Levels of specific glycans significantly distinguish lymph node-positive from lymph node-negative breast cancer patients.

One of the most urgent requirements in breast cancer is the development of a blood-based test for early detection and prognosis. Previously published results found a significant difference between specific glycan levels in patients with advanced breast cancer and healthy controls. The aim of this investigation was to address a more clinically relevant problem, i.e., whether the measurement of specific glycans could identify women with aggressive disease at an early stage. In order to reduce potential bias in this study, blood samples from patients were collected, stored and analyzed in a similar manner. Agalactosyl biantennary glycans (FA2) and glycans containing the sialyl Lewis x epitope (A3F1G1 and A2F1G1) were measured using high throughput normal-phase high-performance liquid chromatography in combination with exoglycosidase digestions in sera from 52 patients with early breast cancer (21 with lymph node-negative and 20 with lymph node-positive disease) and 134 women with benign breast disease. The combined levels of the glycans were significantly higher in patients with lymph node metastases compared to women without these metastases. Lymph node status is the single most important determinant of survival in early stage breast cancer. As high levels of these glycans were associated with nodal metastases, their measurement may provide a new non-invasive approach to determining prognosis in women with newly diagnosed breast cancer.

A strategy to reveal potential glycan markers from serum glycoproteins associated with breast cancer progression.

Aberrant glycosylation on glycoproteins that are either presented on the surface or secreted by cancer cells is a potential source of disease biomarkers and provides insights into disease pathogenesis. N-Glycans of the total serum glycoproteins from advanced breast cancer patients and healthy individuals were sequenced by HPLC with fluorescence detection coupled with exoglycosidase digestions and mass spectrometry. We observed a significant increase in a trisialylated triantennary glycan containing alpha1,3-linked fucose which forms part of the sialyl Lewis x epitope. Following digestion of the total glycan pool with a combination of sialidase and beta-galactosidase, we segregated and quantified a digestion product, a monogalactosylated triantennary structure containing alpha1,3-linked fucose. We compared breast cancer patients and controls and detected a 2-fold increase in this glycan marker in patients. In 10 patients monitored longitudinally, we showed a positive correlation between this glycan marker and disease progression and also demonstrated its potential as a better indicator of metastasis compared to the currently used biomarkers, CA 15-3 and carcinoembryonic antigen (CEA). A pilot glycoproteomic study of advanced breast cancer serum highlighted acute-phase proteins alpha1-acid glycoprotein, alpha1-antichymotrypsin, and haptoglobin beta-chain as contributors to the increase in the glycan marker which, when quantified from each of these proteins, marked the onset of metastasis in advance of the CA 15-3 marker. These preliminary findings suggest that specific glycans and glycoforms of proteins may be candidates for improved markers in the monitoring of breast cancer progression.

Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG.

Ovarian cancer is the fourth most common cancer in women in the Western world. In a pilot scale study, we highlight changes in the total serum glycome of patients with advanced ovarian cancer that might shed insight into disease pathogenesis. These changes include increases in levels of core fucosylated, agalactosyl biantennary glycans (FA2) and sialyl Lewis x (SLe(x)). To investigate further which proteins contribute to these alterations, we developed technology to analyze simultaneously the glycosylation of protein glycoforms contained in single spots excised from a 2D gel (<1 ng protein). The acute-phase proteins, haptoglobin, alpha1-acid glycoprotein, and alpha1-antichymotrypsin from patients contained elevated levels of subsets of glycoforms containing SLe(x). We also established that IgG heavy chains from patients contained twice the level of FA2 compared with healthy controls. Serum CA125 is the only biomarker that is used routinely, and there is a need for complementary markers that will improve both sensitivity and specificity. There was some preliminary indication that combinations of changes in the serum glycome might improve the separation of ovarian cancer and benign tumors; however, a larger study using data receiver operating characteristic curves will be required to draw any firm conclusions.

Abnormal glycosylation with hypersialylated O-glycans in patients with Sialuria.

Sialuria is an inborn error of metabolism characterized by coarse face, hepatomegaly and recurrent respiratory tract infections. The genetic defect in this disorder results in a loss of feedback control of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase by CMP-N-acetylneuraminic acid (CMP-NeuAc) resulting in a substantial overproduction of cytoplasmic free sialic acid. This study addresses fibroblast CMP-NeuAc levels and N- and O-glycan sialylation of serum proteins from Sialuria patients. CMP-NeuAc levels were measured with HPLC in fibroblasts. Isoelectric focusing (IEF) of serum transferrin and of apolipoprotein C-III (apoC-III) was performed on serum of three Sialuria patients. Isoforms of these proteins can be used as specific markers for the biosynthesis of N- and core 1 O-glycans. Furthermore, total N- and O-linked glycans from serum proteins were analyzed by HPLC. HPLC showed a clear overproduction of CMP-NeuAc in fibroblasts of a Sialuria patient. Minor changes were found for serum N-glycans and hypersialylation was found for core 1 O-glycans on serum apoC-III and on total serum O-glycans in Sialuria patients. HPLC showed an increased ratio of disialylated over monosialylated core 1 O-glycans. The hypersialylation of core 1 O-glycans is due to the increase of NeuAcalpha2,6-containing structures (mainly NeuAcalpha2-3Galbeta1-3[NeuAcalpha2-6]GalNAc). This may relate to KM differences between GalNAc-alpha2,6-sialyltransferase and alpha2,3-sialyltransferases. This is the first study demonstrating that the genetic defect in Sialuria results in a CMP-NeuAc overproduction. Subsequently, increased amounts of alpha2,6-linked NeuAc were found on serum core 1 O-glycans from Sialuria patients. N-glycosylation of serum proteins seems largely unaffected. Sialuria is the first metabolic disorder presenting with hypersialylated O-glycans.