Heterogeneity of glycan biomarker clusters as an indicator of recurrence in pancreatic cancer.

Introduction: Outcomes following tumor resection vary dramatically among patients with pancreatic ductal adenocarcinoma (PDAC). A challenge in defining predictive biomarkers is to discern within the complex tumor tissue the specific subpopulations and relationships that drive recurrence. Multiplexed immunofluorescence is valuable for such studies when supplied with markers of relevant subpopulations and analysis methods to sort out the intra-tumor relationships that are informative of tumor behavior. We hypothesized that the glycan biomarkers CA19-9 and STRA, which detect separate subpopulations of cancer cells, define intra-tumoral features associated with recurrence. Methods: We probed this question using automated signal thresholding and spatial cluster analysis applied to the immunofluorescence images of the STRA and CA19-9 glycan biomarkers in whole-block sections of PDAC tumors collected from curative resections. Results: The tumors (N = 22) displayed extreme diversity between them in the amounts of the glycans and in the levels of spatial clustering, but neither the amounts nor the clusters of the individual and combined glycans associated with recurrence. The combined glycans, however, marked divergent types of spatial clusters, alternatively only STRA, only CA19-9, or both. The co-occurrence of more than one cluster type within a tumor associated significantly with disease recurrence, in contrast to the independent occurrence of each type of cluster. In addition, intra-tumoral regions with heterogeneity in biomarker clusters spatially aligned with pathology-confirmed cancer cells, whereas regions with homogeneous biomarker clusters aligned with various non-cancer cells. Conclusion: Thus, the STRA and CA19-9 glycans are markers of distinct and co-occurring subpopulations of cancer cells that in combination are associated with recurrence. Furthermore, automated signal thresholding and spatial clustering provides a tool for quantifying intra-tumoral subpopulations that are informative of outcome.

Bioorthogonal Chemical Labeling Probes Targeting Sialic Acid Isomers for N-Glycan MALDI Imaging Mass Spectrometry of Tissues, Cells, and Biofluids.

Sialic acid isomers attached in either α2,3 or α2,6 linkage to glycan termini confer distinct chemical, biological, and pathological properties, but they cannot be distinguished by mass differences in traditional mass spectrometry experiments. Multiple derivatization strategies have been developed to stabilize and facilitate the analysis of sialic acid isomers and their glycoconjugate carriers by high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry workflows. Herein, a set of novel derivatization schemes are described that result in the introduction of bioorthogonal click chemistry alkyne or azide groups into α2,3- and α2,8-linked sialic acids. These chemical modifications were validated and structurally characterized using model isomeric sialic acid conjugates and model protein carriers. Use of an alkyne-amine, propargylamine, as the second amidation reagent effectively introduces an alkyne functional group into α2,3-linked sialic acid glycoproteins. In tissues, serum, and cultured cells, this allows for the detection and visualization of N-linked glycan sialic acid isomers by imaging mass spectrometry approaches. Formalin-fixed paraffin-embedded prostate cancer tissues and pancreatic cancer cell lines were used to characterize the numbers and distribution of alkyne-modified α2,3-linked sialic acid N-glycans. An azide-amine compound with a poly(ethylene glycol) linker was evaluated for use in histochemical staining. Formalin-fixed pancreatic cancer tissues were amidated with the azide amine, reacted with biotin-alkyne and copper catalyst, and sialic acid isomers detected by streptavidin-peroxidase staining. The direct chemical introduction of bioorthogonal click chemistry reagents into sialic acid-containing glycans and glycoproteins provides a new glycomic tool set to expand approaches for their detection, labeling, visualization, and enrichment.

Heterogeneity of Glycan Biomarker Clusters as an Indicator of Recurrence in Pancreatic Cancer.

Outcomes following tumor resection vary dramatically among patients with pancreatic cancer. A challenge in defining predictive biomarkers is to discern within the complex tumor tissue the specific subpopulations and relationships that drive recurrence. Multiplexed immunofluorescence is valuable for such studies when supplied with markers of relevant subpopulations and analysis methods to sort out the intra-tumor relationships that are informative of tumor behavior. We hypothesized that the glycan biomarkers CA19-9 and STRA, which detect separate subpopulations of cancer cells, define intra-tumoral features associated with recurrence. We probed this question using automated signal thresholding and spatial cluster analysis applied to the immunofluorescence images of the STRA and CA19-9 glycan biomarkers in whole-block tumor sections. The tumors (N = 22) displayed extreme diversity between them in the amounts of the glycans and in the levels of spatial clustering, but neither the amounts nor the clusters of the individual and combined glycans associated with recurrence. The combined glycans, however, marked divergent types of spatial clusters, alternatively only STRA, only CA19-9, or both. The co-occurrence of more than one cluster type within a tumor associated significantly with disease recurrence, in contrast to the independent occurrence of each type of cluster. In addition, intra-tumoral regions with heterogeneity in biomarker clusters spatially aligned with pathology-confirmed cancer cells, whereas regions with homogeneous biomarker clusters aligned with various non-cancer cells. Thus, the STRA and CA19-9 glycans are markers of distinct and co-occurring subpopulations of cancer cells that in combination are associated with recurrence. Furthermore, automated signal thresholding and spatial clustering provides a tool for quantifying intra-tumoral subpopulations that are informative of outcome.

CarboGrove: a resource of glycan-binding specificities through analyzed glycan-array datasets from all platforms.

Glycan arrays continue to be the primary resource for determining the glycan-binding specificity of proteins. The volume and diversity of glycan-array data are increasing, but no common method and resource exist to analyze, integrate, and use the available data. To meet this need, we developed a resource of analyzed glycan-array data called CarboGrove. Using the ability to process and interpret data from any type of glycan array, we populated the database with the results from 35 types of glycan arrays, 13 glycan families, 5 experimental methods, and 19 laboratories or companies. In meta-analyses of glycan-binding proteins, we observed glycan-binding specificities that were not uncovered from single sources. In addition, we confirmed the ability to efficiently optimize selections of glycan-binding proteins to be used in experiments for discriminating between closely related motifs. Through descriptive reports and a programmatically accessible Application Programming Interface, CarboGrove yields unprecedented access to the wealth of glycan-array data being produced and powerful capabilities for both experimentalists and bioinformaticians.

Imaging Mass Spectrometry and Lectin Analysis of N-Linked Glycans in Carbohydrate Antigen-Defined Pancreatic Cancer Tissues.

The early detection of pancreatic ductal adenocarcinoma (PDAC) is a complex clinical obstacle yet is key to improving the overall likelihood of patient survival. Current and prospective carbohydrate biomarkers carbohydrate antigen 19-9 (CA19-9) and sialylated tumor-related antigen (sTRA) are sufficient for surveilling disease progression yet are not approved for delineating PDAC from other abdominal cancers and noncancerous pancreatic pathologies. To further understand these glycan epitopes, an imaging mass spectrometry (IMS) approach was used to assess the N-glycome of the human pancreas and pancreatic cancer in a cohort of patients with PDAC represented by tissue microarrays and whole-tissue sections. Orthogonally, these same tissues were characterized by multiround immunofluorescence that defined expression of CA19-9 and sTRA as well as other lectins toward carbohydrate epitopes with the potential to improve PDAC diagnosis. These analyses revealed distinct differences not only in N-glycan spatial localization across both healthy and diseased tissues but importantly between different biomarker-categorized tissue samples. Unique sulfated biantennary N-glycans were detected specifically in normal pancreatic islets. N-glycans from CA19-9-expressing tissues tended to be biantennary, triantennary, and tetra-antennary structures with both core and terminal fucose residues and bisecting GlcNAc. These N-glycans were detected in less abundance in sTRA-expressing tumor tissues, which favored triantennary and tetra-antennary structures with polylactosamine extensions. Increased sialylation of N-glycans was detected in all tumor tissues. A candidate new biomarker derived from IMS was further explored by fluorescence staining with selected lectins on the same tissues. The lectins confirmed the expression of the epitopes in cancer cells and revealed different tumor-associated staining patterns between glycans with bisecting GlcNAc and those with terminal GlcNAc. Thus, the combination of lectin-immunohistochemistry and lectin-IMS techniques produces more complete information for tumor classification than the individual analyses alone. These findings potentiate the development of early assessment technologies to rapidly and specifically identify PDAC in the clinic that may directly impact patient outcomes.

Detection of Chemotherapy-resistant Pancreatic Cancer Using a Glycan Biomarker, sTRA.

PURPOSE: A subset of pancreatic ductal adenocarcinomas (PDACs) is highly resistant to systemic chemotherapy, but no markers are available in clinical settings to identify this subset. We hypothesized that a glycan biomarker for PDACs called sialylated tumor-related antigen (sTRA) could be used for this purpose. EXPERIMENTAL DESIGN: We tested for differences between PDACs classified by glycan expression in multiple systems: sets of cell lines, organoids, and isogenic cell lines; primary tumors; and blood plasma from human subjects. RESULTS: The sTRA-expressing models tended to have stem-like gene expression and the capacity for mesenchymal differentiation, in contrast to the nonexpressing models. The sTRA cell lines also had significantly increased resistance to seven different chemotherapeutics commonly used against pancreatic cancer. Patients with primary tumors that were positive for a gene expression classifier for sTRA received no statistically significant benefit from adjuvant chemotherapy, in contrast to those negative for the signature. In another cohort, based on direct measurements of sTRA in tissue microarrays, the patients who were high in sTRA again had no statistically significant benefit from adjuvant chemotherapy. Furthermore, a blood plasma test for the sTRA glycan identified the PDACs that showed rapid relapse following neoadjuvant chemotherapy. CONCLUSIONS: This research demonstrates that a glycan biomarker could have value to detect chemotherapy-resistant PDAC in clinical settings. This capability could aid in the development of stratified treatment plans and facilitate biomarker-guided trials targeting resistant PDAC.

Biomarkers and Strategy to Detect Preinvasive and Early Pancreatic Cancer: State of the Field and the Impact of the EDRN.

Patients afflicted with pancreatic ductal adenocarcinoma (PDAC) face a dismal prognosis, but headway could be made if physicians could identify the disease earlier. A compelling strategy to broaden the use of surveillance for PDAC is to incorporate molecular biomarkers in combination with clinical analysis and imaging tools. This article summarizes the components involved in accomplishing biomarker validation and an analysis of the requirements of molecular biomarkers for disease surveillance. We highlight the significance of consortia for this research and highlight resources and infrastructure of the Early Detection Research Network (EDRN). The EDRN brings together the multifaceted expertise and resources needed for biomarker validation, such as study design, clinical care, biospecimen collection and handling, molecular technologies, and biostatistical analysis, and studies coming out of the EDRN have yielded biomarkers that are moving forward in validation. We close the article with an overview of the current investigational biomarkers, an analysis of their performance relative to the established benchmarks, and an outlook on the current needs in the field. The outlook for improving the early detection of PDAC looks promising, and the pace of further research should be quickened through the resources and expertise of the EDRN and other consortia.See all articles in this CEBP Focus section, "NCI Early Detection Research Network: Making Cancer Detection Possible."

Advances in Tools to Determine the Glycan-Binding Specificities of Lectins and Antibodies.

Proteins that bind carbohydrate structures can serve as tools to quantify or localize specific glycans in biological specimens. Such proteins, including lectins and glycan-binding antibodies, are particularly valuable if accurate information is available about the glycans that a protein binds. Glycan arrays have been transformational for uncovering rich information about the nuances and complexities of glycan-binding specificity. A challenge, however, has been the analysis of the data. Because protein-glycan interactions are so complex, simplistic modes of analyzing the data and describing glycan-binding specificities have proven inadequate in many cases. This review surveys the methods for handling high-content data on protein-glycan interactions. We contrast the approaches that have been demonstrated and provide an overview of the resources that are available. We also give an outlook on the promising experimental technologies for generating new insights into protein-glycan interactions, as well as a perspective on the limitations that currently face the field.

A Novel Mass Spectrometry Platform for Multiplexed N-Glycoprotein Biomarker Discovery from Patient Biofluids by Antibody Panel Based N-Glycan Imaging.

A new platform for N-glycoprotein analysis from serum that combines matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) workflows with antibody slide arrays is described. Antibody panel based (APB) N-glycan imaging allows for the specific capture of N-glycoproteins by antibodies on glass slides and N-glycan analysis in a protein-specific and multiplexed manner. Development of this technique has focused on characterizing two abundant and well-studied human serum glycoproteins, alpha-1-antitrypsin and immunoglobulin G. Using purified standard solutions and 1 µL samples of human serum, both glycoproteins can be immunocaptured and followed by enzymatic release of N-glycans. N-Glycans are detected with a MALDI FT-ICR mass spectrometer in a concentration-dependent manner while maintaining specificity of capture. Importantly, the N-glycans detected via slide-based antibody capture were identical to that of direct analysis of the spotted standards. As a proof of concept, this workflow was applied to patient serum samples from individuals with liver cirrhosis to accurately detect a characteristic increase in an IgG N-glycan. This novel approach to protein-specific N-glycan analysis from an antibody panel can be further expanded to include any glycoprotein for which a validated antibody exists. Additionally, this platform can be adapted for analysis of any biofluid or biological sample that can be analyzed by antibody arrays.

The sTRA Plasma Biomarker: Blinded Validation of Improved Accuracy Over CA19-9 in Pancreatic Cancer Diagnosis.

PURPOSE: The CA19-9 biomarker is elevated in a substantial group of patients with pancreatic ductal adenocarcinoma (PDAC), but not enough to be reliable for the detection or diagnosis of the disease. We hypothesized that a glycan called sTRA (sialylated tumor-related antigen) is a biomarker for PDAC that improves upon CA19-9. EXPERIMENTAL DESIGN: We examined sTRA and CA19-9 expression and secretion in panels of cell lines, patient-derived xenografts, and primary tumors. We developed candidate biomarkers from sTRA and CA19-9 in a training set of 147 plasma samples and used the panels to make case-control calls, based on predetermined thresholds, in a 50-sample validation set and a blinded, 147-sample test set. RESULTS: The sTRA glycan was produced and secreted by pancreatic tumors and models that did not produce and secrete CA19-9. Two biomarker panels improved upon CA19-9 in the training set, one optimized for specificity, which included CA19-9 and 2 versions of the sTRA assay, and another optimized for sensitivity, which included 2 sTRA assays. Both panels achieved statistical improvement (P < 0.001) over CA19-9 in the validation set, and the specificity-optimized panel achieved statistical improvement (P < 0.001) in the blinded set: 95% specificity and 54% sensitivity (75% accuracy), compared with 97%/30% (65% accuracy). Unblinding produced further improvements and revealed independent, complementary contributions from each marker. CONCLUSIONS: sTRA is a validated serological biomarker of PDAC that yields improved performance over CA19-9. The new panels may enable surveillance for PDAC among people with elevated risk, or improved differential diagnosis among patients with suspected pancreatic cancer.

The CA19-9 and Sialyl-TRA Antigens Define Separate Subpopulations of Pancreatic Cancer Cells.

Molecular markers to detect subtypes of cancer cells could facilitate more effective treatment. We recently identified a carbohydrate antigen, named sTRA, that is as accurate a serological biomarker of pancreatic cancer as the cancer antigen CA19-9. We hypothesized that the cancer cells producing sTRA are a different subpopulation than those producing CA19-9. The sTRA glycan was significantly elevated in tumor tissue relative to adjacent pancreatic tissue in 3 separate tissue microarrays covering 38 patients. The morphologies of the cancer cells varied in association with glycan expression. Cells with dual staining of both markers tended to be in well-to-moderately differentiated glands with nuclear polarization, but exclusive sTRA staining was present in small clusters of cells with poor differentiation and large vacuoles, or in small and ill-defined glands. Patients with higher dual-staining of CA19-9 and sTRA had statistically longer time-to-progression after surgery. Patients with short time-to-progression (<2 years) had either low levels of the dual-stained cells or high levels of single-stained cells, and such patterns differentiated short from long time-to-progression with 90% (27/30) sensitivity and 80% (12/15) specificity. The sTRA and CA19-9 glycans define separate subpopulations of cancer cells and could together have value for classifying subtypes of pancreatic adenocarcinoma.

A Gastric Glycoform of MUC5AC Is a Biomarker of Mucinous Cysts of the Pancreas.

Molecular indicators to specify the risk posed by a pancreatic cyst would benefit patients. Previously we showed that most cancer-precursor cysts, termed mucinous cysts, produce abnormal glycoforms of the proteins MUC5AC and endorepellin. Here we sought to validate the glycoforms as a biomarker of mucinous cysts and to specify the oligosaccharide linkages that characterize MUC5AC. We hypothesized that mucinous cysts secrete MUC5AC displaying terminal N-acetylglucosamine (GlcNAc) in either alpha or beta linkage. We used antibody-lectin sandwich assays to detect glycoforms of MUC5AC and endorepellin in cyst fluid samples from three independent cohorts of 49, 32, and 66 patients, and we used monoclonal antibodies to test for terminal, alpha-linked GlcNAc and the enzyme that produces it. A biomarker panel comprising the previously-identified glycoforms of MUC5AC and endorepellin gave 96%, 96%, and 87% accuracy for identifying mucinous cysts in the three cohorts with an average sensitivity of 92% and an average specificity of 94%. Glycan analysis showed that MUC5AC produced by a subset of mucinous cysts displays terminal alpha-GlcNAc, a motif expressed in stomach glands. The alpha-linked glycoform of MUC5AC was unique to intraductal papillary mucinous neoplasms (IPMN), whereas terminal beta-linked GlcNAc was increased in both IPMNs and mucinous cystic neoplasms (MCN). The enzyme that synthesizes alpha-GlcNAc, A4GNT, was expressed in the epithelia of mucinous cysts that expressed alpha-GlcNAc, especially in regions with high-grade dysplasia. Thus IPMNs secrete a gastric glycoform of MUC5AC that displays terminal alpha-GlcNAc, and the combined alpha-GlcNAc and beta-GlcNAc glycoforms form an accurate biomarker of mucinous cysts.

Characterizing Protein Glycosylation through On-Chip Glycan Modification and Probing.

Glycans are critical to protein biology and are useful as disease biomarkers. Many studies of glycans rely on clinical specimens, but the low amount of sample available for some specimens limits the experimental options. Here we present a method to obtain information about protein glycosylation using a minimal amount of protein. We treat proteins that were captured or directly spotted in small microarrays (2.2 mm × 2.2 mm) with exoglycosidases to successively expose underlying features, and then we probe the native or exposed features using a panel of lectins or glycan-binding reagents. We developed an algorithm to interpret the data and provide predictions about the glycan motifs that are present in the sample. We demonstrated the efficacy of the method to characterize differences between glycoproteins in their sialic acid linkages and N-linked glycan branching, and we validated the assignments by comparing results from mass spectrometry and chromatography. The amount of protein used on-chip was about 11 ng. The method also proved effective for analyzing the glycosylation of a cancer biomarker in human plasma, MUC5AC, using only 20 µL of the plasma. A glycan on MUC5AC that is associated with cancer had mostly 2,3-linked sialic acid, whereas other glycans on MUC5AC had a 2,6 linkage of sialic acid. The on-chip glycan modification and probing (on-chip GMAP) method provides a platform for analyzing protein glycosylation in clinical specimens and could complement the existing toolkit for studying glycosylation in disease.

Glycans related to the CA19-9 antigen are elevated in distinct subsets of pancreatic cancers and improve diagnostic accuracy over CA19-9.

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.

Multiplexed autoantigen microarrays identify HLA as a key driver of anti-desmoglein and -non-desmoglein reactivities in pemphigus.

Patients with pemphigus vulgaris (PV) harbor antibodies reactive against self-antigens expressed at the surface of keratinocytes, primarily desmoglein (Dsg) 3 and, to a lesser extent, Dsg1. Conventionally, only antibodies targeting these molecules have been thought to contribute to disease pathogenesis. This notion has been challenged by a growing pool of evidence that suggests that antibodies toward additional targets may play a role in disease. The aims of this study were to (i) establish high-throughput protein microarray technology as a method to investigate traditional and putative autoantibodies (autoAbs) in PV and (ii) use multiplexed protein array technology to define the scope and specificity of the autoAb response in PV. Our analysis demonstrated significant IgG reactivity in patients with PV toward the muscarinic acetylcholine receptor subtypes 3, 4, and 5 as well as thyroid peroxidase. Furthermore, we found that healthy first- and second-degree relatives of patients with PV express autoAbs toward desmoglein and non-Dsg targets. Our analysis also identified genetic elements, particularly HLA, as key drivers of autoAb expression. Finally, we show that patients with PV exhibit significantly reduced IgM reactivity toward disease-associated antigens relative to controls. The use of protein microarrays to profile the autoAb response in PV advanced the current understanding of disease and provided insight into the complex relationship between genetics and disease development.

Definitive Characterization of CA 19-9 in Resectable Pancreatic Cancer Using a Reference Set of Serum and Plasma Specimens.

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.

Segment and fit thresholding: a new method for image analysis applied to microarray and immunofluorescence data.

Experiments involving the high-throughput quantification of image data require algorithms for automation. A challenge in the development of such algorithms is to properly interpret signals over a broad range of image characteristics, without the need for manual adjustment of parameters. Here we present a new approach for locating signals in image data, called Segment and Fit Thresholding (SFT). The method assesses statistical characteristics of small segments of the image and determines the best-fit trends between the statistics. Based on the relationships, SFT identifies segments belonging to background regions; analyzes the background to determine optimal thresholds; and analyzes all segments to identify signal pixels. We optimized the initial settings for locating background and signal in antibody microarray and immunofluorescence data and found that SFT performed well over multiple, diverse image characteristics without readjustment of settings. When used for the automated analysis of multicolor, tissue-microarray images, SFT correctly found the overlap of markers with known subcellular localization, and it performed better than a fixed threshold and Otsu's method for selected images. SFT promises to advance the goal of full automation in image analysis.

Upregulation of glycans containing 3' fucose in a subset of pancreatic cancers uncovered using fusion-tagged lectins.

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.

Glycan motif profiling reveals plasma sialyl-lewis x elevations in pancreatic cancers that are negative for sialyl-lewis A.

The sialyl-Lewis A (sLeA) glycan forms the basis of the CA19-9 assay and is the current best biomarker for pancreatic cancer, but because it is not elevated in ∼25% of pancreatic cancers, it is not useful for early diagnosis. We hypothesized that sLeA-low tumors secrete glycans that are related to sLeA but not detectable by CA19-9 antibodies. We used a method called motif profiling to predict that a structural isomer of sLeA called sialyl-Lewis X (sLeX) is elevated in the plasma of some sLeA-low cancers. We corroborated this prediction in a set of 48 plasma samples and in a blinded set of 200 samples. An antibody sandwich assay formed by the capture and detection of sLeX was elevated in 13 of 69 cancers that were not elevated in sLeA, and a novel hybrid assay of sLeA capture and sLeX detected 24 of 69 sLeA-low cancers. A two-marker panel based on combined sLeA and sLeX detection differentiated 109 pancreatic cancers from 91 benign pancreatic diseases with 79% accuracy (74% sensitivity and 78% specificity), significantly better than sLeA alone, which yielded 68% accuracy (65% sensitivity and 71% specificity). Furthermore, sLeX staining was evident in tumors that do not elevate plasma sLeA, including those with poorly differentiated ductal adenocarcinoma. Thus, glycan-based biomarkers could characterize distinct subgroups of patients. In addition, the combined use of sLeA and sLeX, or related glycans, could lead to a biomarker panel that is useful in the clinical diagnosis of pancreatic cancer. Précis: This paper shows that a structural isomer of the current best biomarker for pancreatic cancer, CA19-9, is elevated in the plasma of patients who are low in CA19-9, potentially enabling more comprehensive detection and classification of pancreatic cancers.

Exploring the specificities of glycan-binding proteins using glycan array data and the GlycoSearch software.

The glycan array is a powerful tool for investigating the specificities of glycan-binding proteins. By incubating a glycan-binding protein on a glycan array, the relative binding to hundreds of different oligosaccharides can be quantified in parallel. Based on these data, much information can be obtained about the preference of a glycan-binding protein for specific subcomponents of oligosaccharides or motifs. In many cases, the analysis and interpretation of glycan array data can be time consuming and imprecise if done manually. Recently we developed software, called GlycoSearch, to facilitate the analysis and interpretation of glycan array data based on the previously developed methods called Motif Segregation and Outlier Motif Analysis. Here we describe the principles behind the software, the use of the software, and an example application. The automated, objective, and precise analysis of glycan array data should enhance the value of the data for a broad range of research applications.