Evaluation of Blood Soluble CD26 as a Complementary Biomarker for Colorectal Cancer Screening Programs.

Fecal hemoglobin immunodetection (FIT) in combination with endoscopy has been implemented to reduce mortality from colorectal cancer (CRC), although there are issues that can be improved in relation to participation rates. We studied whether the blood biomarker soluble-CD26 (sCD26), related at least in part to the immune system and inflammation, and/or its dipeptidyl peptidase enzyme activity (DPP4), could help reduce false positives. In a cohort of 1703 individuals who underwent colonoscopy and had a serum sample, sCD26 and DPP4 activity showed statistically significant differences regarding sex and age. According to the colonoscopy findings, sCD26 and DPP4 activity progressively decreased in advanced adenomas and CRC, with statistically significant differences, even between both groups; 918 of them had a FIT result (n = 596 positive cases) with approximately 70% of these (n = 412) false positives. With cut-offs of 440 ng/mL for sCD26, 42 mU/mL for DPP4, and 11 ng/mU for their ratio, the combined information of the three biomarkers (at least positive for one biomarker) identified almost all advanced adenomas and CRC cases in the FIT cohort with approximately half of the false positives compared to FIT. A sequential testing strategy with FIT and our blood biomarker test is proposed.

Postoperative serum levels of sCD26 for surveillance in colorectal cancer patients.

One of the main aims of the follow-up after curative resection of colorectal cancer is the early detection and treatment of tumor recurrence. We previously demonstrated decreased preoperative soluble CD26 (sCD26) levels in serum from colorectal cancer patients. We extended now the study to investigate if sCD26 levels in postoperative serum serve as marker of recurrence of the disease during surveillance. Soluble sCD26 was measured in pre- and postoperative serum samples of 43 patients with primary colorectal cancer. Carcinoembryonic antigen, carbohydrate antigen 19.9 and 72.4 levels were also measured during surveillance. The average follow-up period was 41.8 ± 20.8 months. sCD26 levels during follow-up showed well-defined patterns in patients without disease (n = 28), and in patients with tumor persistence (n = 2), local recurrence (n = 3) or distant metastasis (n = 10). Disease-free patients showed stable levels between 460-850 ng/mL during follow-up, while high (over 850 ng/mL) and unstable sCD26 levels were found before recurrence was diagnosed. The mean maximum/minimum sCD26 ratios during surveillance were 1.52, 2.12 and 2.63 for patients with no recurrence, local recurrence and metastasis, respectively (p = 0.005). From the cut-off obtained from a receiver operator characteristics (ROC) curve built with the maximum/minimum sCD26 ratios and the upper and lower cut-offs of sCD26, we were able to discriminate patients with and without recurrent disease. We propose that the measurement of serum sCD26 during the follow-up of patients diagnosed of colorectal cancer could be valuable for the early detection of local and distant recurrence. A large, randomized, prospective trial should be performed to confirm our findings.

Proteomics for discovery of candidate colorectal cancer biomarkers.

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in Europe and other Western countries, mainly due to the lack of well-validated clinically useful biomarkers with enough sensitivity and specificity to detect this disease at early stages. Although it is well known that the pathogenesis of CRC is a progressive accumulation of mutations in multiple genes, much less is known at the proteome level. Therefore, in the last years many proteomic studies have been conducted to find new candidate protein biomarkers for diagnosis, prognosis and as therapeutic targets for this malignancy, as well as to elucidate the molecular mechanisms of colorectal carcinogenesis. An important advantage of the proteomic approaches is the capacity to look for multiple differentially expressed proteins in a single study. This review provides an overview of the recent reports describing the different proteomic tools used for the discovery of new protein markers for CRC such as two-dimensional electrophoresis methods, quantitative mass spectrometry-based techniques or protein microarrays. Additionally, we will also focus on the diverse biological samples used for CRC biomarker discovery such as tissue, serum and faeces, besides cell lines and murine models, discussing their advantages and disadvantages, and summarize the most frequently identified candidate CRC markers.

Decreased expression of alpha-L-fucosidase gene FUCA1 in human colorectal tumors.

In previous studies we described a decreased alpha-L-fucosidase activity in colorectal tumors, appearing as a prognostic factor of tumoral recurrence. The aim of this work was to extend the knowledge about tissue alpha-L-fucosidase in colorectal cancer by quantifying the expression of its encoding gene FUCA1 in tumors and healthy mucosa. FUCA1 mRNA levels were measured by RT-qPCR in paired tumor and normal mucosa tissues from 31 patients. For the accuracy of the RT-qPCR results, five candidate reference genes were validated in those samples. In addition, activity and expression of alpha-L-fucosidase in selected matched tumor and healthy mucosa samples were analyzed. According to geNorm and NormFinder algorithms, RPLP0 and HPRT1 were the best reference genes in colorectal tissues. These genes were used for normalization of FUCA1 expression levels. A significant decrease of more than 60% in normalized FUCA1 expression was detected in tumors compared to normal mucosa (p = 0.002). Moreover, a gradual decrease in FUCA1 expression was observed with progression of disease from earlier to advanced stages. These findings were confirmed by Western blot analysis of alpha-L-fucosidase expression. Our results demonstrated diminished FUCA1 mRNA levels in tumors, suggesting that expression of tissue alpha-L-fucosidase could be regulated at transcriptional level in colorectal cancer.

Changes on the Caco-2 secretome through differentiation analyzed by 2-D differential in-gel electrophoresis (DIGE).

Colorectal cancer is still a major health burden worldwide, and its diagnosis has not improved in recent years due to a lack of appropriate diagnostic serum markers. Aiming to find new diagnostic proteins, we applied the proteomic DIGE technology to analyze changes in the secretome before/after differentiation of the colon adenocarcinoma Caco-2 cell line, an accepted in vitro model to study colorectal tumorigenesis. When the secretomes from undifferentiated (tumor-like) and differentiated cells (resembling healthy enterocytes) were compared, we found 96 spots differentially expressed. After MS/MS analysis, 22 spots corresponding to 15 different proteins were identified. Principal component analysis demonstrated these 22 spots could serve as a discriminatory panel between the tumor-like and normal-like cells. Among the identified proteins, the translationally-controlled tumor protein (TCTP), the transforming growth factor-beta-induced protein ig-h3 (TGFßIp), and the Niemann-Pick disease type C2 protein (NPC2) are interesting candidates for future studies focused on their utility as serum biomarkers of colorectal cancer.

Selection of putative colorectal cancer markers by applying PCA on the soluble proteome of tumors: NDK A as a promising candidate.

Aiming to find new tumor markers for colorectal cancer (CRC), we applied proteomic methodologies to compare the soluble sub-proteome of healthy and tumoral colorectal mucosa. Out of 91 differentially expressed proteins, 23 were selected by principal component analysis (PCA) as the major contributors to the overall difference detected. After MS/MS analysis, 16 proteins were identified. From those, we chose 14-3-3-zeta/delta, retinoblastoma-binding protein 4 (RBBP-4), DJ-1, and nucleoside diphosphate kinase A (NDK A) for further studies, on the basis of their levels and known implication in cancer. Specific immunodetection demonstrated only the NDK A levels allowed to differentiate healthy mucosa from tumor tissue in all the patients. Hence, we used the colon cancer cell line Caco-2 to study the relationship between NDK A and colon cell tumorigenesis, finding it over-expressed in undifferentiated (tumor-like) cells regarding the differentiated ones. Noticeably, we also found increased levels of the NDK A in the secretome of tumor-like cells and, as expected, indications of higher levels of NDK A in the serum of CRC patients. In conclusion, the four validated proteins could constitute a panel of tissue markers for CRC, being the NDK A the most interesting candidate for further serum biomarker studies.

Serum CD26 is related to histopathological polyp traits and behaves as a marker for colorectal cancer and advanced adenomas.

BACKGROUND: Serum CD26 (sCD26) levels were previously found diminished in colorectal cancer (CRC) patients compared to healthy donors, suggesting its potential utility for early diagnosis. Therefore we aimed to estimate the utility of the sCD26 as a biomarker for CRC and advanced adenomas in a high-risk group of patients. The relationship of this molecule with polyp characteristics was also addressed. METHODS: sCD26 levels were measured by ELISA in 299 symptomatic and asymptomatic patients who had undergone a colonoscopy. Patients were diagnosed as having no colorectal pathology, non-inflammatory or inflammatory bowel disease, polyps (hyperplastic, non-advanced and advanced adenomas) or CRC. RESULTS: At a 460 ng/mL cut-off, the sCD26 has a sensitivity and specificity of 81.8% (95% CI, 64.5-93.0%) and 72.3% (95% CI, 65.0-77.2%) for CRC regarding no or benign colorectal pathology. Clinicopathological analysis of polyps showed a relationship between the sCD26 and the grade of dysplasia and the presence of advanced adenomas. Hence, a 58.0% (95% CI, 46.5-68.9%) sensitivity detecting CRC and advanced adenomas was obtained, with a specificity of 75.5% (95% CI, 68.5-81.0%). CONCLUSIONS: Our preliminary results show that measurement of the sCD26 is a non-invasive and reasonably sensitive assay, which could be combined with others such as the faecal occult blood test for the early diagnosis and screening of CRC and advanced adenomas. Additional comparative studies in average-risk populations are necessary.

Soluble CD26 levels and its association to epidemiologic parameters in a sample population.

INTRODUCTION: Previous studies have suggested the use of soluble CD26 (sCD26) as a tumour marker for the detection of colorectal cancer (CRC) and advanced adenomas. The aim of this study was to assess the sCD26 concentration in a large cohort to evaluate its association to epidemiologic parameters and CRC-related symptoms/pathologies. SUBJECTS AND METHODS: Serum samples were collected from 2,754 putatively healthy individuals with ages ranging from 30-65 years, and with personal or familial history of polyps, CRC and/or CR symptoms. sCD26 levels were measured by ELISA. RESULTS: No association was found between the sCD26 concentration and age (< 50 and 50), the personal or familial history of polyps or CRC, rectal bleeding, haemorrhoids or diverticula. However, sCD26 was related to non-inflammatory benign pathologies (excluding rectal bleeding, changes in bowel habits, haemorrhoids, diverticula) and to inflammatory benign pathologies. DISCUSSION: Our results confirm that the sCD26 can be easily offered and evaluated in a large cohort. Additionally, the validation of sCD26 as a tumour marker for screening and case-finding purposes requires a further comparison with an established non-invasive test like the faecal occult blood.

How the measurements of a few serum markers can be combined to enhance their clinical values in the management of cancer.

The number of potential molecular markers is constantly increasing. However, for most malignancies there is no simple test to detect early-stage tumours that is useful for screening purposes because most biomarkers have poor sensitivity or specificity, or other clinical value. One approach to increase their value is to measure several biomarkers at a time. The additional information should always yield a test more able to distinguish between patients and healthy individuals, and ideally between different kinds of tumour. Our work in colorectal, lung, and head and neck cancer, illustrates the evolution of this idea. A test in which ELISAs for key serum markers are arrayed based on immunoblot technology or flow cytometric beads is suggested because these techniques are more transferable to practical application in clinical decision making. Moreover, the multivariate data obtained from such a test can easily be managed with statistical methods already developed in the fields of genomics and proteomics.

Identification of hydrophobic proteins as biomarker candidates for colorectal cancer.

Nowadays, colorectal cancer is one of the major causes of cancer death in Western countries. Due to the lack of biomarkers with clinical utility for this pathology, and considering that membrane and hydrophobic proteins have not been studied in depth, we performed a prefractionation of colorectal tissues prior to two-dimensional gel electrophoresis in order to identify hydrophobic proteins differentially expressed in colorectal cancer patients. Fractions enriched in hydrophobic proteins were obtained from healthy mucosa and tumor tissue by a specific extraction method based on temperature-dependent phase partitioning with Triton X-114. Proteins were separated by two-dimensional gel electrophoresis and gels were silver-stained, scanned and compared using the PDQuest software. Those spots presenting significantly different abundance were submitted to mass spectrometry for protein identification. Alterations in the expression of cytoskeletal proteins, including a decrease of vimentin and the absence of desmin, were found. We also detected alterations in antioxidant and transport proteins, chaperones, and in two isoforms of the calcium-binding protein S100A6. On the other hand, vimentin was chosen to corroborate the electrophoretic results by specific immunodetection. Most of the altered proteins have been related to cellular membranes, many of them to lipid rafts microdomains in the plasma membrane, and they have also been implicated in the control of cell proliferation, apoptosis, or metastasis. In conclusion, all the proteins found altered in colorectal tumor samples could be considered as candidates for future studies focused on their utility as markers for colorectal diagnosis and prognosis, or as targets for colorectal cancer therapy.

Differential expression of serum clusterin isoforms in colorectal cancer.

Clusterin is an enigmatic protein altered in tumors of colorectal cancer patients. Because there is no information available about serum clusterin regarding this pathology, we applied proteomic techniques to analyze its isoforms in donors and patients. First we separated serum proteins through concanavalin A, obtaining a fraction with non- and O-glycosylated proteins (FI) and a second fraction enriched in N-glycoproteins (FII) wherein clusterin was supposed to elute on the basis of its glycosylation. Surprisingly analysis of the FI fraction revealed the existence of an unexpected and aberrantly N-glycosylated clusterin that was overexpressed in patients and comprised at least five isoforms with different isoelectric points. On the other hand, two-dimensional electrophoretic analysis of the clusterin eluted in FII detected one isoform that was increased and 15 isoforms that were decreased or absent in serum of patients. Finally immunoquantification by slot blot showed that in total serum and in FI the clusterin levels were significantly increased in patients, whereas in FII there was no significant variation. Therefore, serum clusterin and some of its isoforms could have a potential value as colorectal tumor markers and are interesting subjects for biomarker studies.

Application of relative warp analysis to the evaluation of two-dimensional gels in proteomics: studying isoelectric point and relative molecular mass variation.

We propose a geometric-morphometrics method (relative warp analysis) to be used in proteomic comparisons. This approach was applied to a dataset from a comparison between 5 controls and 5 patients with colorectal cancer disease published elsewhere. The spots in the 2-D maps were used as landmarks in a morphometric study, and the differences in shape (spot distribution) among them were obtained. The shape variables were used to compare controls and patients. These components mostly ignore random or experimental effects affecting all the proteins in any of the two dimensions studied. Furthermore, the method allows the researcher to find those proteins which contributed the most to the local shape component detected. Applying this approach, we detected variations in the position (isoelectric point and/or relative molecular mass) of some spots that may reflect differences in the amino acidic sequence or post-translational modifications.

Preoperative serum alpha-L-fucosidase activity as a prognostic marker in colorectal cancer.

OBJECTIVES: The aim of this study was to examine the prognostic value of the preoperative serum alpha-L-fucosidase (AFU) activity in colorectal cancer and to assess whether it could add prognostic information that Dukes' stages do not give. METHODS: A postoperative follow-up of 137 colorectal cancer patients was performed, and survival analyses were carried out to evaluate the impact of AFU activity on disease-free survival. Dukes' stage classification, preoperative serum carcinoembryonic antigen levels and six other clinicopathological features of the patients were also analysed. RESULTS: In previous studies, we have stressed the diagnostic value of AFU activity in preoperatively obtained serum from colorectal cancer patients. In the present work, we have found that the enzymatic activity of serum AFU was not related to the Dukes' stage of the primary tumour, but it was associated with the type of metastasis and recurrence of the disease. The mean value of preoperative serum AFU activity was higher in patients with distant metastases than in those with lymph node or peritoneal metastases, or without metastasis (p = 0.034). After a mean postoperative follow-up period of 22 months, three groups of patients with different recurrence rates could be distinguished (p = 0.0014). Similar results were found when only patients in Dukes' stage B (p = 0.0439) or C (p = 0.0122) were considered. CONCLUSIONS: According to our findings, serum AFU activity appears to be a good prognostic factor of tumour recurrence in colorectal carcinoma. Furthermore, patients in Dukes' stage B or C at high or very high risk of tumour recurrence could be spotted.

Lectin isolation and detection of N-glycoproteins bearing sialic acid and L-fucose residues in human colorectal mucosa and in adenocarcinoma biopsies.

A method to improve the reactivity to specific lectins of N-glycoproteins isolated from human colorectal mucosa and from adenocarcinoma biopsies was developed using a combination of techniques. Total protein extracts were subjected to affinity chromatography, using the immobilised lectin Concanavalin A coupled to Sepharose, by fast performance liquid chromatography (FPLC). N-glycoprotein enriched fractions were resolved by SDS-PAGE, transferred to PVDF membranes and incubated with various lectins. Digoxigenin-conjugated SNA I and MAA lectins were used to detect sialic acid residues. Biotin-conjugated UEA I lectin was used to detect L-fucose residues. By this method, lectin-binding N-glycoproteins were found in a broad relative molecular mass (Mr) range (from 47 to 205 kDa). No tissue-specific N-glycoproteins were observed when human colorectal mucosa and adenocarcinoma samples were compared.