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.

Certain experiments involve the high-throughput quantification of image data, thus requiring 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 multi-color, 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.

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 × 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 nanograms. 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.

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.

Summary Patients with Gardner’s syndrome are increasingly found to have polyps on routine upper gastrointestinal endoscopy, and their risk of developing periampullary carcinoma is between 3-12%. We report a 45 year old man with Gardner’s syndrome who presented with periampullary carcinoma 5 years after colectomy. Review of the literature amassed another 21 cases of periampullary carcinoma in patients with Gardner’s syndrome.

A 45-year-old man presented with a 1 week history of progressive painless jaundice. An ultrasound examination at the referring hospital indicated distal common bile duct (CBD) obstruction. Six years previously he had noticed swelling around both mandibular angles and an orthopantomograph had shown two rounded radio-opaque masses in these areas (Fig. 1). One year later, the patient had a colectomy with an ileo-rectal anastomosis.