Combination of urinary free-glycan markers for the diagnosis of various malignant tumors

Many urinary free-glycan markers were identified in our previous studies. Here, the clinical utility of these markers was examined. Urine samples taken from 120 healthy subjects and 503 patients with various malignant tumors were analyzed. Four lactose-core glycans containing N-glycolylglucosamine (GlcNGc) were synthesized and used as internal standards (ISs). Free-glycans were isolated using a two-step column purification procedure, pyridylaminated, and subjected to LC-selected reaction monitoring. Assay validation was performed using four ISs and eight reference glycans. Twelve markers composed of three sialyl lactose-core glycans, three sulfated glycans, four Gn1-core free-N-glycans, and two glycans with unusual structures were selected to investigate their effectiveness for cancer diagnosis. Markers were simultaneously measured and the relative area ratio (marker/IS) quantified. This method was shown to be accurate and precise by repeated analysis of calibrators and quality control samples in urine. Receiver operating characteristic curve analyses revealed that individual markers were not sufficient for highly accurate diagnosis. However, a combination of four markers resulted in higher area under curves of 0.910, 0.867, and 0.914 for gastric, colorectal, and pancreatic cancers, respectively. Moreover, levels of these markers were elevated in various other malignancies. These analyses demonstrated high clinical utility of the free-glycan markers.