Serum sCD26 for colorectal cancer screening in family-risk individuals: comparison with faecal immunochemical test.

BACKGROUND: The development of specific screening programs for individuals with a family history of colorectal cancer (CRC) is a priority. This study evaluates the diagnostic performance of serum soluble CD26 (sCD26) in family-risk individuals and compares this marker with the faecal immunochemical test for the detection of advanced neoplasia (AN) (CRC or advanced adenomas; AA). METHODS: Five hundred and sixteen asymptomatic individuals with at least one first-degree relative with CRC were included. Serum sCD26 was measured in all the individuals who also underwent a colonoscopy (53 AA and four cancer cases were found) and a faecal immunochemical test. RESULTS: Setting specificity to 90% and 95%, respectively, sCD26 showed a sensitivity of 39.6% and 28.3% for AA, and of 42.1% and 28.1% for AN. The combination of sCD26 and the faecal test detected AA and AN with a 52.8% and 56.1% sensitivity, corresponding to 93.5% specificity. CONCLUSIONS: The combination of serum sCD26 and the faecal blood test could result a valuable strategy for detecting AN in familial-risk CRC screening.

Calprotectin: a novel biomarker for the diagnosis of pleural effusion.

BACKGROUND: Novel non-invasive biomarkers for the precise diagnosis of malignancy in pleural effusion (PE) are needed. The aim of this study was to determine the diagnostic accuracy of calprotectin for predicting malignancy in patients with exudative PE. METHODS: Calprotectin concentration was measured in 156 individuals diagnosed with exudative PE (67 malignant and 89 benign). Calprotectin accuracy for discriminating between malignant and benign PE was evaluated using receiver operating characteristic (ROC) curves. Univariate and multivariate logistic regression were performed to test the association between calprotectin levels and malignant PE. RESULTS: Calprotectin levels were significantly lower in malignant pleural fluid (257.2 ng ml(-1), range: 90.7-736.4) than benign effusions (2627.1 ng ml(-1), range: 21-9530.1). The area under the curve was 0.963. A cutoff point of ≤ 736.4 ng ml(-1) rendered a sensitivity of 100%, with a specificity of 83.15%, which could prove useful to delimit those patients with negative cytology tests that should be referred for more invasive diagnostic procedures. Logistic regression demonstrated a strong association between calprotectin and malignancy (adjusted OR 663.14). CONCLUSION: Calprotectin predicts malignancy in pleural fluid with high accuracy and could be a good complement to cytological methods.

Secreted clusterin in colon tumor cell models and its potential as diagnostic marker for colorectal cancer.

We studied the specific changes of the secreted protein clusterin and its cytoplasmic precursor regarding colorectal tumorigenesis, using in vitro differentiation of Caco-2 cells. In tumor-like stage, we observed an overexpression of both precursor and secreted clusterin, corroborated in the cell line SW-480. Noticeably, SW-620 cells (from a tumoral node, thus with metastatic capacity) did not show overexpression of either precursor or secreted clusterin, suggesting a downregulation related to local metastasis. We further investigated clusterin in serum, finding a significant increase in colorectal cancer patients, with 81% sensitivity, 79% specificity, and an area under the ROC curve of 0.85.

Absence of activating mutations in the EGFR kinase domain in Spanish head and neck cancer patients.

The discovery of kinase domain mutations in the epidermal growth factor receptor gene (EGFR) in never-smoker patients, associated with an increased sensitivity to tyrosine kinase inhibitors (TKIs) such as gefitinib or erlotinib, has been one of the most relevant findings ever in non-small cell lung carcinomas (NSCLC). Since treatment with TKIs has furthermore shown a clinical benefit in head and neck squamous cell carcinoma (HNSCC) patients, we hypothesized that these mutations could also be present in this neoplasia. Current studies looking for EGFR mutations in HNSCC are limited and results are still controversial. In this work, we screened for EGFR tyrosine kinase mutations in tumour DNA obtained from 31 Spanish patients with HNSCC by PCR-single-strand conformational polymorphism analysis. None of the patients displayed a somatic EGFR mutation, previously described in NSCLC, but other DNA sequence variations were found in 9 of 31 HNSCC patients. Accordingly, activating EGFR mutations in HNSCC patients seem to be a rare event in Spanish patients, suggesting that there is little room for the administration of TKIs in HNSCC based on the presence of these mutations. Additional investigations about EGFR amplification are indicated to establish a potential relationship between EGFR overexpression and the response to anti-EGFR therapies.

Fucose levels in sera and in tumours of colorectal adenocarcinoma patients.

Fucose levels were determined in normal and tumour-derived tissues from patients with colorectal adenocarcinoma. Total, free and bound fucose were significantly higher (P < 0.001) in the tumoral tissue. Comparison of serum fucose levels between patients with colorectal cancer and control subjects indicated no statistically significant differences in total, free and bound fucose expressed as nmol/ml serum. However, when total and bound fucose were normalized to total protein (nmol/mg protein), an elevation in both parameters was found in colorectal cancer patients, although only that for total fucose was statistically significant (P < 0.001). A clear association between fucose content and clinical stage of patients according to the Dukes' classification can not be established, although patients at stage C showed higher levels of fucose both in serum and tissue. The current investigation provides direct evidence of a relationship between the elevation of fucose and the presence of tumour in colorectal cancer patients.

Sialic acid levels in serum and tissue from colorectal cancer patients.

Sialic acid levels were determined in serum and in both normal and tumour-derived tissues from 30 patients with colorectal cancer. Total sialic acid (TSA), bound sialic acid (BSA),TSA normalised to total protein (TSA/TP) and BSA normalised to total protein (BSA/TP) were significantly higher (P < 0.0001) in sera from patients than in normal subjects. We found a trend of increasing serum sialic acid levels (TSA/TP and BSA/TP) as the malignancy became more severe (i.e. Dukes' stages A to C). Comparison of sialic acid levels between normal and tumour-derived colorectal tissues indicated no statistically significant differences in TSA, BSA or FSA (free sialic acid) levels between both tissues; however, TSA/TP and BSA/TP values were significantly decreased (P < 0.05) in the tumoral tissue. In this study, the possible relation between serum and tumour sialic acid levels in colorectal cancer patients was investigated. Our results showed that in these patients there was no correlation between serum BSA and tumour BSA levels.

Purification and characterization of acid beta-D-galactosidase from rabbit spleen.

beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.

Clinical significance of preoperative serum sialic acid levels in colorectal cancer: utility in the detection of patients at high risk of tumor recurrence.

This study was conducted to evaluate the significance of preoperative serum sialic acid levels in the diagnosis and prognosis of colorectal cancer (CRC). Total sialic acid (TSA) was determined by the thiobarbituric acid method and normalized to total protein (TP). A postoperative follow-up of CRC patients classified as Dukes' stages A, B or C was performed and survival analysis was carried out to evaluate the impact of sialic acid levels on tumor recurrence. Our diagnostic studies indicate that TSA/TP is a better marker than either TSA or carcinoembryonic antigen (CEA), especially for the detection of CRC patients at an early stage. At a cutoff of 30.90 nmol/mg of protein, TSA/TP showed a sensitivity of 85% with a specificity of 97% to discriminate CRC patients from healthy donors. In survival analysis, both TSA and TSA/TP were found to be significant prognostic factors for tumor recurrence in CRC. Furthermore, TSA/TP could distinguish patients at high risk of recurrence within Dukes' stage B and in multivariate analysis it was identified as the best independent prognostic factor. According to our results, preoperative serum TSA/TP content could supply additional information to that provided by Dukes' stage about the prognosis of CRC patients.

Nonradioactive immunoquantification of alpha-L-fucosidase protein in human colon tissues.

alpha-L-Fucosidase is a glycosidase involved in the degradation of fucoglycoconjugates and has a diagnostic significance because it has been described to be altered in several known diseases. However, in vitro studies on enzymatic activities may not reflect the real protein levels in tissues. This paper describes a simple method to quantify alpha-L-fucosidase protein levels in human crude extracts, combining the slot-blot technique and a nonradioactive immunoassay. Taking advantage of the similarities in different mammalian fucosidases, a polyclonal antiserum was raised against commercial purified alpha-L-fucosidase from bovine kidney that cross-reacted with the human colon enzyme. The method is able to detect as little as 0.75 ng alpha-L-fucosidase. To illustrate the direct application of this technique, we analysed and quantified alpha-L-fucosidase protein levels in 18 human colon crude samples. This technique could prove useful in clinical pathology, allowing fast and accurate measurement of alpha-L-fucosidase in crude extracts.

On the identification of biomarkers for non-small cell lung cancer in serum and pleural effusion.

The current imperative need for new biomarkers of non-small cell lung cancer (NSCLC) prompted us to compare the proteome of serum and pleural effusion samples from cancer patients with those with benign lung diseases as pneumonia or tuberculosis. Samples were prefractionated through affinity chromatography prior to 2D-DIGE to detect proteins with altered expression in cancer patients. Overall, we identified more potential biomarkers in pleural effusion, which is closer to the affected organ, than in serum. Nevertheless, in both cases principal component analysis demonstrated that the pattern of significantly altered proteins discriminates between disease groups. The biomarker candidates comprise proteins increased in malignant pleural effusions as gelsolin and the metalloproteinase inhibitor 2, and others with lower levels as S100-A8 and S100-A9. The most interesting protein was the pigment epithelium-derived factor (PEDF), which is related to angiogenesis inhibition, and was significantly overexpressed both in serum and pleural effusion from NSCLC patients. More than 12 PEDF isoforms were specifically immunodetected in both fluids in 2-D blots, most of them overexpressed in NSCLC. Thus, further validation would be ideally directed to quantify individual PEDF isoforms, as it may be only one or some of them the ones altered in the cancer process.

Alteration of the serum levels of the epidermal growth factor receptor and its ligands in patients with non-small cell lung cancer and head and neck carcinoma.

Serum levels of the soluble epidermal growth factor receptor (sEGFR) and its ligands epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha) and amphiregulin (AR) were measured in healthy donors and patients with non-small cell lung cancer (NSCLC) and head and neck carcinoma (HNC). In NSCLC, we found sEGFR and EGF levels significantly lowered in patients with respect to healthy donors. In HNC patients, significantly diminished levels were found in the case of sEGFR, EGF and also AR. In both malignancies, no significant association was found between the serum levels of the molecules and the patients' gender, age or smoking habit. Only a significant association was found between the decrease of sEGFR and the absence of distant metastasis in NSCLC and the tumour stage in HNC. The most interesting result was that combining sEGFR and EGF, sensitivities of 88% in NSCLC and 100% in HNC were reached without losing specificity (97.8% in both cases). The use of discriminant analysis and logistic regression improved the sensitivity for NSCLC and the specificity for HNC. These data demonstrate a potentially interesting value of the serum levels of sEGFR and EGF, especially when combined, as markers for NSCLC and HNC.

Cholinesterases are down-expressed in human colorectal carcinoma.

The aberrations of cholinesterase (ChE) genes and the variation of ChE activity in cancerous tissues prompted us to investigate the expression of ChEs in colorectal carcinoma. The study of 55 paired specimens of healthy (HG) and cancerous gut (CG) showed that acetylcholinesterase (AChE) activity fell by 32% and butyrylcholinesterase (BuChE) activity by 58% in CG. Abundant AChE-H, fewer AChE-T, and even fewer AChE-R and BuChE mRNAs were observed in HG, and their content was greatly diminished in CG. The high level of the AChE-H mRNA explains the abundance of AChE-H subunits in HG, which as glycosylphosphatidylinositol (GPI)-anchored amphiphilic AChE dimers (G2(A)) and monomers (G1(A)) account for 69% of AChE activity. The identification of AChE-T and BuChE mRNAs justifies the occurrence in gut of A12, G4(H) and PRiMA-containing G4(A) AChE forms, besides G4(H), G4(A) and G1(H) BuChE. The down-regulation of ChEs might contribute to gut carcinogenesis by increasing acetylcholine availability and over-stimulating muscarinic receptors.

Molecular cloning and in vivo expression of a precursor to rat mitochondrial aspartate aminotransferase.

A 2.4 kilobase cDNA for rat mitochondrial aspartate aminotransferase (E.C. 2.6.1.1.) was isolated and sequenced. The predicted presequence is 93% homologous to the presequences of the enzyme from pig and mouse. The predicted amino acid sequence of the mature enzyme differs from that determined directly by amino acid sequencing (Huynh, Q.K., Sakakibara, R., Watanabe, T., and Wada, H. (1981) J. Biochem. (Tokyo) 90, 863-875) at 13 amino acids residues. The most important difference is at position 140 where the cDNA encodes a tryptophanyl residue rather than the previously reported glycine. This critical residue is now seen to be conserved in all aspartate aminotransferases. The coding region of this cDNA was inserted into the plasmid cloning vector pKK233-2 and used to stably express an unfused precursor in Escherichia coli JM105.

Heterogeneity of beta-galactosidase from rabbit spleen. Separation and characterization of different forms.

Two forms, I and II, of an acid beta-galactosidase from rabbit spleen were separated by DEAE-cellulose chromatography and then characterized. Both forms of the enzyme showed different heat-stability (form I being heat-labile and form II heat-stable), and different pI (6.7 for form I and 5.3 and 6.7 for form II). Their gel filtration patterns were also different: form I was resolved in a single peak of mol. wt. 75,000, whereas form II was resolved in one or two peaks of mol. wt. 120,000 and greater than 200,000, depending on the pH of elution. However, both forms had similar pH stability and behavior toward alpha-methyl-beta-D-galactopyranoside, alpha-methyl-beta-D-glucopyranoside, urea and KCl. Differences in pH optima, optimal temperature and Km values were not marked.

Preoperative serum CD26 levels: diagnostic efficiency and predictive value for colorectal cancer.

CD26 is an ectoenzyme with dipeptidyl peptidase IV activity expressed on a variety of cell types. Although the function of the high concentration of serum-soluble CD26 (sCD26) is unknown, it may be related to the cleavage of biologically active polypeptides. As CD26 or enzymatic activity levels were previously associated with cancer, we examined the potential diagnostic and prognostic value of preoperative sCD26 measurements by ELISA in colorectal carcinoma patients. We found a highly significant difference between sCD26 levels in healthy donors (mean 559.7 +/- 125.5 microg l(-1)) and cancer patients (mean 261.7 +/- 138.1 microg l(-1)) (P< 0.001). A cut-off at 410 microg l(-1)gave 90% sensitivity with 90% specificity which means that the diagnostic efficiency of sCD26 is higher than that shown by other markers, particularly in patients at early stages. Moreover, sCD26 as a variable is not related with Dukes' stage classification, age, gender, tumour location or degree of differentiation. With a follow-up of 2 years until recurrence, preliminary data show that sCD26 can be managed as a prognostic variable of early carcinoma patients. In addition, the origin of sCD26 is discussed.

N-acetyl-beta-hexosaminidase activity and isoenzymes in human gastric adenocarcinoma.

The enhancement of lysosomal beta-hexosaminidase degradative activity in different human cancer tissues is fairly well documented. Gastric tumors have attracted considerable attention on the basis of their social incidence and clinical recurrence. Here we report a comparative study of beta-hexosaminidase activity and of its isoenzymes beta-hexosaminidase A (HA) and beta-hexosaminidase B (HB) from gastric adenocarcinoma and normal mucosa. Tumor beta-hexosaminidase activity from crude extracts and chromatographically resolved HA and HB forms were analyzed as regards their physicochemical and enzymatic properties and were compared to similar samples obtained from control tissue. The existence of one active site in the beta-hexosaminidase enzyme responsible for both N-acetyl-beta-D-glucosaminidase and N-acetyl-beta-D- galactosaminidase activities was determined. Apart from their relative contributions to beta-hexosaminidase activities, two major differences appeared in tumor HA and HB forms with respect to the corresponding controls: (1) the presence of an atypical heat-stable HB isoenzyme in gastric adenocarcinoma, and (2) a significantly increased Vmax of the HA form acting on both p-nitrophenyl-N-acetyl-beta-D-glucosaminide and p-nitrophenyl-N-acetyl-beta-D-galactosaminide substrates. The results show that the beta-hexosaminidase HA and HB isoenzymes from gastric adenocarcinoma display different patterns of response from the same forms from other human tumors.

Value of the serum alpha-L-fucosidase activity in the diagnosis of colorectal cancer.

OBJECTIVES: The purpose of this study was to assess the value of the serum levels of alpha-L-fucosidase activity in the diagnosis of patients with colorectal cancer. METHODS: Using a fluorometric method we analyzed the alpha-L-fucosidase activity in preoperative sera from 137 colorectal cancer patients and in sera from 232 donors. RESULTS: The enzymatic activity of alpha-L-fucosidase was significantly lower (p <0.001) in patients (4.8+/-3.09 U/ml) than in donors (10.5+/-5.46 U/ml). Using the ROC curve, the ideal cut-off for the diagnostic value of alpha-L-fucosidase activity was determined to be 5.6 U/ml. The diagnostic efficiency for colorectal cancer of alpha-L-fucosidase activity was higher than that observed for carcinoembryonic antigen (cut-off 5.0 ng/ml), especially for tumors at an early stage. CONCLUSIONS: Our results suggest that preoperative serum alpha-L-fucosidase activity may be used as a cheap and easy complementary test, in addition to standard clinical procedures routinely used for the diagnosis of colorectal cancer.

Isolation and properties of a liver mitochondrial precursor protein to aspartate aminotransferase expressed in Escherichia coli.

The precursor to rat liver mitochondrial aspartate aminotransferase has been expressed in Escherichia coli JM105 using the pKK233-2 expression vector. This mammalian natural precursor has been isolated as a soluble dimeric protein. The amino-terminal sequence and the amino acid composition of the isolated protein correspond to those predicted from the inserted cDNA (Mattingly, J. R., Jr., Rodriguez-Berrocal, F. J., Gordon, J., Iriarte, A., and Martinez-Carrion, M. (1987) Biochem. Biophys. Res. Commun. 149, 859-865). The isolated precursor contains bound pyridoxal phosphate and shows catalytic activity with a specific activity equal to that of the mature form of the enzyme. This precursor can also be processed by mitochondria into a form with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of mature enzyme. The isolation of this precursor as a stable and catalytically active entity indicates that the presequence peptide does not necessarily interfere with much of the folding and basic structural properties of the mature protein component.

Human colon sialidase: characterization and activity levels in normal mucosa and colonic adenocarcinoma.

Human colon sialidase has been characterized, and its activity levels in normal mucosa and colonic adenocarcinoma have been determined. Sialidase activity was maximal at pH 5.5, and was unstable with storage at 4 and -20 degrees C. The bulk of activity was pellet-associated, and could not be released with triton X-100 or 3-([3-cholamidopropyl]- dimethylammonio)-1-propanesulfonate. Using 2'-(4-methylumbelliferyl)alpha-D-N-acetylneuraminic acid as substrate, the Km and Vmax values were estimated to be 0.140 mmol/l and 63 mU/g, respectively. Furthermore, an inhibition by substrate concentrations above 1.5 mmol/l was detected. Neuraminic acid caused a competitive inhibition with a Ki of 3.5 mmol/l. A statistically significant increase (p < 0.001) in the sialidase specific activity was found in primary colonic adenocarcinoma (104.20 +/- 8.00 mU/g) compared to that of the normal mucosa (72.50 +/- 7.67 mU/g).

Alpha-L-fucosidase enzyme in the prediction of colorectal cancer patients at high risk of tumor recurrence.

The aim of this study was to examine the prognostic value of the alpha-L-fucosidase enzyme to determine whether it can help in the early recognition of colorectal cancer cases at high risk of tumor recurrence. One hundred and twenty-three colorectal carcinoma patients treated by curative surgery were studied. The alpha-L-fucosidase activity was assayed in the tumor and in normal mucosa from each patient using a fluorometric method. Seven other clinical and pathologic features were also studied. To evaluate the impact of each variable over the disease-free interval, a postoperative 30-month follow-up of patients was performed, and a statistical survival analysis was carried out. The recurrence appearance was higher when the relative decrease of alpha-L-fucosidase activity was more than 52% (log-rank test, P = .0261). The results of this work indicate that alpha-L-fucosidase activity appears to be a good independent prognostic factor of tumoral recurrence in colorectal carcinoma.