry in 60 malignant and 20 normal samples from patients with colorectal adenocarcinoma. Results: There was a significant difference in the mRNA expression levels of NFY-C and RORA between normal and malignant tissues (p < 0.001 and p = 0.015, respectively). The mRNA levels of NFY- C and RORA were also related to the primary site of the tumor (p = 0.05 and p = 0.03, respectively). A 3-year survival benefit was also observed in patients with high expression levels of NFY-C (p = 0.023). There was no correlation between the mRNA levels of NFY-C or RORA and age, gender, grade, stage and relapse of the disease. However, mRNA levels of NFYC of stage B patients were significantly correlated with time to disease progression (p = 0.035). NFY-C protein was detected only in the cytoplasm both in malignant and normal tissues, with strong and weak intensity respectively. NFYC protein expression levels were correlated with mRNA expression levels of NFYC in malignant tissues (p = 0.011) and with the primary site of the tumour (p < 0.001). Conclusions: NFY-C and RORA exhibited elevated levels in colon carcinomas compared to normal tissue samples indicating a possible role for these molecules in colon carcinogenesis. The role of NFY-C and RORA in colorectal cancer warrants further investigation. No significant financial relationships to disclose.

T1717 The Combined Analysis of miRNA and mRNA Expression Levels in Normal and Neoplastic Colorectal Tissues Allows the Identification of Putative miRNA Target Transcripts

G A A b st ra ct s RNA was isolated from 105 specimens and evaluated on Affymetrics GeneChip HG-U133 plus 2.0 microarrays. For proteomic and kinomic analyses, the total tissue protein was extracted from pulled 5-7 samples of each study group. The iTRAQ tagging technology and isoelectrofocusing combined with tandem mass spectrometry (MS) analysis were applied to estimate the relative protein/peptide abundance. A kinase activity was defined as the relative level of phosphorylation of peptide substrates provided by PepChip Kinomics array (Pepscan Presto B.V.). The data were evaluated using pair-wise comparisons, clustering analyses and data decomposition into SVD modes. Results: From 25410 probe sets which passed the filtering procedure according to GCRMA+LVS algorithm, 824 probe sets represented 424 PK genes. From 6162 proteins identified in MS analyses (peptides score ≥ 40), 177 represented PKs. Of them, 157 were also identified by microarray analysis. Altogether, 444 PK transcripts and/or PK proteins were identified. Expression of 64% of transcripts, the amount of 26% of proteins and phosphorylation level of 34% of peptide substrates were changed in Ad, CRC or both tissues, compared to NM. The highest level of transcript, protein and phosphorylation changes were observed in the NM-CRC, Ad-CRC, and NM-Ad paircomparisons, respectively. Low level of correlation between transcript and protein levels was observed, pointing the utility of combined approach for a kinome study. Conclusion: The combination of multiple approaches defined by integrative genomics offers a powerful tool to elucidate changes in the kinome and cell signaling during neoplastic progression to CRC. Supported by PBZ-MNiI-2/1/2005 grant.

The Survivin −31 Snp in Human Colorectal Cancer Correlates with Survivin Splice Variant Expression and Improved Overall Survival

Background: Survivin is involved in the regulation of cell division and survival, two key processes in cancer. The majority of studies on survivin in colorectal cancer (CRC) have focused on protein expression and less is known about the expression of survivin splicing variants or survivin gene polymorphisms in CRC. In the present study, the mRNA levels of the five known isoforms of survivin as well as survivin protein were assessed in matched normal and neoplastic colorectal tissue. Moreover, the 9386C/T and −31G/C polymorphisms were investigated.Methods: Quantitative RT-PCR was used to assess mRNA levels in fresh/frozen tissue samples. Protein levels were immunohistochemically evaluated on formalin-fixed paraffin-embedded tissue sections. Individuals were genotyped using real time PCR.Results: Expression of all 5 survivin splice variants as well as survivin protein was elevated in colorectal carcinomas compared to normal tissue. Specific splice variant expression differentially correlated with clinicopathological parameters. Furthermore, both snps correlated with splice variant levels or their ratios in colorectal carcinomas while the −31G/C snp may be related to CRC development and improved overall survival.Conclusion: Our results support a role of survivin in colorectal carcinogenesis while the −31G/C snp may constitute a marker of survival.

Nfy-c expression in colorectal cancer

e15086 Background: Nuclear factor Y- C (NFY-C) gene encodes one of the three subunits of nuclear factor Y, a highly conserved transcription factor which binds to the promoters of a variety of genes, which are implicated in cell cycle progression, drug metabolism and antigen presentation. The purpose of the current study was to investigate the role of NFY-C in colorectal cancer by evaluating its mRNA expression in both malignant and normal colonic tissue from patients with colorectal carcinomas with and without disease relapse. Methods: Publicly available expression microarray data were analyzed to reveal elevated levels of NFY-C in patients with colorectal carcinoma who relapsed compared to patients who remained disease free. The mRNA levels of NFY-C were evaluated by quantitative RT-PCR in 81 neoplastic colorectal tissue specimens and 23 normal tissue specimens from patients with colorectal cancer who had undergone curative resections at the University Hospital of Patras, Greece, between 1995 and 2005. The mRNA levels were analysed in relation to clinicopathological parameters. Results: No significant difference was found in the expression levels of NFY-C between normal and malignant tissues. The expression levels of NFY-C were not related to age, gender, grade, stage or primary site of the disease. However, a statistically significant difference (p=0.02) was observed in NFY-C levels between patients with and without disease recurrence. More specifically, patients with low NFY-C levels relapsed more often than patients who overexpressed NFY-C. Nevertheless, the expression was not related to time to disease progression. Finally, patients with higher NFY-C expression levels seem to have improved survival, compared to patients with low NFY-C expression levels although the difference was not statistically significant. Conclusions: Expression levels of NFY-C seem to be associated with disease recurrence. The role of NFY-C in colorectal cancer warrants further investigation. No significant financial relationships to disclose.

Discrimination of Healthy and Neoplastic Human Colon Tissues by ex Vivo HR-MAS NMR Spectroscopy and Chemometric Analyses

The metabolic profile of human healthy and neoplastic colorectal tissues was obtained using ex vivo High-Resolution Magic Angle Spinning (HR-MAS) NMR spectroscopy. Principal Components Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) were applied to NMR data in order to highlight the biochemical differences between healthy and neoplastic colorectal tissues. The synergic combination of ex vivo HR-MAS NMR spectroscopy with Multivariate Data Analysis enables discrimination between healthy and tumoral colorectal tissues and identification of the increase of taurine, acetate, lactate, and lipids, and the decrease of polyols and sugars as tumoral characteristics. Moreover, it was found that macroscopically/histologically normal colorectal tissues, collected at least 15 cm from the adenocarcinoma, are characterized by a metabolic pattern quite similar to that typical of tumoral lesions. It was shown that ex vivo HR-MAS NMR spectroscopy, performed on intact specimens, may be of great potentiality in the clinical evaluation of human neoplastic colorectal tissues and that the biochemical data represent the molecular basis for an accurate and noninvasive clinical applications of in vivo NMR spectroscopy.

Leptin Expression Correlates with Favorable Clinicopathologic Phenotype and Better Prognosis in Colorectal Adenocarcinoma

Leptin, the product of the ob gene, is an adipocyte-derived neurohormone that regulates body fat storage and feeding behavior. Some studies have suggested that leptin has growth-factor-like functions in epithelial cells and its abnormal expression may be involved in cancer development and progression. We investigated leptin expression in normal and neoplastic colorectal tissues and its association with clinicopathological features and clinical outcome in colorectal adenocarcinoma patients. Leptin expression was evaluated on the tissue microarray of 44 normal colon mucosal tissues, 44 adenomatous polyps, and 437 colorectal adenocarcinomas by immunohistochemistry. Data were analyzed by chi-square test, one-way analysis of variance (ANOVA), Cox regression hazards model, and log-rank test with Kaplan-Meier curves. Frequency of leptin expression was dramatically increased from normal colonic mucosa (2/44, 4.5%) to adenomas (13/44, 29.5%) and adenocarcinomas (321/437, 73.5%) as neoplastic progression. Interestingly, leptin expression was correlated with favorable tumor features in depth of invasion (p = 0.033), lymph node metastasis (p = 0.019), American Joint Committee on Cancer (AJCC) and Dukes' stage (p = 0.021 and p = 0.005, respectively), differentiation (p = 0.010), and lymphatic invasion (p = 0.003). In univariate survival analysis, patients with leptin-positive adenocarcinoma revealed better overall and disease-free survivals (p = 0.032 and p = 0.004, respectively, log-rank test). In multivariate survival analysis with Cox proportional hazards model, leptin expression was an independent prognostic marker of disease-free survival (p = 0.009). We conclude that leptin was gradually expressed during the normal-adenoma-adenocarcinoma sequence, suggesting an association in colorectal carcinogenesis. In addition, high leptin expression was an indicator of favorable tumor features and better survival of colorectal cancer patients.

Clusterin Expression in Normal Mucosa and Colorectal Cancer

The gene Clusterin is a target for cancer therapy in clinical trials. The indication for intervention is up-regulated Clusterin expression. Clusterin has been reported to be deregulated in multiple cancer types, including colorectal cancer (CRC). However, for CRC the studies have disagreed on whether Clusterin is up- or down-regulated by neoplastic cells. In the present study we sought to clarify the expression and distribution of Clusterin mRNAs and proteins in normal and neoplastic colorectal tissue through laser microdissection, variant-specific real time RT-PCR, immunohistochemistry, immunofluorescence, Western blotting, and array-based transcriptional profiling. At the transcript level we demonstrated the expression of two novel Clusterin transcripts in addition to the known transcript, and at the protein level we demonstrated two Clusterin isoforms. Our analysis of normal epithelial cells revealed that among these, Clusterin was only expressed by rare neuroendocrine subtype. Furthermore our analysis showed that in the normal mucosa the majority of the observed Clusterin protein originated from the stromal compartment. In tumors we found that Clusterin was de novo synthesized by non-neuroendocrine cancer cells in approximately 25% of cases. Moreover we found that the overall Clusterin level in tumors often appeared to be lower than in normal mucosa due to the stromal compartment often being suppressed in tumors. Although Clusterin in normal neuroendocrine cells showed a basal localization, the localization in cancer cells was often apical and in some cases associated with apical secretion. Collectively our results indicate that Clusterin expression is very complex. We conclude that Clusterin expression is associated with neuroendocrine differentiation in normal epithelia and that the Clusterin observed in neoplastic cells is de novo synthesized. The cases with de novo synthesized Clusterin define a distinct subgroup of CRC that may be of clinical importance as anti-Clusterin therapeutics are now in clinical trials.

Identification of differentially expressed proteins in colorectal cancer by proteomics: Down-regulation of secretagogin

To identify proteins with colorectal cancer-specific regulation, comparative 2-DE of individual-matched normal and neoplastic colorectal tissue specimens was performed. We found 15 protein spots with concordantly increased and 20 protein spots with concordantly decreased intensity in tumor tissue (expression regulation more than fivefold). Nine of these proteins were identified by MS/MS. Interestingly, one of the proteins, which exhibited a marked down-regulation in colorectal cancer tissues, was the recently identified endocrine cell-expressed protein secretagogin. The reduction of the secretagogin content in colorectal cancer tissues was confirmed by comparative immunoblotting (n = 17) and RT-PCR (n = 22) as well as by immunohistochemistry (n = 45) of individual-matched neoplastic and normal colorectal tissue specimens. Immunohistochemistry revealed absence of secretagogin-expressing cells in most of the colorectal cancer tissue specimens. However, some colorectal cancers were characterized by secretagogin-expressing cells. In normal mucosa, positively stained cells exhibited a neuroendocrine cell-characteristic morphology and mucosal location. In colorectal cancer tissues, secretagogin-expressing cells were characterized by a malignant morphology. Our findings might represent the basis for the clinical application of secretagogin as a biomarker for a distinct subgroup of colorectal cancers.

EphB2 is a Prognostic Factor in Colorectal Cancer

A receptor tyrosine kinase for ephrin ligands, EphB2 is expressed in colorectal cancer and has been proposed as a target for immunoconjugate therapy. The aim of this study was to perform a detailed histologic analysis of EphB2 expression in normal and neoplastic colorectal tissues. In addition, we sought to evaluate EphB2 expression as a prognostic factor in colorectal cancer. Expression of EphB2 was examined in normal colon (n = 28), colorectal cell lines (n = 20), colorectal adenomas (n = 148), primary cancers (n = 28), and metastases (n = 39) using immunohistochemistry. In addition, a series of primary cancers and matched normal (n = 342) with outcome data were profiled in tissue microarrays. The intensity of EphB2 expression was assessed in the entire series by immunohistochemistry, and in a subset by in situ hybridization. Overall survival and recurrence-free survival were correlated with EphB2 protein expression in retrospective subset analyses. Epithelial EphB2 expression was shown at all stages of colorectal tumorigenesis, including the base of all normal crypts, 77% of adenomas, 82% of primary cancers, and 64% of metastases. Although homogeneous expression was observed in adenomas, the pattern of staining was focal (mean 25%) in most malignant lesions. Patients whose tumor stained 2+ for EphB2 expression (versus 0/1+) exhibited significantly prolonged overall survival: mean duration of survival, 2,514 versus 1,044 days; hazard ratio, 0.45; 95% confidence interval, 0.18 to 0.95 (P = 0.035). In summary, EphB2 is expressed in normal crypts, colorectal adenomas, primary cancers, and metastases. High levels of EphB2 expression are associated with a longer mean duration of survival in colorectal cancer.

Differential Expression of the AP-1 Transcription Factor Family Members in Human Colorectal Epithelial and Neuroendocrine Neoplasms

We immunohistochemically examined 75 human colorectal neoplasms (adenoma, 27; adenocarcinoma, 24; neuroendocrine carcinoma, 24) for the expression of activator protein (AP)-1 family proteins. Nuclear and cytoplasmic expression levels of c-Jun and Fra-1 proteins were markedly elevated in adenomas, adenocarcinomas and neuroendocrine carcinomas compared with nonneoplastic colorectal epithelial cells. JunB also was overexpressed in these tumors but with a predominantly cytoplasmic staining pattern. Overexpression of Fra-2 was evident in carcinomas but less frequent in adenomas. Expression levels of JunD and c-Fos were high in nonneoplastic colorectal epithelial cells and remained so in neoplasms. FosB was undetectable in nonneoplastic and neoplastic colorectal tissues. Neuroendocrine carcinomas exhibited an AP-1 expression profile similar to adenocarcinomas except for infrequent overexpression of c-Jun in poorly differentiated variants. Hierarchical clustering separated the majority of malignant from benign tumors based on AP-1 expression patterns. AP-1 transcription factor family members are expressed differentially in nonneoplastic and neoplastic colorectal tissues. Up-regulation of c-Jun and Fra-1 is an early event in human colorectal tumorigenesis. Overexpression of Fra-2 may participate in tumor progression.