High-Throughput Mass Spectrometry Analysis of N-Glycans and Protein Markers after FUT8 Knockdown in the Syngeneic SW480/SW620 Colorectal Cancer Cell Model.

Disruption of the glycosylation machinery is a common feature in many types of cancer, and colorectal cancer (CRC) is no exception. Core fucosylation is mediated by the enzyme fucosyltransferase 8 (FucT-8), which catalyzes the addition of α1,6-l-fucose to the innermost GlcNAc residue of N-glycans. We and others have documented the involvement of FucT-8 and core-fucosylated proteins in CRC progression, in which we addressed core fucosylation in the syngeneic CRC model formed by SW480 and SW620 tumor cell lines from the perspective of alterations in their N-glycosylation profile and protein expression as an effect of the knockdown of the FUT8 gene that encodes FucT-8. Using label-free, semiquantitative mass spectrometry (MS) analysis, we found noticeable differences in N-glycosylation patterns in FUT8-knockdown cells, affecting core fucosylation and sialylation, the Hex/HexNAc ratio, and antennarity. Furthermore, stable isotopic labeling of amino acids in cell culture (SILAC)-based proteomic screening detected the alteration of species involved in protein folding, endoplasmic reticulum (ER) and Golgi post-translational stabilization, epithelial polarity, and cellular response to damage and therapy. This data is available via ProteomeXchange with identifier PXD050012. Overall, the results obtained merit further investigation to validate their feasibility as biomarkers of progression and malignization in CRC, as well as their potential usefulness in clinical practice.

Core Fucosylation Mediated by the FucT-8 Enzyme Affects TRAIL-Induced Apoptosis and Sensitivity to Chemotherapy in Human SW480 and SW620 Colorectal Cancer Cells.

Epithelial cells can undergo apoptosis by manipulating the balance between pro-survival and apoptotic signals. In this work, we show that TRAIL-induced apoptosis can be differentially regulated by the expression of α(1,6)fucosyltransferase (FucT-8), the only enzyme in mammals that transfers the α(1,6)fucose residue to the pentasaccharide core of complex N-glycans. Specifically, in the cellular model of colorectal cancer (CRC) progression formed using the human syngeneic lines SW480 and SW620, knockdown of the FucT-8-encoding FUT8 gene significantly enhanced TRAIL-induced apoptosis in SW480 cells. However, FUT8 repression did not affect SW620 cells, which suggests that core fucosylation differentiates TRAIL-sensitive premetastatic SW480 cells from TRAIL-resistant metastatic SW620 cells. In this regard, we provide evidence that phosphorylation of ERK1/2 kinases can dynamically regulate TRAIL-dependent apoptosis and that core fucosylation can control the ERK/MAPK pro-survival pathway in which SW480 and SW620 cells participate. Moreover, the depletion of core fucosylation sensitises primary tumour SW480 cells to the combination of TRAIL and low doses of 5-FU, oxaliplatin, irinotecan, or mitomycin C. In contrast, a combination of TRAIL and oxaliplatin, irinotecan, or bevacizumab reinforces resistance of FUT8-knockdown metastatic SW620 cells to apoptosis. Consequently, FucT-8 could be a plausible target for increasing apoptosis and drug response in early CRC.

Inhibition of α(1,6)fucosyltransferase: Effects on Cell Proliferation, Migration, and Adhesion in an SW480/SW620 Syngeneic Colorectal Cancer Model.

The present study explored the impact of inhibiting α(1,6)fucosylation (core fucosylation) on the functional phenotype of a cellular model of colorectal cancer (CRC) malignization formed by the syngeneic SW480 and SW620 CRC lines. Expression of the FUT8 gene encoding α(1,6)fucosyltransferase was inhibited in tumor line SW480 by a combination of shRNA-based antisense knockdown and Lens culinaris agglutinin (LCA) selection. LCA-resistant clones were subsequently assayed in vitro for proliferation, migration, and adhesion. The α(1,6)FT-inhibited SW480 cells showed enhanced proliferation in adherent conditions, unlike their α(1,6)FT-depleted SW620 counterparts, which displayed reduced proliferation. Under non-adherent conditions, α(1,6)FT-inhibited SW480 cells also showed greater growth capacity than their respective non-targeted control (NTC) cells. However, cell migration decreased in SW480 after FUT8 knockdown, while adhesion to EA.hy926 cells was significantly enhanced. The reported results indicate that the FUT8 knockdown strategy with subsequent selection for LCA-resistant clones was effective in greatly reducing α(1,6)FT expression in SW480 and SW620 CRC lines. In addition, α(1,6)FT impairment affected the proliferation, migration, and adhesion of α(1,6)FT-deficient clones SW480 and SW620 in a tumor stage-dependent manner, suggesting that core fucosylation has a dynamic role in the evolution of CRC.

Haptoglobin expression in human colorectal cancer.

Aims and experimental design. The acute-phase protein haptoglobin (Hp) has been recently detected in colorectal cancer (CRC) tissue, where its expression correlates with metastasis. Recently, we identified Hp as a CDw75 antigen-expressing protein in colorectal tissue. To deepen the knowledge of this protein in CRC, we studied the expression of Hp in healthy and tumour tissue specimens from 62 CRC patients by immunohistochemistry and Western blotting, as well as in the Caco-2 and HT-29 CRC cell lines by quantitative PCR, immunofluorescence microscopy and flow cytometry. Results and discussion. Hp immuno-positive staining was absent in the 18 healthy colorectal specimens analysed, whereas it was observed in 24% (15/62) of the tumour specimens as cytoplasmic granules within cancer cells. Furthermore, Hp expression in CRC was associated with Dukes' stage and the presence of metastasis in our population of study. In vitro cultured Caco-2 and HT-29 cells expressed mRNA for Hp and the protein was detected at the cell surface. Conclusions. This study confirms the expression of Hp in CRC, both in vivo and in vitro, and provides further evidence of its association with disease progression and metastasis.

Identification of proteins with the CDw75 epitope in human colorectal cancer.

The CDw75 epitope is an α(2,6) sialylated antigen overexpressed in colorectal cancer (CRC), where its expression correlates with the progression of the disease. The CDw75 epitope is located mainly in N-glycoproteins, whose identity remains unknown. The aim of the present study was to identify proteins with the CDw75 epitope as a strategy to deepen the understanding of molecular pathogenesis of CRC and to identify novel biomarkers for this disease. For this purpose, a two-dimensional electrophoresis approach was employed. Protein spots in the gels were matched to the corresponding CDw75 positive spots in the immunoblotted polyvinylidene difluoride membranes, and further identification of the protein species was performed by mass spectrometry. Additionally, one-dimensional western blotting experiments were performed to verify the expression of these candidate proteins in the colorectal tissue and their coincidence in molecular mass with the CDw75-positive bands. The findings of the present study indicate that haptoglobin and the keratins 8 (K8) and 18 (K18) are proteins with the CDw75 epitope in the colorectal tissue from CRC patients and also suggest novel functions and cellular locations for these proteins in the colorectal tissue and in relation to CRC.

Screening of enzymatic synthesis and expression of Lewis determinants in human colorectal carcinoma.

BACKGROUND: Although colorectal carcinogenesis has been intensively studied, the published investigations do not provide a consistent description of how different carbohydrate determinants of colorectal epithelium are modified in colorectal cancer (CRC). OBJECTIVE: This study is an attempt to characterize the terminal fucosylation steps responsible for the synthesis of mono- Le(a)/Le(x)- and difucosylated -Le(b)/Le(y)- Lewis antigens in healthy and tumour CRC tissue. METHODS: An immunohistochemical study of Lewis antigens' expression was undertaken, along with screening of the fucosyltransferase (FT) activities involved in their synthesis, on healthy and tumour samples from 18 patients undergoing CRC. RESULTS: Analysis of alpha(1,2/3/4)FT activities involved in the sequential fucosylation of cores 1 and 2 showed significant increases in tumour tissue. Expressed as microU/mg and control vs. tumour activity (pfrom Wilcoxon's test), the FT activities for Le(a)/Le(b) synthesis were: lacto-N-biose alpha(1,2)/alpha(1,4)FT, 65.4 ± 19.0 vs. 186 ± 35.1 (p< 0.005); lacto-N-fucopentaose 1 alpha(1,4)FT, 64.9 ± 11.9 vs. 125.4 ± 20.7 (p< 0.005); Le(a) alpha(1,2)FT, 56.2 ± 7.2 vs. 130.5 ± 15.6 (p< 0.001). Similarly, for Le(x)/Le(y) synthesis were: N-acetyllactosamine alpha(1,2)-/alpha(1,3)FT, 53.4 ± 12.2 vs. 108.1 ± 18.9 (p< 0.001); 2'-Fucosyl-N-acetyllactosamine alpha(1,3)FT, 61.3 ± 10.7 vs. 126.4 ± 22.9 (p< 0.001); 2'-Fucosyllactose alpha(1,3)FT, 38.9 ± 10.9 vs. 143.6 ± 28.9 (p< 0.001); 2'-Methyllactose alpha(1,3)FT, 30.9 ± 4.8 vs. 66.1 ± 8.1 (p< 0.005); and Le(x) alpha(1,2)FT, 54.3 ± 11.9 vs. 88.2 ± 14.4 (p< 0.001). Immunohistochemical Le(y) expression was increased (p< 0.01 according to Wilcoxon's test) in tumour tissue, with 84.6% of specimens being positive: 7.7% weak, 15.4% moderate and 61.5% high intensity. CONCLUSIONS: Results suggest the activation of the biosynthesis pathways of mono- and difucosylated Lewis histo-blood antigens in tumour tissue from CRC patients, leading to the overexpression of Le(y), probably at the expense of Le(x).

Screening of enzymatic synthesis and expression of Lewis determinants in human colorectal carcinoma

BACKGROUND: Although colorectal carcinogenesis has been intensively studied, the published investigations do not provide a consistent description of how different carbohydrate determinants of colorectal epithelium are modified in colorectal cancer (CRC). OBJECTIVE: This study is an attempt to characterize the terminal fucosylation steps responsible for the synthesis of mono- (Lea/Lex) and difucosylated (Leb/Ley) Lewis antigens in healthy and tumour CRC tissue. METHODS: An immunohistochemical study of Lewis antigens' expression was undertaken, along with screening of the fucosyltransferase (FT) activities involved in their synthesis, on healthy and tumour samples from 18 patients undergoing CRC. RESULTS: Analysis of a(1,2/3/4)FT activities involved in the sequential fucosylation of cores 1 and 2 showed significant increases in tumour tissue. Expressed as mU/mg and control vs. tumour activity (p from Wilcoxon's test), the FT activities for Lea/ Leb synthesis were: lacto-N-biose a(1,2)/a(1,4)FT, 65.4 ± 19.0 vs. 186 ± 35.1 (p < 0.005); lacto-N-fucopentaose 1 a(1,4)FT, 64.9 ± 11.9 vs. 125.4 ± 20.7 (p < 0.005); Lea a(1,2)FT, 56.2 ± 7.2 vs. 130.5 ± 15.6 (p < 0.001). Similarly, for Lex/Ley synthesis were: N-acetyllactosamine a(1,2)-/a(1,3)FT, 53.4 ± 12.2 vs. 108.1 ± 18.9 (p < 0.001); 2'-Fucosyl-N-acetyllactosamine a(1,3)FT, 61.3 ± 10.7 vs. 126.4 ± 22.9 (p < 0.001); 2'-Fucosyllactose a(1,3)FT, 38.9 ± 10.9 vs. 143.6 ± 28.9 (p < 0.001); 2'-Methyllactose a(1,3) FT, 30.9 ± 4.8 vs. 66.1 ± 8.1 (p < 0.005); and Lex a(1,2)FT, 54.3 ± 11.9 vs. 88.2 ± 14.4 (p < 0.001). Immunohistochemical Ley expression was increased (p < 0.01 according to Wilcoxon's test) in tumour tissue, with 84.6% of specimens being positive: 7.7% weak, 15.4% moderate and 61.5% high intensity. CONCLUSIONS: Results suggest the activation of the biosynthesis pathways of mono- and difucosylated Lewis histo-blood antigens in tumour tissue from CRC patients, leading to the overexpression of Ley, probably at the expense of Lex

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FX enzyme and GDP-L-Fuc transporter expression in colorectal cancer.

AIMS: Fucosylation is regulated by fucosyltransferases, the guanosine diphosphate-L-fucose (GDP-L-Fuc) synthetic pathway, and the GDP-L-fucose transporter (GDP-L-Fuc Tr). We have reported previously an increased level of α(1,6)fucosyltransferase activity and expression in colorectal cancer (CRC). The present study aimed to analyse the expression profiles of the FX enzyme and GDP-L-Fuc Tr in a cohort of operated CRC patients to elucidate their role in α(1,6)fucosylation in this neoplasm. METHODS AND RESULTS: We assessed the immunohistochemical expression of FX and GDP-L-Fuc Tr in a series of tumour samples and healthy tissues from CRC specimens. FX expression was observed in 58 of 91 (63.7%) tumours and 23 of 28 (82.1%) corresponding healthy samples. GDP-L-Fuc Tr expression was detected in 86 of 102 (84.3%) colorectal tumours, and 13 of 27 (48.1%) healthy tissue specimens. The expression of GDP-L-Fuc Tr was statistically higher in tumours than in healthy tissues (P < 0.001). A correlation was found between FX and GDP-L-Fuc Tr expression in tumour samples (P = 0.003). CONCLUSION: GDP-L-Fuc Tr overexpression in the tumour tissue of CRC patients suggests that GDP-L-Fuc transport to the Golgi apparatus may be an important factor associated with increased α(1,6)fucosylation in CRC.

Identification of α(1,6)fucosylated proteins differentially expressed in human colorectal cancer.

BACKGROUND: A universal hallmark of cancer cells is the change in their glycosylation phenotype. One of the most frequent alterations in the normal glycosylation pattern observed during carcinogenesis is the enhancement of α(1,6)linked fucose residues of glycoproteins, due to the up-regulation of the α(1,6)fucosyltransferase activity. Our previous results demonstrated the specific alteration of this enzyme activity and expression in colorectal cancer, suggesting its implication in tumour development and progression. METHODS: In the current work we combined a LCA-affinity chromatography with SDS-PAGE and mass spectrometry in order to identify α(1,6)fucosylated proteins differentially expressed in colorectal cancer. This strategy allowed the identification of a group of α(1,6)fucosylated proteins candidates to be involved in CRC malignancy. RESULTS: The majority of the identified proteins take part in cell signaling and interaction processes as well as in modulation of the immunological response. Likewise, we confirmed the increased expression of GRP94 in colorectal cancer tissue and the significant down-regulation of the IgGFcBP expression in tumour cells. CONCLUSION: All these results validate the importance of core-fucosylated proteins profile analysis to understand the mechanisms which promote cancer onset and progression and to discover new tumour markers or therapeutic targets.

Disease-free survival of colorectal cancer patients in relation to CDw75 antigen expression.

OBJECTIVE: CDw75 is an α(2,6)-sialylated antigen associated with a poor prognosis in gastric cancer. In the present study, we examined if CDw75 expression in colorectal cancer (CRC) predicts tumour recurrence. Besides, we evaluated CDw75 expression in different colorectal tissue specimens to clarify their role in tumour development and progression. METHODS: We analyzed CDw75 expression in 34 specimens of healthy disease-free colorectal mucosa, 19 specimens of inflammatory colorectal mucosa, 73 colorectal adenomas, 35 specimens of healthy tissue and 101 specimens of tumoural tissue from CRC patients. RESULTS: None of the healthy disease-free and inflammatory colorectal mucosa specimens showed the presence of the epitope. CDw75 was expressed in 26% of the colorectal adenomas. In healthy and tumoural tissue from CRC patients, CDw75 was detected in 22.9% and 82.2% of the specimens, respectively. CDw75 expression in tumoural tissue was correlated with growth pattern (p = 0.044), Dukes stage (p = 0.002), TNM stage (p = 0.020) and distant metastasis (p = 0.005). Survival analysis showed that CDw75 expression is not associated with tumour recurrence. CONCLUSION: CDw75 expression in CRC is not a prognostic factor for predicting disease-free survival. Nevertheless, CDw75 expression may be a good marker of tumour progression and of the malignant potential of CRC.

α(1,6)Fucosyltransferase expression is an independent prognostic factor for disease-free survival in colorectal carcinoma.

We previously reported that α(1,6)fucosyltransferase (Enzyme class 2.4.1.68) activity and expression are increased in colorectal cancer, suggesting a role for this enzyme in tumor development and progression. However, the possible impact of α(1,6)fucosyltransferase activity or expression on clinical outcomes in colorectal cancer patients has never been studied. Thus, the present study was conducted to determine the value of α(1,6)fucosyltransferase as a prognostic factor for colorectal cancer. α(1,6)Fucosyltransferase expression was analyzed using immunohistochemistry in 141 colorectal tumors, and α(1,6)fucosyltransferase activity was determined in 39 tumors. A complete standardized follow-up of patients was documented until the end of the observation period of 5 years or patient death. Univariate analysis demonstrated the absence of a correlation between enzyme activity and disease evolution. However, in patients with moderate or strong α(1,6)fucosyltransferase expression, a significant decrease in the overall (P = .04) and disease-free (P = .03) survival rates was observed. In addition, when local and distant disease recurrence were considered separately, enzyme expression was found to correlate with local tumor recurrences (P = .01). Furthermore, multivariate analysis showed that α(1,6)fucosyltransferase expression has independent value for predicting tumor recurrences and, specifically, local recurrences. These findings suggest that α(1,6)fucosyltransferase expression may be a good indicator of poor prognosis in colorectal cancer and, therefore, a helpful tool to choose the most effective treatment.

Synthesis and expression of CDw75 antigen in human colorectal cancer.

BACKGROUND: Increased ST6Gal I activity has been associated with the alpha(2,6)sialylation enhancement of membrane glycoconjugates observed in metastatic colorectal carcinomas (CRC). Siaalpha(2,6)Galbeta(1,4)GlcNAc sequence, known as CDw75, is a sialylated carbohydrate determinant generated by the ST6Gal I. This epitope has been reported to be associated with the progression of gastric and colorectal tumours, hence there are only a few conclusive studies to date. METHODS: By radioisotopic techniques we evaluated the ST6Gal I activity in healthy, transitional and tumour tissues from 43 patients with CRC. By immunohistochemistry we assessed the CDw75 expression in 25 colorectal adenomas, 43 tumours, 13 transitional and 28 healthy tissues of CRC patients. RESULTS: ST6Gal I activity was likewise found to be statistically higher in tumour tissue respect to healthy tissue from CRC patients. CDw75 expression was positive in 20% of colorectal adenomas. Furthermore, 70% of tumour specimens and 8.3% of transitional specimens were positive for CDw75 expression, whereas none of the healthy ones showed the presence of the epitope. CONCLUSION: The major contribution of this study is the inclusion of data from transitional tissue and the analysis of CDw75 antigen expression in CRC and in colorectal adenomas, little known so far. ST6Gal I activity and CDw75 antigen expression were increased in CRC. Although their comparison did not reach the statistical significance, a great extent of patients showed both, an enhanced tumour ST6Gal I activity and an increased CDw75 expression in the tumour tissue. So, these two variables may play a role in malignant transformation. The expression of CDw75 in colorectal adenomas suggests that this antigen may be a tumour marker in CRC.

Superoxide dismutase and catalase: tissue activities and relation with age in the long-lived species Margaritifera margaritifera.

Free radicals are extremely reactive and produce damage and modify cell functions. Furthermore, superoxide dismutase and catalase are believed to play a key role in the enzymatic defence of the cells. Indeed, some authors have argued that reduced free-radical damage could explain increased longevity. Margaritifera margaritifera is one of the longest-lived animals in the world (up to 100-200 years). Furthermore, this organism may serve as a useful model for gerontologists interested in exploring the mechanisms that promote long life and the slowing of senescence. The present study estimated for the first time individual enzymatic activity for superoxide dismutase isozymes (Cu,Zn-SOD and Mn-SOD) and catalase in tissue preparations of gills, digestive glands and mantles of two natural populations of M. margaritifera. Superoxide dismutase activities showed significant differences in the tissues analysed of specimens from the same river and in specimens from different rivers for the same tissue. Catalase activity levels also showed significant variation, but differences among tissues, within tissues or between rivers were of relatively little interest. We failed to find any relationship between individual enzymatic activities and the age estimated for each mussel. Indeed, the wide variation found in activity levels can be principally interpreted as an adaptation to the unpredictable and changing nature of freshwater natural habitats.

Expression and enzyme activity of alpha(1,6)fucosyltransferase in human colorectal cancer.

Changes in enzyme activity and the expression levels of alpha(1,6)fucosyltransferase [alpha(1,6)FT] have been reported in certain types of malignant transformations. To develop a better understanding of the role of alpha(1,6)FT in human colorectal carcinoma (CRC), we analysed the enzyme activity in healthy and tumour tissues. alpha(1,6)FT activity was considerably higher in tumour tissue than in healthy tissue and was related to gender, lymph node metastasis, type of growth and tumour stage. We also observed a significant increase in the alpha(1,6)FT expression in tumour tissues as compared to healthy and transitional tissues, inflammatory lesions and adenomas. The immunohistochemical expression in tumour tissues was correlated with the degree of infiltration through the intestinal wall. Finally, a statistical correlation was found between enzyme activity and expression obtained by Western blot in colorectal tumours when compared in the same patient. All these findings demonstrate an alteration of alpha(1,6)FT activity and expression in CRC.

Elevation of ST6Gal I activity in malignant and transitional tissue in human colorectal cancer.

OBJECTIVES: The aim of the present study was to investigate the activity of CMP-NeuAc:Galbeta(1,4)GlcNAc sialyltransferase (ST6Gal I) in colorectal cancer (CRC). METHODS: ST6Gal I activity was determined in healthy, transitional and tumor tissues from the same patient using asialotransferrin and N-acetyllactosamine as acceptors. RESULTS: ST6Gal I activities with asialotransferrin (n = 85) and N-acetyllactosamine (n = 40) as acceptors were statistically significantly enhanced in CRC tissue compared with healthy mucosa from the same patient (p = 0.001). Using transitional tissue (n = 27), enhancement versus healthy tissue was observed (p < 0.05). A positive correlation was found between ST6Gal I activity with N-acetyllactosamine and asialotransferrin in healthy (n = 32), tumorous (n = 32) and transitional tissue (n = 27), supporting the fact that the same enzyme was detected using both acceptors. Furthermore, we studied the relationship between some patients' clinicopathological features and ST6Gal I activity. Although the differences were not statistically significant, the levels of ST6Gal I activity in tumorous and transitional tissues varied with the histological grade of the tumor; however, we failed to find a correlation with the AJCC tumor classification. CONCLUSIONS: This work reports enhanced ST6Gal I activity in tumor and transitional tissues from CRC patients. However, our overall results suggest that ST6Gal I activity is not indicative of the patient's outcome.

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.

Clinical interest of the combined use of serum CD26 and alpha-L-fucosidase in the early diagnosis of colorectal cancer.

The purpose of this study was to assess if the combination of CD26 and alpha-L-fucosidase has a role in the diagnosis of colorectal cancer, paying particular attention to the stages in which the tumour is not yet disseminated. CD26 concentration and alpha-L-fucosidase activity were determined in sera from 110 colorectal cancer patients and 46 donors. The combination of CD26 and alpha-L-fucosidase showed a specificity of 100% with a sensitivity of 64% in the diagnosis of colorectal cancer. Interestingly, the combination of both markers had a sensitivity of 75% in the stage I at the highest specificity (100%), providing also high sensitivity levels for the other non-disseminated stages (66% for stages II and III). In conclusion, the combined use of CD26 and alpha-L-fucosidase offers high sensitivity with high specificity in the diagnosis of colorectal cancer, especially at the earliest stage (TNM I).