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

TLDR

Aberrant glycosylation of cancer cell–derived glycoproteins offers potential biomarkers and insights into disease pathogenesis. The authors sequenced total serum N‑glycans from advanced breast cancer patients and controls using HPLC–fluorescence with exoglycosidase digestions and mass spectrometry, then digested the glycan pool with sialidase and β‑galactosidase to isolate and quantify a monogalactosylated tri‑antennary structure bearing α1,3‑linked fucose. Patients with advanced breast cancer exhibited a two‑fold increase in a trisialylated tri‑antennary glycan containing α1,3‑linked fucose, which correlated positively with disease progression and outperformed CA 15‑3 and CEA as a metastasis indicator; pilot glycoproteomic analysis linked this marker to acute‑phase proteins and suggested that specific glycans could serve as superior progression biomarkers.

Abstract

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 α1,3-linked fucose which forms part of the sialyl Lewis x epitope. Following digestion of the total glycan pool with a combination of sialidase and β-galactosidase, we segregated and quantified a digestion product, a monogalactosylated triantennary structure containing α1,3-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 α1-acid glycoprotein, α1-antichymotrypsin, and haptoglobin β-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.