Serum Of Colorectal Cancer Patients Research Articles

Psychoactive drugs influence brain-derived neurotrophic factor and neurotrophin 4/5 levels in the serum of colorectal cancer patients.

Previous studies have reported the association between brain-derived neurotrophic factor (BDNF) and tumor development in numerous cancers. However, the accurate implication of the two specific ligands of tropomyosin kinase B receptor, BDNF and neurotrophic factor 4 (NT4/5), has not been studied in colorectal cancer (CRC) patients. The present study investigated the significance of serum BDNF and the NT4/5 in association with the intake of psychoactive drugs in CRC patients. Soluble BDNF and NT4 in the serum were assessed by ELISA. Although no correlation of BDNF and NT4 with the CRC stage was identified, a positive correlation was found between NT4 and the intake of psychoactive drugs (P=0.0457). For BDNF, a correlation was found in particular with the intake of benzodiazepine (P=0.0221). As BDNF and NT4/5 are implicated in the response of psychoactive treatments applied to manage depression, which frequently occurs in cancer patients, they cannot be used as prognostic or diagnostic markers for CRC in these patients. However, high expression of BDNF and NT4 was significantly associated with better survival. Therefore, these NTs may be used as markers for monitoring depression or predicting survival in CRC patients.

Circulating microRNA-1290 as a novel diagnostic and prognostic biomarker in human colorectal cancer

Circulating microRNAs (miRNAs) are attracting major interest as potential non-invasive biomarkers for colorectal cancer (CRC). This study aimed to identify a novel serum miRNA biomarker for the early detection and/or evaluating prognosis of CRC patients. Comprehensive miRNA array analysis was carried out using serum samples from patients with colorectal neoplasia and healthy controls. Next, to verify whether the candidate miRNA possessed a secretory potential, we screened miRNA expression levels in culture medium from 2 CRC cell lines, followed by serum analysis from 12 stage IV CRC, 12 adenoma, and 12 control subjects. Thereafter, we validated expression of candidate miRNAs in 179 primary CRC tissues, as well as serum samples from an independent cohort of 211 CRCs, 56 adenomas, and 57 control subjects. Through microarray analysis, we identified significantly higher levels of miRNA-1290 (miR-1290) in serum from patients with colorectal adenomas and cancers. We verified miR-1290 overexpression in serum of CRC patients in a training cohort. In the validation cohort, serum miR-1290 levels were significantly up-regulated in patients with colorectal adenomas (P < 0.0001) and cancers (P < 0.0001). Serum miR-1290 levels could robustly distinguish adenoma [area under the curve (AUC) = 0.718] and CRC patients (AUC = 0.830) from normal subjects. High miR-1290 expression in serum and tissue was significantly associated with tumor aggressiveness and poor prognosis. Moreover, serum miR-1290 levels were an independent prognostic factor [hazard ratio (HR) = 4.51; 95% confidence interval (CI) = 1.23-23.69; P = 0.0096] and an independent predictor for tumor recurrence (hazard ratio = 3.92; 95% confidence interval = 1.11-25.14; P = 0.032) in CRC. Serum miR-1290 is a novel biomarker for early detection, recurrence, and prognosis in CRC.

Decreased expression of LncRNA SLC25A25-AS1 promotes proliferation, chemoresistance, and EMT in colorectal cancer cells

Increasing evidence suggests that long non-coding RNAs (lncRNAs) are aberrantly expressed in colorectal cancer (CRC); however, only few CRC-related lncRNAs have been characterized. In this study, we aimed to dig out potential dysregulated lncRNAs that are highly involved in CRC development. Using a lncRNA-mining approach, we performed lncRNA expression profiling in a large CRC cohort from Gene Expression Ominus (GEO), GSE39582 test series (N=585). We identified 31 downregulated lncRNAs and 16 upregulated lncRNAs from the GSE39582 test series patients (566 tumor patients and 19 normal controls). The reliability of lncRNA expression profiles was further confirmed by RT-qPCR in carcinoma tissues and paired adjacent normal tissues from 30 CRC patients, also in the serum from 109 CRC patients, and 99 normal individuals. We demonstrated that the expression of SLC25A25-AS1, which has not been reported previously, was significantly decreased in both the tumor tissues (27 out of 30) and serum of CRC patients. SLC25A25-AS1 overexpression significantly inhibited proliferation and colony formation in colorectal cancer cell lines, and downregulation of SLC25A25-AS1 obviously enhanced chemoresistance and promoted EMT process in vitro associated with Erk and p38 signaling pathway activation. Therefore, SLC25A25-AS1 was determined to play a tumor suppressive role in CRC. Our results might provide a lncRNA-based target for CRC treatment.

Abstract 968: Circulating microRNA-1290 as a novel diagnostic and prognostic biomarker in human colorectal cancer

Abstract Background &amp; Aims: MicroRNAs (miRNAs) play a crucial role in diverse cellular biological processes such as differentiation, proliferation, migration, invasion and survival. MiRNA expression patterns are found to be frequently dysregulated in human tumors, which is currently being exploited both from a basic science perspective and for its clinical usefulness as disease biomarkers. Circulating miRNAs are attracting major interest as potential noninvasive biomarkers for colorectal cancer (CRC). This study aimed to identify a novel serum miRNA biomarker for the early detection and/or evaluating prognosis of CRC patients. Methods: Comprehensive miRNA array analysis was performed using serum samples from patients with colorectal neoplasia (CRC: n = 3, adenoma: n = 3) and healthy controls (n = 3). Next, to verify whether the candidate miRNA possessed a secretory potential, we screened miRNA expression levels in culture medium from 2 CRC cell lines, followed by serum analysis from 12 stage IV CRC, 12 adenoma and 12 control subjects. Thereafter, we validated expression of candidate miRNAs in 179 primary CRC tissues, as well as serum samples from an independent cohort of 211 CRCs, 56 adenomas, and 57 control subjects. Results: Through microarray analysis, we identified significantly higher levels of miRNA-1290 (miR-1290) in serum from patients with colorectal adenomas and cancers. We verified miR-1290 overexpression in serum of CRC patients in a training cohort. In the validation cohort, serum miR-1290 levels were significantly upregulated in patients with colorectal adenomas (P &amp;lt; 0.0001) and cancers (P &amp;lt; 0.0001). Serum miR-1290 levels could robustly distinguish adenoma (area under the curve [AUC] = 0.718) and CRC patients (AUC = 0.830) from normal subjects. Serum miR-1290 levels significantly diminished after surgical resection of the primary tumor in the same patients’ serum specimens (P &amp;lt; 0.0001). In addition, we observed a statistically significant positive correlation of miR-1290 expression levels between primary CRC tissues and matched serum samples (ρ = 0.482; P &amp;lt; 0.0001). High miR-1290 expression in serum and tissue was significantly associated with tumor aggressiveness and poor prognosis. Moreover, serum miR-1290 levels were an independent prognostic factor in CRC patients (hazard ratio = 4.51; 95% confidence interval = 1.23-23.69; P = 0.0096). Conclusions: Serum miR-1290, which might be secreted from primary CRC, is a novel diagnostic and prognostic biomarker in CRC. Citation Format: Yuji Toiyama, Hiroki Imaoka, Hiroyuki Fujikawa, Junichiro Hiro, Susumu Saigusa, Koji Tanaka, Yasuhiro Inoue, Yasuhiko Mohri, Takao Mori, Toshio Kato, Ajay Goel, Masato Kusunoki. Circulating microRNA-1290 as a novel diagnostic and prognostic biomarker in human colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 968.

The growth differentiation factor-15 (GDF-15) can be useful in the detection of distant metastases in sera of colorectal cancer patients

Purpose: Growth differentiation factor-15 (GDF- 15) protein belongs to a transforming growth factor-β family which determines the growth and differentiation of cells. In cancers, GDF-15 influences on the proliferation, differentiation, viability, migration and invasiveness of cancer cells. The aim of our study was to evaluate the expression of GDF-15 in the tissue and its levels in sera of patients with colorectal cancer. Materials and methods: The level of GDF-15 in the sera of 55 patients diagnosed with colorectal cancer was determined using the ELISA method whereas expression of this protein was performed by immunohistochemical method. Results: The mean value of GDF-15 levels in the sera of patients with colorectal cancer was significantly higher than in healthy control group (p&lt;0.001). The expression of GDF-15 in the tissue was weak, moderate and strong in 23.6%, 15.7% and 60.7% cases, respectively. Statistical analysis showed that the expression of GDF-15 correlated with patients’ age (p&lt;0.005) and non-mucinous type of cancer (p&lt;0.001). The high GDF-15 levels in the serum was associated with tumor size (p&lt;0.01) and distant metastases (p&lt;0.05). Conclusions: According to our results, we postulate that the level of GDF-15 in serum can be use to assess the metastatic behavior of colorectal cancer

Multiplex determination of serological signatures in the sera of colorectal cancer patients using hydrogel biochips.

Colorectal cancer (CRC) is the third most common malignancy in industrialized countries. Despite the advances in diagnostics and development of new drugs, the 5‐year survival remains only 60–65%. Our approach to early diagnostics of CRC is based on the determination of serological signatures with an array of hemispherical hydrogel cells containing immobilized proteins and oligosaccharides (glycochip). The compounds immobilized on the glycochip include tumor‐associated glycans (SiaTn, Tn, TF, LeC, LeY, SiaLeA, and Manβ1‐4GlcNAcβ) and antibodies against human immunoglobulins IgG, IgA, and IgM. The glycochip detects antibodies against tumor‐associated glycans in patients’ sera. The simultaneous measurement of the levels of immunoglobulins enhances the diagnostic impact of the signatures. In this work, we found previously unreported increase in antibodies against oligosaccharide Manβ1‐4GlcNAcβ in patients with CRC. In parallel with these experiments, we determined the levels of oncomarkers carcinoembryonic antigen (CEA), cancer antigen (CA) 19–9, CA 125, CA 15–3, human chorionic gonadotropin (HCG), and alpha‐fetoprotein (AFP) using another gel‐based biochip with immobilized antibodies (oncochip) developed earlier in our laboratory. In total, 69 samples from healthy donors, 33 from patients with colorectal carcinoma, and 27 from patients with inflammatory bowel diseases were studied. The use of combined signatures of antiglycan antibodies and oncomarkers provides much better predictive value than the conventional measurement of oncomarkers CEA and CA 19–9. Positive predictive value of CRC diagnoses using together glycochip and oncochip reached 95% with the sensitivity and specificity 88% and 98%, respectively. Thus, the combination of antibody profiling with detection of conventional oncomarkers proved to be a promising tool in diagnostics of CRC.

Lectin-based protein microarray analysis of differences in serum alpha-2-macroglobulin glycosylation between patients with colorectal cancer and persons without cancer.

Glycosylation is co- and posttranslational modifications affecting proteins. The glycopattern changes are associated with changes in biological function and are involved in many diseases including cancer. We present the lectin-based protein microarray method enabling determination of differences in protein glycosylation. The method involves isolation of targeted protein from samples by immunoprecipitation, spotting of protein from multiple samples into arrays on a microarray slide, incubation with set of biotinylated lectins, the reaction with fluorescent conjugate of streptavidin, and detection of fluorescent intensities by microarray scanner. Lectin-based protein microarray was applied in investigation of differences in alpha-2-macroglobulin (α2M) glycosylation isolated from sera samples of healthy persons and patients with colorectal cancer (CC). From 14 lectins used in analysis, statistically significant differences (Student's t-test, P < 0.05) between two groups of samples (persons without cancer and CC patients) were found for 5 of them. α2M molecules isolated from sera of CC patients have higher content of α2,6 sialic acid, N-acetylglucosamine and mannose residues, and tri-/tetraantennary complex type high-mannose N-glycans. A novel lectin-based protein microarray developed and described can serve as a suitable analytical technique for sensitive, simple, fast, and high-throughput determination of differences in protein glycosylation isolated from serum or other samples.

Expressions and its clinical significance of microRNA-21 and microRNA-146a in colorectal neoplasms

Objective To investigate the differences of miRNA-21 and miRNA-146a expression between colorectal neoplasms tissues and serum of the patients with colorectal neoplasm, and the clinical significance was analyzed. Methods The endoscopic biopsy tissues and serum samples of 100 colorectal cancer (CRC), 80 colorectal adenoma (CRA) patients and 65 healthy controls were collected. The expressions of miRNA-21 and miRNA-146a were detected by quantitative real time polymerase chain reaction. The relationship between the expression and clinicopathological features were analyzed. And then, the diagnostic value of expression difference of serum miRNA in colorectal neoplasm were evaluated. The Mann-Whitney U test and Kruskal-Wallis test were performed for comparisons between groups, and the correlation of miRNA expression between tissues and serum was analyzed by Spearman test. Results The expressions of miRNA-21 in the tissues of CRC and CRA were 8.573±0.898 and 7.746±1.183, respectively, which were significantly higher than that of healthy controls 6.160±0.835 (U=120.129 and 33.230, both P 0.05). The expression of miRNA-146a in the tissues and serum of CRC patients were both lower than those of CRA patients (U=73.809 and 21.123, both P<0.01). The degree of decreased expression of miRNA-146a in CRC patients was correlated with TNM staging and tumor differentiation degree, however there was no correlation between the expression in CRA and clinical features. According to receiver operating characteristic (ROC) curve analysis, the AUC of miRNA-21, miRNA-146a and a combination of them in CRC and health individuals was 0.889, 0.791 and 0.863, respectively; in CRA and health individuals was 0.784, 0.692 and 0.761, respectively; in CRC and CRA was 0.705, 0.820 and 0.713, respectively. Conclusion The different expressions of miRNA-21 and miRNA-146a had potential values in early detection of colorectal cancer. Key words: microRNA; microRNA-21; microRNA-146a; Colorectal neoplasms; Tissues; Serum; Real-time polymerase chain reaction

Affinity proteomics led identification of vimentin as a potential biomarker in colon cancers: insights from serological screening and computational modelling.

Proteomic analysis using multiplex affinity reagents is perhaps the most reliable strategy to capture differentially expressed proteins that are slightly or immensely modified. In addition to expressional variation, it is comprehensively evident that the immunogenicity of a protein can be a deciding factor for instigating an inflammation afflicted-carcinogenesis. Considering both these factors, a simple and systematic strategy was designed to capture the immunogenic cancer biomarkers from sera of colorectal cancer patients. The affinity reagent, in the form of an antibody repertoire against the secretome of the HT29 cell line was used to grade the sera samples on the basis of the degree of immuno-reactivity and to capture differentially expressed antigens from the patient sera. Following affinity based 2DE-MALDI-TOF; the proteins were identified as (1) soluble vimentin; and (2) TGF-beta-inhibited membrane-associated protein (PP16B), in colon cancer sera and (3) keratin, type II cytoskeletal protein in rectal cancer sera. Pathway reconstruction and protein-protein networking of identified proteins predicted only Vimentin to be physically and genetically engaged in close proximity with the most established colorectal cancer associated tumorigenic pathways. Furthermore, our findings suggest that a possible surface stoichiometric shift in the structure of protein could be due to mutations in the coding sequence of Vimentin that may elicit its enhanced secretion possibly due to protein-hyperphosphorylation. Of the three proteins identified, only Vimentin showed higher expression in sera of colon cancer patients alone. Thus, it could be argued that vimentin might help in predicting individuals at higher risk of developing colon cancers. Our data are therefore suggestive of using vimentin as an antigen for tumor vaccination in an autologous set-up for colon cancers.

Identification of specific proteins in colorectal cancer patients with Schistosoma mansoni infection as a possible biomarker for the treatment of this infection

ObjectiveTo identify the associated proteins or antigens in serum of colorectal cancer patients with and without the chronic Schistosoma mansoni (S. mansoni) infection in Sudan. MethodsThe study included 93 patients with colorectal cancer and/or S. mansoni infection. A clinical database of colorectal cancer patients was documented by pathologist and information of S. mansoni infection was collected through stool analysis. Blood samples were collected from the identified patients. Geographical distribution of patients and etiological factors of colorectal cancer and S. mansoni infection were also discussed in this study. The proteins extracted from serum samples of patients using Tri Reagent were electrophoretically separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Specific protein band was then subjected to matrix laser desorption ionisation-time assistance-of-flight mass spectrometry analysis. ResultsStool analysis documented that 40 patients were colorectal cancer patients infected with S. mansoni, 26 patients were diagnosed with colorectal cancer only, and 27 patients were infected with S. mansoni only. Approximately 27% of participating patients with colorectal cancer and S. mansoni infection in this study came from Gezira State. A specific protein band was identified from the serum samples of colorectal cancer patients with S. mansoni infection on the separated protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The specific protein band was then analyzed by matrix laser desorption ionisation-time assistance-of-flight mass spectrometry and identified as a clusterin protein. ConclusionsThe study showed that the colorectal cancer patients infected with S. mansoni expressed clusterin protein, indicating that clusterin protein may be a potential biomarker to aid in the treatment of colorectal cancer patients infected with S. mansoni in the future.

Multiplex profiling of tumor‐associated proteolytic activity in serum of colorectal cancer patients

The monitoring of tumor-associated protease activity in blood specimens has recently been proposed as new diagnostic tool in cancer research. In this paper, we describe the screening of a peptide library for identification of reporter peptides (RPs) that are selectively cleaved in serum specimens from colorectal cancer patients and investigate the benefits of RP multiplexing. A library of 144 RPs was constructed that contained amino acid sequences of abundant plasma proteins. Proteolytic cleavage of RPs was monitored with MS. Five RPs that were selectively cleaved in serum specimens from tumor patients were selected for further validation in serum specimens of colorectal tumor patients (n = 30) and nonmalignant controls (n = 60). RP spiking and subsequent quantification of proteolytic fragments with LC-MS showed good reproducibility with CVs always below 26%. The linear discriminant analysis and PCA revealed that a combination of RPs for diagnostic classification is superior to single markers. Classification accuracy reached 88% (79/90) when all five markers were combined. Functional protease profiling with RPs might improve the laboratory-based diagnosis, monitoring and prognosis of malignant disease, and has to be evaluated thoroughly in future studies.

Hydrazide functionalized core-shell magnetic nanocomposites for highly specific enrichment of N-glycopeptides.

In view of the biological significance of glycosylation for human health, profiling of glycoproteome from complex biological samples is highly inclined toward the discovery of disease biomarkers and clinical diagnosis. Nevertheless, because of the existence of glycopeptides at relatively low abundances compared with nonglycosylated peptides and glycan microheterogeneity, glycopeptides need to be highly selectively enriched from complex biological samples for mass spectrometry analysis. Herein, a new type of hydrazide functionalized core-shell magnetic nanocomposite has been synthesized for highly specific enrichment of N-glycopeptides. The nanocomposites with both the magnetic core and the polymer shell hanging high density of hydrazide groups were prepared by first functionalization of the magnetic core with polymethacrylic acid by reflux precipitation polymerization to obtain the Fe3O4@poly(methacrylic acid) (Fe3O4@PMAA) and then modification of the surface of Fe3O4@PMAA with adipic acid dihydrazide (ADH) to obtain Fe3O4@poly(methacrylic hydrazide) (Fe3O4@PMAH). The abundant hydrazide groups toward highly specific enrichment of glycopeptides and the magnetic core make it suitable for large-scale, high-throughput, and automated sample processing. In addition, the hydrophilic polymer surface can provide low nonspecific adsorption of other peptides. Compared to commercially available hydrazide resin, Fe3O4@PMAH improved more than 5 times the signal-to-noise ratio of standard glycopeptides. Finally, this nanocomposite was applied in the profiling of N-glycoproteome from the colorectal cancer patient serum. In total, 175 unique glycopeptides and 181 glycosylation sites corresponding to 63 unique glycoproteins were identified in three repeated experiments, with the specificities of the enriched glycopeptides and corresponding glycoproteins of 69.6% and 80.9%, respectively. Because of all these attractive features, we believe that this novel hydrazide functionalized core-shell magnetic nanocomposite will shed new light on the profiling of N-glycoproteome from complex biological samples in high throughput.

Diagnostic significance of TIMP-1 level in serum and its immunohistochemical expression in colorectal cancer patients.

Tissue inhibitor of metalloproteinase-1 (TIMP-1) inhibits the ability of cancer cells to metastasize, but it can also stimulate cancer development. The aim of this study was to assess the level of TIMP-1 in serum and its expression in patients with colorectal cancer (CRC). The study group consisted of 43 patients diagnosed with colorectal cancer and 24 healthy volunteers. The level of TIMP-1 was assessed by the ELISA method while the expression of this protein was performed immunohistochemically. The concentration of TIMP-1 in the sera of colorectal cancer patients was significantly higher than in the healthy control group (p = 0.004). Higher level of TIMP-1 in the sera correlated with female gender (p = 0.045), tumor location in colon (p = 0.016), poorly differentiated tumor (p = 0.034) and higher platelet count in whole blood (p < 0.004). A positive reaction of the protein in cancer cells was observed in 31 cases and was found to correlate negatively with its reaction in peritumoral stroma (p < 0.001). According to this study, TIMP-1 protein may play an important role in cancer development. The assessment of this molecule in serum and tissue can be useful at the time of diagnosis and can help us to understand the nature of colorectal pathogenesis.

TLR4 is essential for dendritic cell activation and anti-tumor T-cell response enhancement by DAMPs released from chemically stressed cancer cells

The combination of immunotherapy and chemotherapy is regarded as a promising approach for the treatment of certain types of cancer. However, the underlying mechanisms need to be fully investigated to guide the design of more efficient protocols for cancer chemoimmunotherapy. It is well known that danger-associated molecular patterns (DAMPs) can activate immune cells, including dendritic cells (DCs), via Toll-like receptors (TLRs); however, the role of DAMPs released from chemical drug-treated tumor cells in the activation of the immune response needs to be further elucidated. Here, we found that colorectal cancer (CRC) cells treated with oxaliplatin (OXA) and/or 5-fluorouracil (5-Fu) released high levels of high-mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70). After OXA/5-Fu therapy, the sera of CRC patients also exhibited increased levels of HMGB1 and HSP70, both of which are well-known DAMPs. The supernatants of dying CRC cells treated with OXA/5-Fu promoted mouse and human DC maturation, with upregulation of HLA-DR, CD80 and CD86 expression and enhancement of IL-1β, TNF-α, MIP-1α, MIP-1β, RANTES and IP-10 production. Vaccines composed of DCs pulsed with the supernatants of chemically stressed CRC cells induced a more significant IFN-γ-producing Th1 response both in vitro and in vivo. However, the supernatants of chemically stressed CRC cells failed to induce phenotypic maturation and cytokine production in TLR4-deficient DCs, indicating an essential role of TLR4 in DAMP-induced DC maturation and activation. Furthermore, pulsing with the supernatants of chemically stressed CRC cells did not efficiently induce an IFN-γ-producing Th1 response in TLR4-deficient DCs. Collectively, these results demonstrate that DAMPs released from chemically stressed cancer cells can activate DCs via TLR4 and enhance the induction of an anti-tumor T-cell immune response, delineating a clinically relevant immuno-adjuvant pathway triggered by DAMPs.

Immunohistochemical expression of MMP-7 protein and its serum level in colorectal cancer

The study objective was to determine the presence of MMP-7 in cancer tissue in correlation with its serum level in patients diagnosed with colorectal cancer (CRC). In 45 patients with CRC, MMP-7 expression was assessed immunohistochemically on FFPE slides in tumours (N = 37) and in the corresponding surgical margin sample. MMP-7 serum level was measured preoperatively. The expression of MMP-7 in cancer tissue was much stronger as compared to the normal intestinal mucosa. Also the level of MMP-7 in the serum of CRC patients was higher than in healthy subjects (N = 24) (p < 0.01). The tumour located in the colon showed higher expression of MMP-7 than CRCs located in the rectum (p < 0.05), whereas the higher MMP-7 serum level showed correlation with older age (p = 0.005), tumour size less than 5 cm (p < 0.05), higher Dukes' stage (p < 0.05) and distant metastases (p < 0.05). The increased serum level of MMP-7 in CRC patients may indicate the presence of distant metastases.

Hyaluronidases and their inhibitors in the serum of colorectal carcinoma patients

Colorectal cancer is the third most commonly diagnosed type of cancer. Hyaluronan is involved in this malignancy. Moreover, hyaluronidases – its degrading enzymes – display controversial roles regarding their involvement in tumor development. HYAL-1 is the major tumor derived hyaluronidase. The aim of the study was the determination and evaluation of hyaluronidase levels in serum of colorectal cancer patients, before and after surgery, with a view to assessing its potential role as a tumor marker for recurrence. By zymography and Western blotting, it was confirmed that HYAL-1 was the only hyaluronidase present in samples. Quantification of its activity indicated a statistically significant decrease in samples seven days postoperatively, compared with the respective ones before surgery. HYAL-1 levels before surgery were significantly reduced in comparison with healthy samples and samples one year postoperatively. Hyaluronidase inhibitor activity was demonstrated under mild alkaline conditions via reverse zymography. A statistically significant increase was observed in samples seven days postoperatively, when compared with samples before surgery. HYAL-1 levels in sera of colorectal cancer patients were lower than those of healthy population, possibly because of the local accumulation of the enzyme in tumor microenvironment. A gradual elevation up to one year postoperatively to reach healthy levels might indicate a role of HYAL-1 levels in cancer.

Serum miR-21 as a Diagnostic and Prognostic Biomarker in Colorectal Cancer

The oncogenic microRNAs (miRNAs) miR-21 and miR-31 negatively regulate tumor-suppressor genes. Their potential as serum biomarkers has not been determined in human colorectal cancer (CRC). To determine whether miR-21 and miR-31 are secretory miRNAs, we screened expression in medium from 2 CRC cell lines, which was followed by serum analysis from 12 CRC patients and 12 control subjects. We validated expression of candidate miRNAs in serum samples from an independent cohort of 186 CRC patients, 60 postoperative patients, 43 advanced adenoma patients, and 53 control subjects. We analyzed miR-21 expression in 166 matched primary CRC tissues to determine whether serum miRNAs reflect expression in CRC. Patient survival analyses were performed by Kaplan-Meier analyses and Cox regression models. All statistical tests were two-sided. Although miR-21 was secreted from CRC cell lines and upregulated in serum of CRC patients, no statistically significant differences were observed in serum miR-31 expression between CRC patients and control subjects. In the validation cohort, miR-21 levels were statistically significantly elevated in preoperative serum from patients with adenomas (P < .001) and CRCs (P < .001). Importantly, miR-21 expression dropped in postoperative serum from patients who underwent curative surgery (P < .001). Serum miR-21 levels robustly distinguished adenoma (area under the curve [AUC] = 0.813; 95% confidence interval [CI] = 0.691 to 0.910) and CRC (AUC = 0.919; 95% CI = 0.867 to 0.958) patients from control subjects. High miR-21 expression in serum and tissue was statistically significantly associated with tumor size, distant metastasis, and poor survival. Moreover, serum miR-21 was an independent prognostic marker for CRC (hazard ratio = 4.12; 95% CI = 1.10 to 15.4; P = .03). Serum miR-21 is a promising biomarker for the early detection and prognosis of CRC.

Circulating miR-17-3p, miR-29a, miR-92a and miR-135b in serum: Evidence against their usage as biomarkers in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the world. Therefore, there is a high demand for cost-effective and non-invasive biomarkers that would enable an early detection of asymptomatic and curable disease with high sensitivity and specificity. The main objective of this study was to investigate the potential of circulating miRNAs as biomarkers of CRC. Total RNA enriched for small RNAs was isolated from 100~sera of patients with CRC and 30 sera of healthy donors. The expression levels of miR-17-3p, miR-29a, miR-92a and miR-135b were determined using quantitative real-time PCR. The average expression levels of particular miRNAs were normalized to miR-16 levels and statistically evaluated. Using Mann-Whitney U test, no significant differences were observed in miR-17-3p (P=0.18), miR-29a (P=0.14) and miR-92a (P=0.60) levels between sera of CRC patients and controls. The levels of miR-135b in serum were too low to be quantified accurately. Subsequently, we tried to correlate expression levels of analyzed miRNAs to clinical-pathological features of CRC patients. Only levels of mir-29a were correlated with the clinical stage (P=0.04). Expression levels of the other miRNAs were correlated neither with the clinical stage, nor with the grade. Interestingly, our results are contradictory to previous studies performed on the CRC patients from Chinese population, providing an evidence against usage of serum miR-17-3p, miR-29a, miR-92a and miR-135b as new biomarkers for early detection of CRC.

Elevated levels of mitochondrial mortalin and cytosolic HSP70 in blood as risk factors in patients with colorectal cancer

Mortalin/GRP75 is a ubiquitous mitochondrial chaperone related to the cytosolic heat shock protein 70 (HSP70). It protects cells from senescence and apoptosis and is overexpressed in cancer cells. Cell resistance to complement-dependent cytotoxicity depends on mortalin and during complement attack mortalin is released from cells. Our goal was to determine whether cancer patients have circulating mortalin in blood. The significance of mortalin in blood to survival prospects of colorectal cancer patients was evaluated. Occurrence of extracellular soluble HSP70 (sHSP70) is documented. We developed a sensitive ELISA for mortalin. The association between mortalin level and survival was subjected to the Cox proportional hazards analysis (univariate and multivariate analyses). Mortalin concentration in serum of colorectal cancer patients was 10-214 ng/ml. Survival data of the patients were known from an earlier study of sHSP70 in these samples. Cox regression analysis indicated that high mortalin (>60 ng/ml) is a risk factor for shorter survival. Serum levels of sHSP70 and mortalin in patients were independent variables. Concurrence of high sHSP70 and mortalin was associated with rapid disease progression (HR = 4, 2.04-8.45, p < 0.001). Addition of high sHSP70 and mortalin to a baseline model of age, sex and TNM stage, significantly (p < 0.001) enhanced the risk score to 8 (3.26-20.46). This is the first demonstration of circulating mortalin in cancer patients. Analysis of mortalin in blood, and even more so of mortalin and sHSP70, adds a high prognostic value to the TNM stage and will identify colorectal cancer patients at high risk of poor survival.

Correlation Between Auto-antibodies to Survivin and MUC1 Variable Number Tandem Repeats in Colorectal Cancer

The aim of this study was to investigate the frequency and correlation between auto-antibodies to survivin and MUC1 variable number tandem repeats (VNTR) in colorectal cancer (CRC), which can provide valuable information for the design of immunotherapeutic vaccines for this disease. Enzyme-linked immunosorbent assays (ELISA) were used to examine the level of auto-antibodies against survivin and MUC1 VNTR in the serum of 135 CRC patients and 95 healthy volunteers. Using mean absorbance+2 standard deviations (SD) of the healthy samples as a cut-off value, the positive rates of survivin and MUC1 VNTR auto- antibodies in CRC were 31.1% and 18.5%, respectively. Altogether, the survivin and MUC1 VNTR positive samples accounted for 36.3% of the CRC patients, and 7.4% were positive for both. A significant positive correlation was found between levels of specific antibodies against survivin and MUC1 VNTR in the serum of CRC patients (r=0.3652, P<0.0001), suggesting that vaccines against both targets would elicit immune responses more effectively.