A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.

Recent research is uncovering unexpected ways that glycans contribute to biology, as well as new strategies for combatting disease using approaches involving glycans. To make full use of glycans for clinical applications, we need more detailed information on the location, nature, and dynamics of glycan expression in vivo. Such studies require the use of specimens acquired directly from patients. Effective studies of clinical specimens require low-volume assays, high precision measurements, and the ability to process many samples. Assays using affinity reagents—lectins and glycan-binding antibodies—can meet these requirements, but further developments are needed to make the methods routine and effective. Recent advances in the use of glycan-binding proteins could meet that need. The advances involve improved determination of specificity using glycan arrays; the availability of databases for mining and analyzing glycan array data; lectin engineering methods; and the ability to quantitatively interpret lectin measurements. Here we describe many of the challenges and opportunities involved in the application of these new approaches to the study of biological samples. The new tools hold promise for developing methods to improve the outcomes of patients afflicted with diseases characterized by aberrant glycan expression.

Background and Aims The CA19-9 antigen is the current best biomarker for pancreatic cancer, but it is not elevated in about 25% of pancreatic cancer patients at a cutoff that gives a 25% false-positive rate. We hypothesized that antigens related to the CA19-9 antigen, which is a glycan called sialyl-Lewis A (sLeA), are elevated in distinct subsets of pancreatic cancers. Methods We profiled the levels of multiple glycans and mucin glycoforms in plasma from 200 subjects with either pancreatic cancer or benign pancreatic disease, and we validated selected findings in additional cohorts of 116 and 100 subjects, the latter run blinded and including cancers that exclusively were early-stage. Results We found significant elevations in two glycans: an isomer of sLeA called sialyl-Lewis X, present both in sulfated and non-sulfated forms; and the sialylated form of a marker for pluripotent stem cells, type 1 N-acetyl-lactosamine. The glycans performed as well as sLeA as individual markers and were elevated in distinct groups of patients, resulting in a 3-marker panel that significantly improved upon any individual biomarker. The panel gave 85% sensitivity and 90% specificity in the combined discovery and validation cohorts, relative to 54% sensitivity and 86% specificity for sLeA; and it gave 80% sensitivity and 84% specificity in the independent test cohort, as opposed to 66% sensitivity and 72% specificity for sLeA. Conclusions Glycans related to sLeA are elevated in distinct subsets of pancreatic cancers and yield improved diagnostic accuracy over CA19-9.

The validation of candidate biomarkers often is hampered by the lack of a reliable means of assessing and comparing performance. We present here a reference set of serum and plasma samples to facilitate the validation of biomarkers for resectable pancreatic cancer. The reference set includes a large cohort of stage I-II pancreatic cancer patients, recruited from 5 different institutions, and relevant control groups. We characterized the performance of the current best serological biomarker for pancreatic cancer, CA 19–9, using plasma samples from the reference set to provide a benchmark for future biomarker studies and to further our knowledge of CA 19–9 in early-stage pancreatic cancer and the control groups. CA 19–9 distinguished pancreatic cancers from the healthy and chronic pancreatitis groups with an average sensitivity and specificity of 70–74%, similar to previous studies using all stages of pancreatic cancer. Chronic pancreatitis patients did not show CA 19–9 elevations, but patients with benign biliary obstruction had elevations nearly as high as the cancer patients. We gained additional information about the biomarker by comparing two distinct assays. The two CA 9–9 assays agreed well in overall performance but diverged in measurements of individual samples, potentially due to subtle differences in antibody specificity as revealed by glycan array analysis. Thus, the reference set promises be a valuable resource for biomarker validation and comparison, and the CA 19–9 data presented here will be useful for benchmarking and for exploring relationships to CA 19–9.

The fucose post-translational modification is frequently increased in pancreatic cancer, thus forming the basis for promising biomarkers, but a subset of pancreatic cancer patients does not elevate the known fucose-containing biomarkers. We hypothesized that such patients elevate glycan motifs with fucose in linkages and contexts different from the known fucose-containing biomarkers. We used a database of glycan array data to identify the lectins CCL2 to detect glycan motifs with fucose in a 3’ linkage; CGL2 for motifs with fucose in a 2’ linkage; and RSL for fucose in all linkages. We used several practical methods to test the lectins and determine the optimal mode of detection, and we then tested whether the lectins detected glycans in pancreatic cancer patients who did not elevate the sialyl-Lewis A glycan, which is upregulated in ~75% of pancreatic adenocarcinomas. Patients who did not upregulate sialyl-Lewis A, which contains fucose in a 4’ linkage, tended to upregulate fucose in a 3’ linkage, as detected by CCL2, but they did not upregulate total fucose or fucose in a 2’ linkage. CCL2 binding was high in cancerous epithelia from pancreatic tumors, including areas negative for sialyl-Lewis A and a related motif containing 3’ fucose, sialyl-Lewis X. Thus glycans containing 3’ fucose may complement sialyl-Lewis A to contribute to improved detection of pancreatic cancer. Furthermore, the use of panels of recombinant lectins may uncover details about glycosylation that could be important for characterizing and detecting cancer.