In situ proteomic analysis by MALDI imaging identifies ubiquitin and thymosin-β4 as markers of malignant intraductal pancreatic mucinous neoplasms.

Vinciane Rebours, Julie Le Faouder, Samira Laouirem, Mounya Mebarki, Miguel Albuquerque, Jean-Michel Camadro, Thibaut Léger, Philippe Ruszniewski, Philippe Lévy, Valérie Paradis, Pierre Bedossa, Anne Couvelard

Journal: Pancreatology : official journal of the International Association of Pancreatology (IAP) ... [et al.] 2014;14(2):117-24

PMID: 24650966

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Abstract

PURPOSE

Intraductal pancreatic mucinous neoplasms (IPMN) are precancerous cystic lesions. The aim was to investigate the in situ IPMN proteome using MALDI (Matrix-Assisted Laser Desorption/Ionisation) imaging and to characterize biomarkers associated with the grade of dysplasia.

EXPERIMENTAL DESIGN

Frozen human Branch duct -IPMN sections were selected according to dysplasia and proteomic analyses were performed by MALDI imaging to obtain mass spectra distribution. The most discriminating peaks were identified using tissue extraction and nanoLC-ESI-MS/MS. Identified peaks were validated in independent series of IPMN by immunochemistry on surgical specimens (tissue-microarrays (TMA), n = 45) and endoscopic ultrasound fine-needle aspiration (EUS FNA) samples (n = 25).

RESULTS

BD-IPMN samples with low (n = 10) and high (n = 10) grades of dysplasia were analyzed. Differential spectra of proteins were found in the two groups with significantly different intensities (n = 15). The two peaks (intense in high grade IPMN) (m/z 8565 and 4747) were characterized as the monomeric ubiquitin (Mascot score = 319.22) and an acetylated fragment of thymosin-β4 (2-42) (Omssa score = 1.37 E-9). Validation on TMA and EUS FNA samples confirmed that ubiquitin was overexpressed in high grade dysplasia (p = 0.04 and p = 0.0004). Thymosin-β4 expression was confirmed on TMA by immunohistochemistry on high grade IPMN (p = 0.011).

CONCLUSION

Ubiquitin and thymosin-β4 are overexpressed in IPMN with high grade dysplasia. Positive immunochemical staining on EUS-FNA material is a major argument in support of preventive resection.

Address: Pancreatology Department, Beaujon Hospital, AP-HP, Clichy, Paris-Diderot University, France; Inserm U773-CRB3, Paris-Diderot University, Paris, France. Electronic address: [email protected].; Inserm U773-CRB3, Paris-Diderot University, Paris, France.; Inserm U773-CRB3, Paris-Diderot University, Paris, France; Pathology Department, Beaujon Hospital, AP-HP, Clichy, Paris-Diderot University, France.; Mass Spectrometry Facility, Jacques Monod Institute, UMR7592, Paris-Diderot University - CNRS, Paris, France; Molecular and Cellular Pathology Program, Jacques Monod Institute, UMR7592, Paris-Diderot University - CNRS, Paris, France.; Mass Spectrometry Facility, Jacques Monod Institute, UMR7592, Paris-Diderot University - CNRS, Paris, France.; Pancreatology Department, Beaujon Hospital, AP-HP, Clichy, Paris-Diderot University, France; Inserm U773-CRB3, Paris-Diderot University, Paris, France.; Pancreatology Department, Beaujon Hospital, AP-HP, Clichy, Paris-Diderot University, France.; Inserm U773-CRB3, Paris-Diderot University, Paris, France; Pathology Department, Bichat Hospital, AP-HP, Paris-Diderot University, France.

DOI: 10.1016/j.pan.2013.12.001

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MeSH Terms: Aged, Amino Acid Sequence, Biomarkers, Tumor, Female, Humans, Hydrolysis, Immunohistochemistry, Male, Middle Aged, Molecular Sequence Data, Neoplasms, Cystic, Mucinous, and Serous, Pancreatic Ducts, Pancreatic Neoplasms, Proteomics, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Thymosin, Trypsin, Ubiquitin