Serum IgA1 shows increased levels of α2,6-linked sialic acid in breast cancer.

The lectin Helix pomatia agglutinin (HPA) recognizes altered glycosylation in solid cancers and the identification of HPA binding partners in tumour tissue and serum is an important aim. Among the many HPA binding proteins, IgA1 has been reported to be the most abundant in liver metastases. In this study, the glycosylation of IgA1 was evaluated using serum samples from patients with breast cancer (BCa) and the utility of IgA1 glycosylation as a biomarker was assessed. Detailed mass spectrometric structural analysis showed an increase in disialo-biantennary N-linked glycans on IgA1 from BCa patients (p < 0.0001: non-core fucosylated; p = 0.0345: core fucosylated) and increased asialo-Thomsen-Friedenreich antigen (TF) and disialo-TF antigens in the O-linked glycan preparations from IgA1 of cancer patients compared with healthy control individuals. An increase in Sambucus nigra binding was observed, suggestive of increased α2,6-linked sialic acid on IgA1 in BCa. Logistic regression analysis showed HPA binding to IgA1 and tumour size to be significant independent predictors of distant metastases (χ 2 13.359; n = 114; p = 0.020) with positive and negative predictive values of 65.7% and 64.6%, respectively. Immunohistochemical analysis of tumour tissue samples showed IgA1 to be detectable in BCa tissue. This report provides a detailed analysis of serum IgA1 glycosylation in BCa and illustrates the potential utility of IgA1 glycosylation as a biomarker for BCa prognostication.

The DietCompLyf study: a prospective cohort study of breast cancer survival and phytoestrogen consumption.

DietCompLyf is a multi-centre prospective study designed to investigate associations between phytoestrogens - naturally occurring plant compounds with oestrogenic properties - and other diet and lifestyle factors with breast cancer recurrence and survival. 3159 women with grades I-III breast cancer were recruited 9-15 months post-diagnosis from 56 UK hospitals. Detailed information on clinico-pathological, diet, lifestyle and quality of life is collected annually up to 5 years. Biological samples have also been collected as a resource for subsequent evaluation. The characteristics of the patients and associations between pre-diagnosis intake of phytoestrogens (isoflavones and lignans; assessed using the EPIC-Norfolk UK 130 question food frequency questionnaire) and breast cancer (i) risk factors and (ii) prognostic factors are described for 1797 women who had complete data for all covariates and phytoestrogens of interest. Isoflavone intakes were higher in the patients who were younger at diagnosis, in the non-smokers, those who had breast-fed and those who took supplements. Lignan intakes were higher in patients with a higher age at diagnosis, in ex-smokers, those who had breast-fed, who took supplements, had a lower BMI at diagnosis, lower age at menarche and were nulliparous. No significant associations between pre-diagnosis phytoestrogen intake and factors associated with improved breast cancer prognosis were observed. The potential for further exploration of the relationship between phytoestrogens and breast cancer recurrence and survival, and for the establishment of evidence to improve dietary and lifestyle advice offered to patients following breast cancer diagnosis using DietCompLyf data is discussed.

A targeted glycoproteomic approach identifies cadherin-5 as a novel biomarker of metastatic breast cancer.

Aberrant glycosylation has long been recognised as a hallmark of cancer, and is increasingly being exploited in biomarker discovery studies. Helix pomatia agglutinin (HPA) is known to bind aberrant glycans associated with metastatic breast cancer, and was used here to isolate glycoproteins from pooled breast cancer serum samples of (i) patients with recurrent breast cancer and (ii) patients with no sign of recurrence 5years after diagnosis of their primary tumour. Pregnancy zone protein, the polymeric immunoglobulin receptor and cadherin-5 emerged as potential markers of metastasis following proteomic identification of HPA binding glycoproteins. ELISAs were developed to verify these findings, and to assess protein glycosylation, in individual patient sera. The cadherin-5 ELISA discriminated serum samples of patients with recurrent breast cancer from those with no sign of recurrence, and analysis of cadherin-5 glycosylation by HPA also showed a significant difference between the two sample groups. The targeted glycoproteomic and validatory approach developed here has shown that when taking into account both the protein levels and HPA binding, serum cadherin-5 discriminated patients with recurrent breast cancer from those with no sign of recurrence with 90% specificity.

Lectin array-based strategies for identifying metastasis-associated changes in glycosylation.

Since 2005, lectin microarray technology has emerged as a relatively simple yet powerful technique for the comprehensive analysis of glycoprotein glycosylation. Lectin microarrays represent a new analytical method that can be used to explore the human glycome, a unique source of markers of diseases including cancer. The lectin microarray technology is a sensitive tool with the potential to allow high-throughput analysis of cancer-associated changes in glycosylation. This chapter describes the generation of a lectin-binding signature associated with metastatic primary breast tumours that have been resected, fixed, and embedded in paraffin. Procedures concerning sample and lectin microarray preparation are explained, alongside experimental considerations and approaches to data analysis.

Lectin microarray profiling of metastatic breast cancers.

Altered protein glycosylation compared with the disease-free state is a universal feature of cancer cells. It has long been established that distinct glycan structures are associated with specific forms of cancer, but far less is known about the complete array of glycans associated with certain tumors. The cancer glycome has great potential as a source of biomarkers, but progress in this field has been hindered by a lack of available techniques for the elucidation of disease-associated glycosylation. In the present study, lectin microarrays consisting of 45 lectins with different binding preferences covering N- and O-linked glycans were coupled with evanescent-field activated fluorescent detection in the glycomic analysis of primary breast tumors and the serum and urine of patients with metastatic breast cancer. A single 50 µm section of a primary breast tumor or <1 µL of breast cancer patient serum or urine was sufficient to detect glycosylation alterations associated with metastatic breast cancer, as inferred from lectin-binding patterns. The high-throughput, sensitive and relatively simple nature of the simultaneous analysis of N- and O-linked glycosylation following minimal sample preparation and without the need for protein deglycosylation makes the lectin microarray analysis described a valuable tool for discovery phase glycomic profiling.

Significant changes in dietary intake and supplement use after breast cancer diagnosis in a UK multicentre study.

The diagnosis of cancer can motivate survivors to alter their lifestyle habits. Healthcare providers need to be aware of what changes patients are likely to make in order to derive more pertinent recommendations; however, few studies have reported pre- and post-diagnostic lifestyle behaviours. Semi-quantitative food frequency questionnaires (FFQs) completed approximately 1 year after diagnosis were used to evaluate dietary intake and supplement use before and after diagnosis in a cohort of 1,560 breast cancer patients participating in the UK, prospective DietCompLyf study. Intake of fruit and vegetables, wholegrains and lean sources of protein increased significantly post-diagnosis (P < 0.05, each). Conversely, after diagnosis consumption of high-fat, high-sugar products, red meat, coffee, some alcoholic drinks and refined grains significantly decreased (P < 0.05, each). Post-diagnostic changes in diet were accompanied by changes in the intake of macronutrients and a number of vitamins and minerals. Supplement use was highly prevalent (56.1%) pre-diagnosis, increasing to 62.8% after diagnosis (P = 0.001). Fish oils, multivitamin and minerals, and evening primrose oil were most often used and the proportion of users significantly increased (P < 0.05, each) after diagnosis. The percentage of women using oestrogenic botanical supplements (OBSs) was small but more than doubled to 8.4% after diagnosis (P < 0.05). British women participating in the DietCompLyf study reported significant changes in dietary intake and supplement use after their breast cancer diagnosis. These findings contribute to our understanding of female cancer survivors' dietary behaviours which is crucial for developing and implementing recommendations.

A sensitive assay to measure biomarker glycosylation demonstrates increased fucosylation of prostate specific antigen (PSA) in patients with prostate cancer compared with benign prostatic hyperplasia.

BACKGROUND: Prostate specific antigen (PSA) measurement is used for the diagnosis of prostate cancer (PCa) but the test lacks specificity due to the number of false positive readings. The glycosylation of PSA is altered in PCa but studies in this area have been limited to few clinical samples and/or require advanced laboratory facilities. An assay to assess PSA glycosylation was established using equipment available in most routine biomedical testing laboratories. METHODS: Serum samples from patients with PCa or benign prostatic hyperplasia (BPH) were used. PSA (range 4-10 ng/ml) was affinity purified, separated and probed with the lectin Ulex europaeus (UEA-1; specific for α1,2 linked fucose). An enzyme-linked immunosorbent lectin assay (ELLA) with colorimetric detection was devised and PSA fucosylation assessed in a further independent set of 26 samples. RESULTS: Free PSA (fPSA) from PCa patients showed a significant increase in fucosylation compared with fPSA from patients with BPH. The ELLA was 92% specific and 69% sensitive for PCa over BPH. In comparison, fPSA measurement was 70% specific and 56% sensitive (threshold set to 25% tPSA) for PCa over BPH. CONCLUSIONS: Changes in glycosylation of PSA were identified using 50 µl of serum with PSA in the range of 4-10 ng/ml, this represents a more specific and sensitive test for PCa based on fucosylation changes of fPSA.

Do phytoestrogens reduce the risk of breast cancer and breast cancer recurrence? What clinicians need to know.

Oestrogen is an important determinant of breast cancer risk. Oestrogen-mimicking plant compounds called phytoestrogens can bind to oestrogen receptors and exert weak oestrogenic effects. Despite this activity, epidemiological studies suggest that the incidence of breast cancer is lower in countries where the intake of phytoestrogens is high, implying that these compounds may reduce breast cancer risk, and possibly have an impact on survival. Isoflavones and lignans are the most common phytoestrogens in the diet. In this article, we present findings from human observational and intervention studies related to both isoflavone and lignan exposure and breast cancer risk and survival. In addition, the clinical implications of these findings are examined in the light of a growing dietary supplement market. An increasing number of breast cancer patients seek to take supplements together with their standard treatment in the hope that these will either prevent recurrence or treat their menopausal symptoms. Observational studies suggest a protective effect of isoflavones on breast cancer risk and the case may be similar for increasing lignan consumption although evidence so far is inconsistent. In contrast, short-term intervention studies suggest a possible stimulatory effect on breast tissue raising concerns of possible adverse effects in breast cancer patients. However, owing to the dearth of human studies investigating effects on breast cancer recurrence and survival the role of phytoestrogens remains unclear. So far, not enough clear evidence exists on which to base guidelines for clinical use, although raising patient awareness of the uncertain effect of phytoestrogens is recommended.

High throughput quantification of phytoestrogens in human urine and serum using liquid chromatography/tandem mass spectrometry (LC-MS/MS).

Phytoestrogens are currently the subject of intense study owing to their potential protective effects against a number of complex diseases. However, in order to investigate the interactions between phytoestrogens and disease state effectively, it is necessary to have analytical methods which are sensitive, reproducible, and require low sample volumes. We report an assay for three isoflavones (daidzein, genistein, and glycitein), two metabolites of daidzein (equol and O-desmethylangolensin), three lignans (secoisolariciresinol, enterodiol, and enterolactone), and one flavanone (naringenin) in human urine and serum. A high throughput of samples has been achieved via the use of 96-well plate sample extraction and liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis incorporating column switching, thus making the assay suitable for use on large sample numbers, such as those found in epidemiological studies. The robustness of the assay was proven via the comparison of data generated on two different LC-MS/MS systems, with and without column switching.

Cancer-associated glycoforms of gelatinase B exhibit a decreased level of binding to galectin-3.

Gelatinase B (MMP-9) and galectin-3 are widely known to participate in tumor cell invasion and metastasis. Glycans derived from MMP-9 expressed in MCF-7 breast cancer and THP-1 myeloid leukemia cells were compared with those from MMP-9 expressed in natural neutrophils. The many O-linked glycans of neutrophil gelatinase B presented a cluster of mainly galactosylated core II structures, 46% of which were ligands for galectin-3; 11% contained two to three N-acetyllactosamine repeating units that are high-affinity ligands for the lectin. The glycan epitopes thus provide MMP-9 with both high-affinity and (presumably) high-avidity interactions with galectin-3. In contrast, the O-glycans released from MMP-9 expressed in MCF-7 and THP-1 cells were predominantly sialylated core I structures. Only 10% of MCF-7 and THP-1 gelatinase B O-glycans were ligands for galectin-3 and contained only a maximum single N-acetyllactosamine repeat. Consistent with the glycan analysis, surface plasmon resonance binding assays indicated that the cancer-associated glycoforms of MMP-9 bound galectin-3 with an affinity and avidity significantly reduced compared with those of the natural neutrophil MMP-9. Galectin-3 exists as a multimer that also binds laminin, providing a means of localizing neutrophil MMP-9 in the extracellular matrix (ECM). The analytical data presented here suggest that MMP-9 glycoforms secreted by tumor cells are unlikely to be tethered at the site of secretion, thus promoting more extensive cleavage of the ECM and providing a rationale for the contribution that gelatinase B makes to cancer cell metastasis.

Short-term phytoestrogen supplementation alters insulin-like growth factor profile but not lipid or antioxidant status.

Phytoestrogens are plant compounds that have been proposed to have a variety of health benefits. The aim of this study was to assess the effects of these compounds on a number of physiological endpoints. Subjects were given a single intake of a phytoestrogen-rich (80 mg total phytoestrogens) supplement containing soy, rye and linseed (Phase 1), followed by a week-long intervention using the same supplement (Phase 2) (80 mg total phytoestrogens daily). A number of biochemical endpoints were assessed including urinary phytoestrogen metabolites, lipids, antioxidant status, DNA damage and insulin-like growth factor-1 (IGF-1) and IGF binding protein-1 (IGFBP-1) and -3 (IGFBP-3). Ten healthy female subjects took part in the study. Excretion of the isoflavones genistein, daidzein and equol in urine increased in both phases of the study. No other endpoint was altered in Phase 1. However, in Phase 2, concentrations of IGF-1 and IGFBP-3 were increased by phytoestrogen supplementation [IGF-1, median (IQ range), baseline 155 (123, 258), postweek 265 (228, 360) ng/ml, P<.05; IGFBP-3, baseline 3725 (3631, 4196), postweek 4420 (4192, 4935) ng/ml, P<.05]. There was no effect of supplementation on lipids or markers of antioxidant status. Short-term phytoestrogen supplementation increases urinary phytoestrogen excretion and increases IGF-1 and IGFBP-3. These results require elucidation in further controlled studies.

The influence of dietary lectins on the cell proliferation of human breast cancer cell lines in vitro.

BACKGROUND: Lectins, carbohydrate proteins, bind non-covalently to glycoconjugate of normal and malignant cells. If used in cell culture, they can influence cellular proliferation. In this study the in vitro effects of six dietary lectins on the cell proliferation of human breast cancer cell lines were investigated. MATERIALS AND METHODS: Cell proliferation was assessed by a colorimetric XTT-based assay kit. Lectin binding was characterized by lectin histochemistry. RESULTS: WGA considerably influenced the cell growth of all tested cell lines (MCF-7, T 47D, HBL 100, BT 20), whereas the effects of PHA-L, SBA and HPA were smaller, began at higher concentrations and were restricted to three cell lines (MCF-7, T 47D and HBL 100 for PHA-L; MCF-7, T 47D and BT 20 for SBA, respectively) and to one cell line (HBL 100 for HPA). STA and PNA had no effect at all. CONCLUSION: The present data suggested that some dietary lectins can inhibit cell growth of human breast cancer cells in vitro. These findings would argue for a protective effect of these plant lectins for breast cancer.