A multiplexed bead assay for profiling glycosylation patterns on serum protein biomarkers of pancreatic cancer

A multiplexed bead‐based immunoassay was developed to simultaneously profile glycosylation patterns of serum proteins to investigate their usefulness as biomarkers for pancreatic cancer. The multiplex assay utilized protein‐specific capture antibodies chemically coupled individually to beads labeled with specific amounts of fluorescent dye. Captured proteins were detected based on the extent and specific type of glycosylation as determined by successive binding of fluorescent lectin probes. Advantages to this technique include the fact that antibodies coupled to the beads had minimal nonspecific binding to the lectins ConA/SNA, avoiding the step of chemically blocking the antibody glycans and the bead assays were performed in a 96‐well filter plate enabling high‐throughput screening applications with improved reproducibility. The assay was tested with ConA and SNA lectins to examine the glycosylation patterns of α‐1‐β glycoprotein (A1BG) and serum amyloid p (SAP) component for use as potential biomarkers for the detection of pancreatic cancer based on the results from prior biomarker studies. The results showed that the SNA response on the captured A1BG protein could distinguish chronic pancreatitis samples from pancreatic cancer with a p ‐value of 0.035 and for the SAP protein with SNA, a p ‐value of 0.026 was found between the signal of normal controls and the pancreatic cancer samples. For the ConA response, a decline in the signal for both proteins in the serum samples was found to distinguish pancreatic cancer from normal controls and renal cell carnoma samples (A1BG, p <0.05; and SAP, p <0.0001).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/87003/1/2028_ftp.pd

Irrelevance of microsatellite instability in the epidemiology of sporadic pancreatic ductal adenocarcinoma

Background and Aims: Pancreatic cancer risk is increased in Lynch syndrome (LS) patients with mismatch repair gene defects predisposing to colonic and extracolonic cancers with microsatellite instability (MSI). However, the frequency of MSI pancreatic cancers has never been ascertained in consecutive, unselected clinical series, and their contribution to the sporadic and inherited burden of pancreatic cancer remains to be established. Aims of the study were to determine the prevalence of MSI in surgically resected pancreatic cancers in a multicentric, retrospective study, and to assess the occurrence of pancreatic cancer in LS. Methods: MS-status was screened by a panel of 5 mononucleotide repeats (Bat26, Bat25, NR-21, NR-24 and NR-27) in 338 consecutive pancreatic ductal adenocarcinoma (PDAC), resected at two Italian and one German referral centres. The personal history of pancreatic cancer was assessed in an independent set of 58 probands with LS and in 138 first degree relatives who had cancers. Results: Only one PDAC (0.3%) showed MSI. This was a medullary type cancer, with hMLH1-deficiency, and no identified germ-line mutation but methylation of hMLH1. Pancreatic cancer occurred in 5 (2.5%) LS patients. Histological sampling was available for 2 cases, revealing PDAC in one case and an ampullary cancer in the other one. Conclusions: MSI prevalence is negligible in sporadic, resected PDAC. Differently, the prevalence of pancreatic cancer is 2.5% in LS patients, and cancers other than PDAC may be encountered in this setting. Surveillance for pancreatic cancer should be advised in LS mutation carriers at referral centers

Matrix-assisted laser desorption/ionization imaging protocol for in situ characterization of tryptic peptide identity and distribution in formalin-fixed tissue

RationaleMatrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry provides the means to map the in situ distribution of tryptic peptides in formalin-fixed clinical tissue samples. The ability to analyze clinical samples is of great importance to further developments in the imaging field. However, there is a requirement in this field of research for additional methods describing the characterization of tryptic peptides by MALDI imaging.Methods and resultsThis protocol gives highly detailed instructions, with examples, for (1) successfully performing tryptic peptide MALDI imaging on formalin-fixed cancer tissue using a MALDI-TOF/TOF MS instrument, (2) tentatively generating identifications through nLC/MS/MS, and (3) validating these identifications by in situ MS/MS of peptides of interest.ConclusionsThis protocol provides a detailed and straightforward description of the methods required for groups new to MALDI imaging to begin analysis of formalin-fixed clinical samples.Ove J. R. Gustafsson, James S. Eddes, Stephan Meding, Shaun R. McColl, Martin K. Oehler and Peter Hoffman