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Quantitative Shotgun Proteomics Unveils Candidate Novel Esophageal Adenocarcinoma (EAC)-specific Proteins

2017; Elsevier BV; Volume: 16; Issue: 6 Linguagem: Inglês

10.1074/mcp.m116.065078

1535-9484

J. Robert O’Neill, HuiSong Pak, Erola Pairo‐Castineira, Vicki Save, Simon Paterson‐Brown, Rudolf Nenutil, Bořivoj Vojtěšek, Ian M. Overton, Alex Scherl, Ted R. Hupp,

Peptidase Inhibition and Analysis

Esophageal cancer is the eighth most common cancer worldwide and the majority of patients have systemic disease at presentation. Esophageal adenocarcinoma (OAC), the predominant subtype in western countries, is largely resistant to current chemotherapy regimens. Selective markers are needed to enhance clinical staging and to allow targeted therapies yet there are minimal proteomic data on this cancer type. After histological review, lysates from OAC and matched normal esophageal and gastric samples from seven patients were subjected to LC MS/MS after tandem mass tag labeling and OFFGEL fractionation. Patient matched samples of OAC, normal esophagus, normal stomach, lymph node metastases and uninvolved lymph nodes were used from an additional 115 patients for verification of expression by immunohistochemistry (IHC).Over six thousand proteins were identified and quantified across samples. Quantitative reproducibility was excellent between technical replicates and a moderate correlation was seen across samples with the same histology. The quantitative accuracy was verified across the dynamic range for seven proteins by immunohistochemistry (IHC) on the originating tissues. Multiple novel tumor-specific candidates are proposed and EPCAM was verified by IHC.This shotgun proteomic study of OAC used a comparative quantitative approach to reveal proteins highly expressed in specific tissue types. Novel tumor-specific proteins are proposed and EPCAM was demonstrated to be specifically overexpressed in primary tumors and lymph node metastases compared with surrounding normal tissues. This candidate and others proposed in this study could be developed as tumor-specific targets for novel clinical staging and therapeutic approaches. Esophageal cancer is the eighth most common cancer worldwide and the majority of patients have systemic disease at presentation. Esophageal adenocarcinoma (OAC), the predominant subtype in western countries, is largely resistant to current chemotherapy regimens. Selective markers are needed to enhance clinical staging and to allow targeted therapies yet there are minimal proteomic data on this cancer type. After histological review, lysates from OAC and matched normal esophageal and gastric samples from seven patients were subjected to LC MS/MS after tandem mass tag labeling and OFFGEL fractionation. Patient matched samples of OAC, normal esophagus, normal stomach, lymph node metastases and uninvolved lymph nodes were used from an additional 115 patients for verification of expression by immunohistochemistry (IHC). Over six thousand proteins were identified and quantified across samples. Quantitative reproducibility was excellent between technical replicates and a moderate correlation was seen across samples with the same histology. The quantitative accuracy was verified across the dynamic range for seven proteins by immunohistochemistry (IHC) on the originating tissues. Multiple novel tumor-specific candidates are proposed and EPCAM was verified by IHC. This shotgun proteomic study of OAC used a comparative quantitative approach to reveal proteins highly expressed in specific tissue types. Novel tumor-specific proteins are proposed and EPCAM was demonstrated to be specifically overexpressed in primary tumors and lymph node metastases compared with surrounding normal tissues. This candidate and others proposed in this study could be developed as tumor-specific targets for novel clinical staging and therapeutic approaches. Esophageal cancer is the sixth leading cause of cancer death worldwide (1.Jemal A. Bray F. Center M.M. Ferlay J. Ward E. Forman D. Global cancer statistics.CA Cancer J. Clin. 2011; 61: 69-90Crossref PubMed Scopus (30255) Google Scholar) and esophageal adenocarcinoma (EAC) 1The abbreviations used are: EAC, Esophageal adenocarcinoma; EGJ, Esophagogastric junction; CT, computed tomography; PET, positron emission tomography; EUS, endoscopic ultrasound; TMT, Tandem Mass Tag; IHC, immunohistochemistry; RT, room temperature; FASP, filter-aided sample preparation; FA, formic acid; dH2O, distilled water; NMWCO, nominal molecular weight cut-off; UPLC, ultra-performance liquid chromatography; TvE, ratio of EAC to normal oesophagus expression; TvG, ratio of EAC to normal gastric expression; ESCC, Esophageal squamous cell carcinoma; LVI, lymphovascular invasion; PNI, perineural invasion. 1The abbreviations used are: EAC, Esophageal adenocarcinoma; EGJ, Esophagogastric junction; CT, computed tomography; PET, positron emission tomography; EUS, endoscopic ultrasound; TMT, Tandem Mass Tag; IHC, immunohistochemistry; RT, room temperature; FASP, filter-aided sample preparation; FA, formic acid; dH2O, distilled water; NMWCO, nominal molecular weight cut-off; UPLC, ultra-performance liquid chromatography; TvE, ratio of EAC to normal oesophagus expression; TvG, ratio of EAC to normal gastric expression; ESCC, Esophageal squamous cell carcinoma; LVI, lymphovascular invasion; PNI, perineural invasion. has become the predominant histological subtype in western countries (2.Lepage C. Rachet B. Jooste V. Faivre J. Coleman M.P. Continuing rapid increase in esophageal adenocarcinoma in England and Wales.Am. J. Gastroenterol. 2008; 103: 2694-2699Crossref PubMed Scopus (214) Google Scholar, 3.Pohl H. Welch H. The role of overdiagnosis and reclassification in the marked increase of esophageal adenocarcinoma incidence.J. Natl. Cancer Inst. 2005; 97: 142-146Crossref PubMed Scopus (1084) Google Scholar). In the UK, 95% of patients diagnosed with EAC will die from metastatic disease and the majority are resistant, at presentation, to current platinum-based chemotherapy regimens (4.Groene O. Cromwell D. Hardwick R. Riley S. Crosby T. Greeenaway K. National Oesophago-Gastric Cancer Audit. Royal College of Surgeons of England, 2012Google Scholar, 5.Allum W.H. Blazeby J.M. Griffin S.M. Cunningham D. Jankowski J.A. Wong R. Association of Upper Gastrointestinal Surgeons of Great Britain and Ireland, the British Society of Gastroenterology and the British Association of Surgical Oncology Guidelines for the management of oesophageal and gastric cancer.Gut. 2011; 60: 1449-1472Crossref PubMed Scopus (446) Google Scholar, 6.O'Neill J.R. Kennedy E.D. Save V. Langdale-Brown B. Wall L. Skipworth R.J.E. Paterson-Brown S. Patients unfit for neoadjuvant therapy may still undergo resection of locally advanced esophageal or esophagogastric junctional cancer with acceptable oncological results.IJS Oncol. 2017; 2: e09Crossref Google Scholar). EAC is frequently associated with both lymphatic and distant metastases yet current staging modalities including computed tomography (CT), positron emission tomography (PET) and endoscopic ultrasound (EUS) are limited in both sensitivity and specificity (5.Allum W.H. Blazeby J.M. Griffin S.M. Cunningham D. Jankowski J.A. Wong R. Association of Upper Gastrointestinal Surgeons of Great Britain and Ireland, the British Society of Gastroenterology and the British Association of Surgical Oncology Guidelines for the management of oesophageal and gastric cancer.Gut. 2011; 60: 1449-1472Crossref PubMed Scopus (446) Google Scholar). Surgical resection only benefits patients with localized disease and carries a 40% risk of major morbidity and 2–3% risk of perioperative mortality (7.Dikken J.L. van Sandick J.W. Allum W.H. Johansson J. Jensen L.S. Putter H. Coupland V.H. Wouters M.W. Lemmens V.E. van de Velde C.J. van der Geest L.G. Larsson H.J. Cats A. Verheij M. Differences in outcomes of oesophageal and gastric cancer surgery across Europe.Br. J. Surg. 2013; 100: 83-94Crossref PubMed Scopus (120) Google Scholar, 8.Fischer C. Lingsma H. Hardwick R. Cromwell D.A. Steyerberg E. Groene O. Risk adjustment models for short-term outcomes after surgical resection for oesophagogastric cancer.Br. J. Surg. 2016; 103: 105-116Crossref PubMed Scopus (25) Google Scholar). The development of accurate noninvasive imaging markers of EAC would enhance clinical staging by allowing the specific detection of locoregional and distant metastases, enabling treatment stratification (9.McElroy M. Kaushal S. Luiken G.A. Talamini M.A. Moossa A.R. Hoffman R.M. Bouvet M. Imaging of primary and metastatic pancreatic cancer using a fluorophore-conjugated anti-CA19–9 antibody for surgical navigation.World J. Surg. 2008; 32: 1057-1066Crossref PubMed Scopus (93) Google Scholar). The normal squamous epithelium-lined esophagus is vulnerable to toxic insult from the esophageal lumen. Indeed, chronic reflux of gastric acid and bile is thought to underlie the development of columnar metaplasia, "Barrett's esophagus", the precursor lesion of EAC (10.Souza R.F. Krishnan K. Spechler S.J. Acid, bile, and CDX: the ABCs of making Barrett's metaplasia.Am. J. Physiol. Gastrointest. Liver Physiol. 2008; 295: 1-218Crossref Scopus (156) Google Scholar). Although the exact molecular mechanisms of Barrett's development and esophageal carcinogenesis remain obscure, the detection and treatment of EAC at an early stage offers the prospect of long term cure with over 80% of patients undergoing surgery for stage I esophageal cancer surviving 5 years (11.Gertler R. Stein H.J. Langer R. Nettelmann M. Schuster T. Hoefler H. Siewert J.R. Feith M. Long-term outcome of 2920 patients with cancers of the esophagus and esophagogastric junction: evaluation of the New Union Internationale Contre le Cancer/American Joint Cancer Committee staging system.Ann. Surg. 2011; 253: 689-698Crossref PubMed Scopus (127) Google Scholar). Intriguingly many of the genetic mutations present in EAC have also been demonstrated in nondysplastic Barrett's epithelium raising the possibility that a transcriptional change such as splicing or RNA-editing, or a post-translational modification is responsible for transformation (12.Weaver J.M. Ross-Innes C.S. Shannon N. Lynch A. Forshew T. Barbera M. Murtaza M. Ong C.A. Lao-Sirieix P. Dunning M.J. Smith L. Smith M.L. Anderson C.L. Carvalho B. O'Donovan M. Underwood T.J. May A.P. Grehan N. Hardwick R. Davies J. Oloumi A. Aparicio S. Caldas C. Eldridge M.D. Edwards P.A. Rosenfeld N. Tavaré S. Fitzgerald R.C. OCCAMS Consortium Ordering of mutations in preinvasive disease stages of esophageal carcinogenesis.Nat. Genet. 2014; 46: 837-843Crossref PubMed Scopus (241) Google Scholar). If such a biomarker could be identified this would offer the possibility of earlier diagnosis and more effective treatment. Characterizing the proteomic changes associated with EAC may also allow novel therapies to be designed. Tumor-specific proteins have been exploited as immunotherapeutic targets in other cancer types by engendering a host response to the cancer (13.McGranahan N. Furness A.J. Rosenthal R. Ramskov S. Lyngaa R. Saini S.K. Jamal-Hanjani M. Wilson G.A. Birkbak N.J. Hiley C.T. Watkins T.B. Shafi S. Murugaesu N. Mitter R. Akarca A.U. Linares J. Marafioti T. Henry J.Y. Van Allen E.M. Miao D. Schilling B. Schadendorf D. Garraway L.A. Makarov V. Rizvi N.A. Snyder A. Hellmann M.D. Merghoub T. Wolchok J.D. Shukla S.A. Wu C.J. Peggs K.S. Chan T.A. Hadrup S.R. Quezada S.A. Swanton C. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade.Science. 2016; 351: 1463-1469Crossref PubMed Scopus (1946) Google Scholar), in some cases leading to durable responses (14.Rosenberg S.A. Yang J.C. Sherry R.M. Kammula U.S. Hughes M.S. Phan G.Q. Citrin D.E. Restifo N.P. Robbins P.F. Wunderlich J.R. Morton K.E. Laurencot C.M. Steinberg S.M. White D.E. Dudley M.E. Durable complete responses in heavily pretreated patients with metastatic melanoma using T-cell transfer immunotherapy.Clin. Cancer Res. 2011; 17: 4550-4557Crossref PubMed Scopus (1525) Google Scholar). To date, no specific markers of EAC have been identified. To identify candidate proteins de novo, expression must be measured using untargeted proteomic methods. Quantitative proteomic methods have now been applied across many cancer tissues. Most previous proteomic studies in EAC, however, have only identified a small number of dysregulated proteins limiting the comparisons that can be made between studies or with other cancers (summarized in Table I). Only one of these previous studies employed a quantitative shotgun proteomic strategy. The authors compared pooled biopsies of EAC, normal esophagus, gastric adenocarcinoma and normal gastric tissue and identified 972 proteins. Although no EAC-specific protein was identified, neutrophil defensin 1, an antimicrobial peptide found in neutrophil granules, was overexpressed in both cancer types relative to normal tissue (15.Singhal R. Carrigan J.B. Wei W. Taniere P. Hejmadi R.K. Forde C. Ludwig C. Bunch J. Griffiths R.L. Johnson P.J. Tucker O. Alderson D. Günther U.L. Ward D.G. MALDI profiles of proteins and lipids for the rapid characterisation of upper GI-tract cancers.J. Proteomics. 2013; 80: 207-215Crossref PubMed Scopus (14) Google Scholar). This may reflect the inflammatory environment associated with these cancers.Table IPublished proteomic studies of OACStudy authorPatientsMethodTissue preparationSquamous samplesGastric samplesBarrett's samplesACC samplesSCC samplesTotal identificationsNumber of proteins dysregulatedZhao226LC-ESI TOF MS, Targeted LC-MS/MSFresh frozen biopsies--66--38 proteinsYoo681LC-ESI TOF MS, Targeted MALDI-MSFresh frozen biopsies---1-22 proteins-Peng2182D Gels, Targeted MALDI-MSFresh frozen biopsies22-8--23 dysregulated gel spots – 22 proteins identifiedLanger69202D Gels, Targeted MALDI- and LC-MS/MSFresh frozen biopsies20-Data for 4 proteins presentedQuaas70477MALDI-MSIFFPE slides from a tissue microarray---30017772 spectral features 13 peptides-Aichler7123MALDI-MSI. Targeted LC-MS/MSFresh frozen biopsies---23-22 spectral features, 6 proteinsElsner7238MALDI-MSI. Targeted LC-MS/MSFresh frozen biopsies--113361 spectral features, 6 proteinsStreitz734MALDI-MSLCM of fresh frozen biopsies--448 spectral featuresSinghal1553 (iTRAQ)MALDI-MS iTRAQ LC-MS/MSFresh frozen biopsies302330-972 proteinsNot described.Abbreviations: EAC, Esophageal Adenocarcinoma; ACC, Adenocarcinoma; SCC, Squamous Cell Carcinoma; LCM, Laser Capture Microdissection. Open table in a new tab Abbreviations: EAC, Esophageal Adenocarcinoma; ACC, Adenocarcinoma; SCC, Squamous Cell Carcinoma; LCM, Laser Capture Microdissection. The comparisons between EAC and normal squamous epithelium in published work reveals many dysregulated proteins, some of which represent proteins associated with glandular differentiation and some associated with carcinogenesis. Glandular-associated proteins may be expressed in gastric and intestinal epithelium and may not represent tractable targets for therapy as toxicity because of intestinal epithelial damage would be expected. It is possible that including columnar epithelium-lined gastric tissue along with squamous and EAC tissue may enable the discrimination of proteins that reflect glandular differentiation from those driving carcinogenesis. Multitissue proteomic profiling has been applied across mouse tissues with relative quantitation using a super-SILAC approach (16.Geiger T. Velic A. Macek B. Lundberg E. Kampf C. Nagaraj N. Uhlen M. Cox J. Mann M. Initial quantitative proteomic map of 28 mouse tissues using the SILAC mouse.Mol. Cell. Proteomics. 2013; 12: 1709-1722Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In this study, snap-frozen biopsies from 28 tissue types were subjected to shotgun proteomics with a spike-in, heavy-labeled mixture of all tissues obtained from the SILAC mouse. By comparing the relative expression of proteins across tissues, tissue-specific expression could be highlighted. The esophagus was not included in this profiling effort although gastrointestinal tissues with columnar epithelia showed similar expression patterns (16.Geiger T. Velic A. Macek B. Lundberg E. Kampf C. Nagaraj N. Uhlen M. Cox J. Mann M. Initial quantitative proteomic map of 28 mouse tissues using the SILAC mouse.Mol. Cell. Proteomics. 2013; 12: 1709-1722Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). This comparative approach has also been employed in a large proteomic study of 30 human tissues by label-free quantification and again tissue-specific expression patterns identified (17.Kim M.S. Pinto S.M. Getnet D. Nirujogi R.S. Manda S.S. Chaerkady R. Madugundu A.K. Kelkar D.S. Isserlin R. Jain S. Thomas J.K. Muthusamy B. Leal-Rojas P. Kumar P. Sahasrabuddhe N.A. Balakrishnan L. Advani J. George B. Renuse S. Selvan L.D. Patil A.H. Nanjappa V. Radhakrishnan A. Prasad S. Subbannayya T. Raju R. Kumar M. Sreenivasamurthy S.K. Marimuthu A. Sathe G.J. Chavan S. Datta K.K. Subbannayya Y. Sahu A. Yelamanchi S.D. Jayaram S. Rajagopalan P. Sharma J. Murthy K.R. Syed N. Goel R. Khan A.A. Ahmad S. Dey G. Mudgal K. Chatterjee A. Huang T.C. Zhong J. Wu X. Shaw P.G. Freed D. Zahari M.S. Mukherjee K.K. Shankar S. Mahadevan A. Lam H. Mitchell C.J. Shankar S.K. Satishchandra P. Schroeder J.T. Sirdeshmukh R. Maitra A. Leach S.D. Drake C.G. Halushka M.K. Prasad T.S. Hruban R.H. Kerr C.L. Bader G.D. Iacobuzio-Donahue C.A. Gowda H. Pandey A. A draft map of the human proteome.Nature. 2014; 509: 575-581Crossref PubMed Scopus (1494) Google Scholar). This biomarker discovery study therefore used a quantitative shotgun proteomic strategy to evaluate protein expression in EAC and adjacent matched normal squamous and gastric tissues from seven patients. By quantifying the relative expression between EAC and normal esophagus and EAC and normal stomach, proteins aberrantly expressed in EAC were identified. The accuracy of this approach was confirmed by immunohistochemistry for multiple candidates and a potential tumor biomarker verified in a cohort of 115 patients with resected EAC and matched normal and metastatic tissues. Fresh frozen biopsies representing macroscopically normal esophagus, normal stomach and esophageal adenocarcinoma tissue were prospectively collected from resection specimens from seven patients undergoing neoadjuvant chemotherapy and attempted curative surgery for locally advanced esophageal and esophagogastric junctional cancer at the Royal Infirmary of Edinburgh between 2010 and 2012. Local institutional ethical and research and development approvals were in place (REC references 06/S1101/16 and 10/S1402/33) (R&D ID 2006/W/PA/01). All patients gave informed consent and participants and their donated samples were de-identified at the time of recruitment. Patients were selected for relative clinical homogeneity with respect to known prognostic variables including lymphatic metastasis and tumor differentiation (18.O'Neill J.R. Stephens N.A. Save V. Kamel H.M. Phillips H.A. Driscoll P.J. Paterson-Brown S. Defining a positive circumferential resection margin in oesophageal cancer and its implications for adjuvant treatment.Br. J. Surg. 2013; 100: 1055-1063Crossref PubMed Scopus (21) Google Scholar). The clinical characteristics of the cohort are presented in Table II.Table IIClinical characteristics of patients donating tissue for proteomic analysisPatient (Pt)Pt44Pt46Pt48Pt51Pt53Pt60Pt61GenderMaleMaleMaleMaleFemaleMaleMaleAge59676541526058HistologyACCACCACCACCACCACCACCLocationEGJ Type IIEso LowerEGJ Type IIEGJ Type IEso LowerEso LowerEso LowerNeoadjuvant therapy2xCF2xCF2xCF2xCF2xCF2xCF2xCFSurgeryILEILEILEILEILEILEILETumor Diameter38 mm70 mm40 mm50 mm83 mm52 mm35 mmPRM and DRM>1 mm>1 mm>1 mm>1 mm>1 mm>1 mm>1 mmDistance to CRM4.2 mm0.0 mm0.0 mm0.3 mm3.0 mm1.0 mm1.0 mmResectionR0R1R1R1R0R0R0DifferentiationModeratePoorPoorPoorPoorPoorPoorLVIYYYYNYYVenous InvasionNNNYNNNPNINYYYNYYT stageypT2ypT4aypT3ypT3ypT2ypT3ypT3N StageypN1ypN3ypN3ypN3ypN1ypN3ypN2Positive nodes28167173Nodes resected27182828232137AJCC StageIIBIIICIIICIIICIIBIIICIIIBMandard TRGVVIVVIVVVAlive at analysisNoNoNoNoYesNoYesOverall survival48.1 months15.3 months10.9 months24.1 months47.6 months (censored)17.8 months49.0 months (censored)Recurrence-free survival34.8 months12.6 months10.1 months10.9 months47.6 months (censored)8.1 months49.0 months (censored)Abbreviations: ACC, Adenocarcinoma, 2xCF-2 cycles of Cisplatin and 5-Fluorouracil; ILE, Ivor-Lewis Esophagectomy; mm–millimetre, AJCC, American Joint Committee on Cancer; EGJ, Esophagogastric Junctional Tumour; PRM, Proximal resection margin; DRM, Distal resection margin; CRM, Circumferential resection margin; LVI, Lymphovascular invasion; PNI, perineural invasion, Y-Yes, N-No; CT, Computed Tomography, Mandard; TRG, Tumour Regression Grade; Eso, Esophagus. Open table in a new tab Abbreviations: ACC, Adenocarcinoma, 2xCF-2 cycles of Cisplatin and 5-Fluorouracil; ILE, Ivor-Lewis Esophagectomy; mm–millimetre, AJCC, American Joint Committee on Cancer; EGJ, Esophagogastric Junctional Tumour; PRM, Proximal resection margin; DRM, Distal resection margin; CRM, Circumferential resection margin; LVI, Lymphovascular invasion; PNI, perineural invasion, Y-Yes, N-No; CT, Computed Tomography, Mandard; TRG, Tumour Regression Grade; Eso, Esophagus. At the commencement of this study, no shotgun proteomic data were available for esophageal adenocarcinoma tissue to inform a power calculation for sample size determination. The sample number was therefore based on previous esophageal discovery-phase proteomic studies or studies in similar tissue types (19.Cai Z. Zhao J.S. Li J.J. Peng D.N. Wang X.Y. Chen T.L. Qiu Y.P. Chen P.P. Li W.J. Xu L.Y. Li E.M. Tam J.P. Qi R.Z. Jia W. Xie D. A combined proteomics and metabolomics profiling of gastric cardia cancer reveals characteristic dysregulations in glucose metabolism.Mol. Cell. Proteomics. 2010; 9: 2617-2628Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 20.Besson D. Pavageau A.H. Valo I. Bourreau A. Bélanger A. Eymerit-Morin C. Moulière A. Chassevent A. Boisdron-Celle M. Morel A. Solassol J. Campone M. Gamelin E. Barré B. Coqueret O. Guette C. A quantitative proteomic approach of the different stages of colorectal cancer establishes OLFM4 as a new nonmetastatic tumor marker.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 21.Peng D. Sheta E.A. Powell S.M. Moskaluk C.A. Washington K. Goldknopf I.L. El-Rifai W. Alterations in Barrett's-related adenocarcinomas: A proteomic approach.Int. J. Cancer. 2008; 122: 1303-1310Crossref PubMed Scopus (31) Google Scholar, 22.Zhao J. Chang A.C. Li C. Shedden K.A. Thomas D.G. Misek D.E. Manoharan A.P. Giordano T.J. Beer D.G. Lubman D.M. Comparative proteomics analysis of Barrett metaplasia and esophageal adenocarcinoma using two-dimensional liquid mass mapping.Mol. Cell. Proteomics. 2007; 6: 987-999Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and the availability of high quality clinical material. Because of the risk of false-positives because of the small sample size, proposed tumor-specific proteins identified by mass spectrometry were additionally verified by immunohistochemistry (IHC) using cores from archival tumors and matched normal and metastatic tissues from an independent cohort of 115 patients with esophageal or EGJ adenocarcinoma (clinical characteristics in supplemental Table S1). The sample processing workflow is summarized in Fig. 1. Fresh tissue biopsies were snap frozen within 30 min of tumor extirpation and maintained in liquid nitrogen or on dry ice until lysis. Frozen sections from each biopsy were reviewed by a consultant histopathologist to confirm the histological diagnosis and, for tumor biopsies, a minimum of 50% tumor cellularity. The published Filter-Aided Sample Preparation (FASP) method was adapted for protein extraction and tryptic digestion from esophagogastric tissue (23.Wiœniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5043) Google Scholar). Biopsies between 30 mg and 60 mg in weight were maintained on dry ice until rapid disruption at room temperature (RT) in low-binding micro-centrifuge tubes containing 1 mm ceramic beads (Matrix D, MP Bio, Santa Ana, CA) by rapid shaking in a bench-top homogenizer (FastPrep-24, MP Bio) for 40 s at 6ms−1. Homogenates were dissolved in FASP lysis buffer (4% w/v SDS, 100 mm Tris/HCl, 100 mm DTT, pH 7.6), mixed for 20 min at RT and sonicated at maximum amplitude, for 30 s, on ice, using a needle sonicator (Bioruptor, Diagenode, Liège, Belgium). Sonicated lysates were heated for 5 min at 95 °C and clarified by centrifugation at 14,000g for 5 min at 20pC before buffer-exchange as per the published FASP protocol (23.Wiœniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5043) Google Scholar). Trypsinization was performed off-column, overnight at 37 °C at a ratio of 100:1 (lysate protein mass : trypsin mass) using sequencing grade modified trypsin (Promega, Madison, WI) as per manufacturer's instructions. Protein concentration was determined using a modified Lowry procedure as per the manufacturer's recommendations (RC-DC, Bio-Rad, Hercules, CA). Tryptic peptides from each tissue sample were independently labeled with one of the 6 Tandem Mass Tag (TMT) reagents (Thermo Scientific, San Jose, CA) in technical duplicate per manufacturer's instructions (Fig. 1). Labeled peptides from a single patient (6 reporter ions) were pooled, desalted on a Macro SpinColumn C18 (Harvard Apparatus, Holliston, MA) and separated into 24 fraction by OFFGEL electrophoresis as previously described (24.Dayon L. Turck N. Kienle S. Schulz-Knappe P. Hochstrasser D.F. Scherl A. Sanchez J.C. Isobaric tagging-based selection and quantitation of cerebrospinal fluid tryptic peptides with reporter calibration curves.Anal. Chem. 2010; 82: 848-858Crossref PubMed Scopus (43) Google Scholar). Dried, desalted peptide fractions were reconstituted in 5% (v/v) acetonitrile, 0.1% (v/v) Formic Acid (FA) in dH2O and ∼0.5 μg loaded onto a homemade, 100 μm internal diameter, 20 mm long trapping column packed with 200 Å, 5 μm Magic C18 AQ (Michrom, Auburn, CA). Trapped peptides were eluted into a 75 μm internal diameter, 150 mm long analytical column packed with 100 Å, 3 μm Magic C18 AQ (Microcom). For ultraperformance liquid chromatography (UPLC), peptides were separated using a variable solvent gradient created by a combination of 0.1% (v/v) FA in dH2O (solvent A) and 0.1% (v/v) FA in acetonitrile (solvent B). The gradient was run as follows: 0–1 mins, 95% (A) and 5% (B), 1–56 mins, 65% (A) and 35% (B), 66–76 mins, 20% (A) and 80% (B) using a flow rate of 220 nL/min. Peptides were analyzed in positive ion mode after electrospray ionisation on an LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific, San Jose, CA). For MS survey scans, the Orbitrap (OT) resolution was set to 60,000 and the ion population was set to 5 × 105 with an m/z window from 400 to 2000. A maximum of three precursor ions with the greatest peak intensities were selected for both collision-induced dissociation (CID) and high-energy C-trap dissociation (HCD) in the LTQ with analysis in the OT. For fragment ion analysis in the LTQ, the ion population was set to 7 × 103 (isolation width of 2 m/z) whereas for detection in the OT, the ion population was set to 2 × 105 (isolation width of 2.5 m/z), with resolution of 7500, first mass at m/z = 100, and maximum injection time of 750 ms. The normalized collision energies were set to 35% for CID and 60% for HCD. Protein identifications were made using the Easyprot platform (v2.3 build 720, Swiss Institute of Bioinformatics) (25.Gluck F. Hoogland C. Antinori P. Robin X. Nikitin F. Zufferey A. Pasquarello C. Fétaud V. Dayon L. Müller M. Lisacek F. Geiser L. Hochstrasser D. Sanchez J.C. Scherl A. EasyProt–an easy-to-use graphical platform for proteomics data analysis.J. Proteomics. 2013; 79: 146-160Crossref PubMed Scopus (59) Google Scholar). Data manipulation was performed using Excel (Version 14.0.6129.5000, Microsoft Office Professional 2010), R (version 2.15.1, General Public License), and custom scripts written in Perl (version 5.18.0, General Public License). Thermo RAW files were converted to peak lists using ReAdW (version 4.3.1, ThermoFinnigan) and CID and HCD spectra were merged for simultaneous identification and quantification as previously described (26.Dayon L. Pasquarello C. Hoogland C. Sanchez J.C. Scherl A. Combining low- and high-energy tandem mass spectra for optimized peptide quantification with isobaric tags.J. Proteomics. 2010; 73: 769-777Crossref PubMed Scopus (96) Google Scholar). Peaklist files were searched against the Uniprot human reference proteome (release 09/01/2013, containing 87,613 entries) using Phenyx® (version 2.6.1, GeneBio) (27.Colinge J. Masselot A. Giron M. Dessingy T. Magnin J. OLAV: towards high-throughput tandem mass spectrometry data identification.Proteomics. 2003; 3: 1454-1463Crossref PubMed Scopus (268) Google Scholar, 28.Vizcaíno J.A. Côté R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. O'Kelly. G. Schoenegger A. Ovelleiro D. Pérez-Riverol Y. Reisinger F. Ríos D. Wang R. Hermjakob H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.Nucleic Acids Res. 2013; 41: D1063-D1069Crossref PubMed Scopus (1595) Google Scholar) with a precursor ion tolerance of 10 parts per million and a fragment ion tolerance of 0.6 Da. Variable peptide modifications included TMT-modified N termini and lysines (additional 229.1629 Da) and oxidized methionines, with carbamidomethylation of cysteines set as a fixed modification. Trypsin was selected as the digestion enzyme, with one potential missed cleavage and a minimum of a single-tryptic terminus, a peptide length of 6 amino acids and a z-score of 4 wer