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Identification of Salivary Biomarkers for Oral Cancer Detection with Untargeted and Targeted Quantitative Proteomics Approaches*[S]

  • Hao-Wei Chu
    Footnotes
    Affiliations
    ‡Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
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  • Kai-Ping Chang
    Footnotes
    Affiliations
    §Department of Otolaryngology-Head & Neck Surgery, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan
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  • Chia-Wei Hsu
    Affiliations
    ¶Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan
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  • Ian Yi-Feng Chang
    Affiliations
    ¶Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan
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  • Hao-Ping Liu
    Affiliations
    ‖Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan
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  • Yi-Ting Chen
    Affiliations
    ‡Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan

    ¶Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan

    **Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan

    ‡‡Kidney Research Center, Department of Nephrology, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan
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  • Chih-Ching Wu
    Correspondence
    To whom correspondence should be addressed: Tel.: 886-3-2118800, ext. 5093
    Affiliations
    ‡Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan

    §Department of Otolaryngology-Head & Neck Surgery, Chang Gung Memorial Hospital, Linkou, Taoyuan, Taiwan

    ¶Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan

    §§Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan

    ¶¶Research Center for Emerging Viral Infections, College of Medicine, Chang Gung University, Taoyuan, Taiwan
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  • Author Footnotes
    * This work was financially supported by grants to C.C.W. from Ministry of Science and Technology (MOST), Taiwan (MOST 107-2320-B-182-009 and MOST 108-2320-B-182-030-MY3), Chang Gung Memorial Hospital (CGMH), Taiwan (CLRPD190018 and BMRPC77), Research Center for Emerging Viral Infections, Chang Gung University, and Molecular Medicine Research Center, Chang Gung University, from Featured Areas Research Center Program within the Framework of Higher Education Sprout Project by Ministry of Education and Ministry of Science and Technology, Taiwan (MOST 107-3017-F-182-001).
    [S] This article contains supplemental material.
    ‡‡‡ These authors have contributed equally to this work.
Open AccessPublished:June 28, 2019DOI:https://doi.org/10.1074/mcp.RA119.001530
      Oral cavity squamous cell carcinoma (OSCC) is one of the most common cancers worldwide. In Taiwan, OSCC is the fifth leading cause of cancer-related mortality and leads to 2800 deaths per year. The poor outcome of OSCC patients is principally ascribed to the fact that this disease is often advanced at the time of diagnosis, suggesting that early detection of OSCC is urgently needed. Analysis of cancer-related body fluids is one promising approach to identify biomarker candidates of cancers. To identify OSCC biomarkers, salivary proteomes of OSCC patients, individuals with oral potentially malignant disorders (OPMDs), and healthy volunteers were comparatively profiled with isobaric tags for relative and absolute quantitation (iTRAQ)-based mass spectrometry (MS). The salivary levels of 67 and 18 proteins in the OSCC group are elevated and decreased compared with that in the noncancerous group (OPMD and healthy groups), respectively. The candidate biomarkers were further selected using the multiple reaction monitoring (MRM)-MS and validated with the immunoassays. More importantly, the higher salivary level of three proteins, complement factor H (CFH), fibrinogen alpha chain (FGA), and alpha-1-antitrypsin (SERPINA1) was correlated with advanced stages of OSCC. Our results indicate that analysis of salivary proteome is a feasible strategy for biomarker discovery, and the three proteins are potential salivary markers for OSCC diagnosis.

      Graphical Abstract

      Oral cancer is one of the leading causes of cancer-related mortality. There were ∼300,000 cases of oral cancer and around 150,000 patients died from oral cancer worldwide in 2018 (
      • Bray F.
      • Ferlay J.
      • Soerjomataram I.
      • Siegel R.L.
      • Torre L.A.
      • Jemal A.
      Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
      ). In Taiwan, oral cancer ranks fifth in terms of deaths for males and leads to 2800 deaths per year (

      Cancer registration system annual report (2017). Department of Statistics, Ministry of Health and Welfare, Executive Yuan, Republic of China (Taiwan). (http://www.mohw.gov.tw/EN/Ministry/Index.aspx),

      ). Oral cavity squamous cell carcinoma (OSCC)
      The abbreviations used are:
      OSCC
      oral cavity squamous cell carcinoma
      APOA1
      apolipoprotein A-I
      APOA2
      apolipoprotein A-II
      AUC
      area under the ROC curve
      CFH
      complement factor H
      CID
      collision-induced dissociation
      DAVID
      Database for Annotation, Visualization and Integrated Discovery
      FGA
      fibrinogen alpha chain
      HCD
      higher-energy collision-induced dissociation
      iTRAQ
      isobaric tags for relative and absolute quantitation
      KEGG
      Kyoto Encyclopedia of Genes and Genomes
      LOD
      limit of detection
      LLOQ
      lower limit of quantitation
      MRM
      multiple reaction monitoring
      OPMD
      oral potentially malignant disorders
      ROC
      receiver operator characteristic
      SERPINA1
      alpha-1-antitrypsin
      SIS
      stable isotope-labeled standard.
      1The abbreviations used are:OSCC
      oral cavity squamous cell carcinoma
      APOA1
      apolipoprotein A-I
      APOA2
      apolipoprotein A-II
      AUC
      area under the ROC curve
      CFH
      complement factor H
      CID
      collision-induced dissociation
      DAVID
      Database for Annotation, Visualization and Integrated Discovery
      FGA
      fibrinogen alpha chain
      HCD
      higher-energy collision-induced dissociation
      iTRAQ
      isobaric tags for relative and absolute quantitation
      KEGG
      Kyoto Encyclopedia of Genes and Genomes
      LOD
      limit of detection
      LLOQ
      lower limit of quantitation
      MRM
      multiple reaction monitoring
      OPMD
      oral potentially malignant disorders
      ROC
      receiver operator characteristic
      SERPINA1
      alpha-1-antitrypsin
      SIS
      stable isotope-labeled standard.
      accounts for more than 90‥ of oral cancers and is associated with chronic irritating habits such as betel quid chewing, alcohol drinking, and smoking (
      • Herrero R.
      • Castellsague X.
      • Pawlita M.
      • Lissowska J.
      • Kee F.
      • Balaram P.
      • Rajkumar T.
      • Sridhar H.
      • Rose B.
      • Pintos J.
      • Fernandez L.
      • Idris A.
      • Sanchez M.J.
      • Nieto A.
      • Talamini R.
      • Tavani A.
      • Bosch F.X.
      • Reidel U.
      • Snijders P.J.
      • Meijer C.J.
      • Viscidi R.
      • Munoz N.
      • Franceschi S.
      • Group I.M.O.C.S.
      Human papillomavirus and oral cancer: the International Agency for Research on Cancer multicenter study.
      ,
      • Ko Y.C.
      • Huang Y.L.
      • Lee C.H.
      • Chen M.J.
      • Lin L.M.
      • Tsai C.C.
      Betel quid chewing, cigarette smoking and alcohol consumption related to oral cancer in Taiwan.
      ). Although the diagnosis and treatment are currently improved, overall survival rates of OSCC patients remain poor in Taiwan (

      Cancer registration system annual report (2017). Department of Statistics, Ministry of Health and Welfare, Executive Yuan, Republic of China (Taiwan). (http://www.mohw.gov.tw/EN/Ministry/Index.aspx),

      ). The major cause of high mortality of the disease is that more than 50‥ of OSCC patients were first diagnosed at its advanced stages, suggesting that early detection of the disease is needed to improve the treatment outcome and reduce the growing burden of OSCC (
      • Tsantoulis P.K.
      • Kastrinakis N.G.
      • Tourvas A.D.
      • Laskaris G.
      • Gorgoulis V.G.
      Advances in the biology of oral cancer.
      ).
      Conventional oral examination following with biopsy of suspected site is current approach for OSCC screening (
      • Epstein J.B.
      • Silverman Jr, S.
      • Epstein J.D.
      • Lonky S.A.
      • Bride M.A.
      Analysis of oral lesion biopsies identified and evaluated by visual examination, chemiluminescence and toluidine blue.
      ,
      • Alawi F.
      Oral cancer screening.
      ). Although the approach has been widely used for decades, its efficacy for OSCC detection remains controversial. Some patients are unable to fully open their mouths for examination. The biopsy is usually acquired from a single site, potentially omitting another cancer sites, especially in patients with multiple types of lesions. In addition, OSCC and several types of oral potentially malignant disorders (OPMDs) have similar appearances, resulting in difficulty to discriminate OPMDs from OSCC (
      • Shugars D.C.
      • Patton L.L.
      Detecting, diagnosing, and preventing oral cancer.
      ). The use of biomarkers in body fluids may represent an effective tool for the early detection of OSCC (
      • Pepe M.S.
      • Etzioni R.
      • Feng Z.
      • Potter J.D.
      • Thompson M.L.
      • Thornquist M.
      • Winget M.
      • Yasui Y.
      Phases of biomarker development for early detection of cancer.
      ). To date, however, no effective biomarker has been approved for the diagnosis and/or prognosis of OSCC.
      Saliva is an emerging specimen for disease diagnosis because it can be harvested easily and non-invasively. OSCC cells are encircled by salivary milieu, and thus it is practicable to detect salivary markers for OSCC screening (
      • Yu J.S.
      • Chen Y.T.
      • Chiang W.F.
      • Hsiao Y.C.
      • Chu L.J.
      • See L.C.
      • Wu C.S.
      • Tu H.T.
      • Chen H.W.
      • Chen C.C.
      • Liao W.C.
      • Chang Y.T.
      • Wu C.C.
      • Lin C.Y.
      • Liu S.Y.
      • Chiou S.T.
      • Chia S.L.
      • Chang K.P.
      • Chien C.Y.
      • Chang S.W.
      • Chang C.J.
      • Young J.D.
      • Pao C.C.
      • Chang Y.S.
      • Hartwell L.H.
      Saliva protein biomarkers to detect oral squamous cell carcinoma in a high-risk population in Taiwan.
      ). More than 100 salivary molecules have been reported as potential biomarkers of OSCC, including proteins, nucleotides (DNA, mRNA, and microRNA), and metabolites (
      • Shah F.D.
      • Begum R.
      • Vajaria B.N.
      • Patel K.R.
      • Patel J.B.
      • Shukla S.N.
      • Patel P.S.
      A review on salivary genomics and proteomics biomarkers in oral cancer.
      ). Previously, we profiled proteomes of saliva from the OSCC patients by means of SDS-PAGE coupled with liquid chromatography (LC)-tandem mass spectrometry (MS). With spectral counting-based label-free quantification, 22 proteins were identified as potential salivary biomarkers of OSCC. Among them, resistin was subjected to further validation using ELISA. The data confirmed that the salivary levels of resistin in the OSCC patients were significantly higher than that in the healthy group (
      • Wu C.C.
      • Chu H.W.
      • Hsu C.W.
      • Chang K.P.
      • Liu H.P.
      Saliva proteome profiling reveals potential salivary biomarkers for detection of oral cavity squamous cell carcinoma.
      ), indicating that analysis of saliva proteome is feasible for discovery of OSCC biomarkers.
      Although numerous studies have been published to search for OSCC biomarkers, few reported protein biomarkers have moved into clinical practice (
      • Yu J.S.
      • Chen Y.T.
      • Chiang W.F.
      • Hsiao Y.C.
      • Chu L.J.
      • See L.C.
      • Wu C.S.
      • Tu H.T.
      • Chen H.W.
      • Chen C.C.
      • Liao W.C.
      • Chang Y.T.
      • Wu C.C.
      • Lin C.Y.
      • Liu S.Y.
      • Chiou S.T.
      • Chia S.L.
      • Chang K.P.
      • Chien C.Y.
      • Chang S.W.
      • Chang C.J.
      • Young J.D.
      • Pao C.C.
      • Chang Y.S.
      • Hartwell L.H.
      Saliva protein biomarkers to detect oral squamous cell carcinoma in a high-risk population in Taiwan.
      ). This failure reflects an insufficient effort to select biomarker candidates from proteome profiling and validate potential biomarkers in adequate samples with suitable methods (
      • Chen C.D.
      • Wang C.L.
      • Yu C.J.
      • Chien K.Y.
      • Chen Y.T.
      • Chen M.C.
      • Chang Y.S.
      • Wu C.C.
      • Yu J.S.
      Targeted proteomics pipeline reveals potential biomarkers for the diagnosis of metastatic lung cancer in pleural effusion.
      ). In this study, we aimed to discover useful salivary biomarkers of OSCC. To this end, the saliva proteomes of the healthy volunteers, the OPMD individuals, and the OSCC patients were comprehensively analyzed with isobaric tags for relative and absolute quantitation (iTRAQ)-based MS. The iTRAQ analyses identified 1838 proteins, among which, 67 and 18 were elevated and decreased in the OSCC patients compared with that in the noncancerous groups, respectively. We then performed the multiple reaction monitoring (MRM)-MS to verify 24 candidates using a small cohort of saliva samples. Three candidate biomarkers (CFH, FGA, and SERPINA1) were further evaluated as OSCC biomarkers using an independent cohort of saliva samples with the sandwich ELISAs. Finally, we identified a marker panel that shows high sensitivity and specificity for OSCC diagnosis.

      DISCUSSION

      Early diagnosis of OSCC can save numerous lives, diminish burden of morbidity arose from treatment of the disease at advanced stage, and lighten economic load of the disease management (
      • Yu J.S.
      • Chen Y.T.
      • Chiang W.F.
      • Hsiao Y.C.
      • Chu L.J.
      • See L.C.
      • Wu C.S.
      • Tu H.T.
      • Chen H.W.
      • Chen C.C.
      • Liao W.C.
      • Chang Y.T.
      • Wu C.C.
      • Lin C.Y.
      • Liu S.Y.
      • Chiou S.T.
      • Chia S.L.
      • Chang K.P.
      • Chien C.Y.
      • Chang S.W.
      • Chang C.J.
      • Young J.D.
      • Pao C.C.
      • Chang Y.S.
      • Hartwell L.H.
      Saliva protein biomarkers to detect oral squamous cell carcinoma in a high-risk population in Taiwan.
      ). However, the current approach to OSCC diagnosis, which includes visual examination of oral cavity succeeded by inspection with biopsy, is sometimes inefficient. Patients with OSCC are sometimes incapable of fully opening their mouths for examination, what is more, the biopsy is customarily depended on a sampling of single site, which probably leads to a false-negative diagnosis, particularly in individual with multiple potentially malignant lesions. Discovery of salivary biomarkers with high efficacy for OSCC detection can greatly ameliorate early diagnosis of OSCC (
      • Wu C.C.
      • Chu H.W.
      • Hsu C.W.
      • Chang K.P.
      • Liu H.P.
      Saliva proteome profiling reveals potential salivary biomarkers for detection of oral cavity squamous cell carcinoma.
      ,
      • Hsu C.W.
      • Yu J.S.
      • Peng P.H.
      • Liu S.C.
      • Chang Y.S.
      • Chang K.P.
      • Wu C.C.
      Secretome profiling of primary cells reveals that THBS2 is a salivary biomarker of oral cavity squamous cell carcinoma.
      ). To this end, the salivary proteome of OSCC patients was quantitatively profiled in this study. The individuals with OPMD were also included here to appraise whether the chronic inflammatory disease in oral cavity could alter the salivary levels of candidate proteins.
      The salivary biomarkers are probably more practical than blood biomarkers for OSCC detection because parts of oral cancer cells are immersed in salivary milieu and saliva specimens can be acquired readily in clinical practice. In line with the speculation, we previously found that the salivary levels of resistin and thrombospondin-2 (THBS2) in OSCC patients are significantly higher than that in the healthy individuals, whereas their serum levels did not differ between the OSCC patients and healthy controls (
      • Wu C.C.
      • Chu H.W.
      • Hsu C.W.
      • Chang K.P.
      • Liu H.P.
      Saliva proteome profiling reveals potential salivary biomarkers for detection of oral cavity squamous cell carcinoma.
      ,
      • Hsu C.W.
      • Yu J.S.
      • Peng P.H.
      • Liu S.C.
      • Chang Y.S.
      • Chang K.P.
      • Wu C.C.
      Secretome profiling of primary cells reveals that THBS2 is a salivary biomarker of oral cavity squamous cell carcinoma.
      ). To improve OSCC diagnosis, we herein identified 24 proteins with the potentials as salivary biomarkers for OSCC diagnosis using the untargeted iTRAQ-based followed by the targeted MRM-based MS analyses (Fig. 1 and Table II). Moreover, the salivary levels of FGA and SERPINA1 have been demonstrated to be useful for detecting the early-stage OSCC in the case-control study (Fig. 3 and supplemental Table S11). To better OSCC diagnosis, the salivary levels of FGA, resistin, SERPINA1, and THBS2 could be simultaneously determined in combination with the oral examination. Further investigations with the same cohorts would be worthy to evaluate whether the ability of OSCC detection could be improved by utilizing a panel consisting of the four salivary proteins.
      Based on the abundances of salivary proteins obtained with the iTRAQ analyses (supplemental Fig. S2), the OSCC patients could be differentiated from the noncancerous group (healthy controls and OPMD individuals). Further, with the proteins differentially expressed in the OSCC group, the biological processes of macromolecule metabolism, blood coagulation, complement activation, acute-phase response, keratinocyte differentiation, and cell-cell adhesion can be highlighted (supplemental Table S5 and S7). The enriched processes are in agreement with the recent findings that the metabolism program (
      • Hsu C.W.
      • Chen Y.T.
      • Hsieh Y.J.
      • Chang K.P.
      • Hsueh P.C.
      • Chen T.W.
      • Yu J.S.
      • Chang Y.S.
      • Li L.
      • Wu C.C.
      Integrated analyses utilizing metabolomics and transcriptomics reveal perturbation of the polyamine pathway in oral cavity squamous cell carcinoma.
      ,
      • Wang H.J.
      • Hsieh Y.J.
      • Cheng W.C.
      • Lin C.P.
      • Lin Y.S.
      • Yang S.F.
      • Chen C.C.
      • Izumiya Y.
      • Yu J.S.
      • Kung H.J.
      • Wang W.C.
      JMJD5 regulates PKM2 nuclear translocation and reprograms HIF-1alpha-mediated glucose metabolism.
      ) and immune response (
      • Winck F.V.
      • Prado Ribeiro A.C.
      • Ramos Domingues R.
      • Ling L.Y.
      • Riano-Pachon D.M.
      • Rivera C.
      • Brandao T.B.
      • Gouvea A.F.
      • Santos-Silva A.R.
      • Coletta R.D.
      • Paes Leme A.F.
      Insights into immune responses in oral cancer through proteomic analysis of saliva and salivary extracellular vesicles.
      ,
      • Perez-Valencia J.A.
      • Prosdocimi F.
      • Cesari I.M.
      • da Costa I.R.
      • Furtado C.
      • Agostini M.
      • Rumjanek F.D.
      Angiogenesis and evading immune destruction are the main related transcriptomic characteristics to the invasive process of oral tongue cancer.
      ) are dysregulated in OSCC tissues, and the expression of proteins related with blood coagulation is altered in head and neck cancers (
      • Peng P.H.
      • Wu C.C.
      • Liu S.C.
      • Chang K.P.
      • Chen C.D.
      • Chang Y.T.
      • Hsu C.W.
      • Chang Y.S.
      • Yu J.S.
      Quantitative plasma proteome analysis reveals aberrant level of blood coagulation-related proteins in nasopharyngeal carcinoma.
      ). In line with the results of process enrichment analysis, the KEGG pathways of complement cascades, ribosome, carbon metabolism, and glycolysis were associated with the proteins with altered levels in the OSCC group (supplemental Table S6 and S8).
      In this study, the iTRAQ-based profiling was used to select the target proteins for the further MRM-based verification. With the usage of the SIS peptides, the salivary proteins could be absolutely quantified using the MRM-MS analysis. However, some of the salivary proteins could not be quantified in the SIS peptide-based MRM assay. To verify more salivary proteins as potential OSCC biomarkers, mTRAQ labeling combined with MRM assay will be applied to comprehensively and relatively quantify target proteins selected by the iTRAQ analysis (
      • Matsumoto M.
      • Matsuzaki F.
      • Oshikawa K.
      • Goshima N.
      • Mori M.
      • Kawamura Y.
      • Ogawa K.
      • Fukuda E.
      • Nakatsumi H.
      • Natsume T.
      • Fukui K.
      • Horimoto K.
      • Nagashima T.
      • Funayama R.
      • Nakayama K.
      • Nakayama K.I.
      A large-scale targeted proteomics assay resource based on an in vitro human proteome.
      ).
      Complement is generally considered as a protective mechanism against the formation of cancers. However, recent studies also indicate a pro-tumorigenic potential of complement in certain cancers (
      • Mamidi S.
      • Hone S.
      • Kirschfink M.
      The complement system in cancer: Ambivalence between tumour destruction and promotion.
      ). CFH can process factor I-mediated C3b cleavage on cell surfaces to regulate complement activation (
      • Mamidi S.
      • Hone S.
      • Kirschfink M.
      The complement system in cancer: Ambivalence between tumour destruction and promotion.
      ,
      • Herbert A.P.
      • Makou E.
      • Chen Z.A.
      • Kerr H.
      • Richards A.
      • Rappsilber J.
      • Barlow P.N.
      Complement evasion mediated by enhancement of captured factor H: Implications for protection of self-surfaces from complement.
      ). The serum level of CFH has been reported to be a diagnostic marker for lung adenocarcinoma (
      • Cui T.
      • Chen Y.
      • Knosel T.
      • Yang L.
      • Zoller K.
      • Galler K.
      • Berndt A.
      • Mihlan M.
      • Zipfel P.F.
      • Petersen I.
      Human complement factor H is a novel diagnostic marker for lung adenocarcinoma.
      ). FGA is the alpha component of fibrinogen, which is cleaved by thrombin to form fibrin when vascular injury (
      • Farrell D.H.
      • Thiagarajan P.
      • Chung D.W.
      • Davie E.W.
      Role of fibrinogen alpha and gamma chain sites in platelet aggregation.
      ). A few studies have suggested that FGA could act as a cancer biomarker. Shi et al. also showed that the plasma level of FGA could be a prognosis marker of HER2-positive breast cancer (
      • Shi Q.
      • Harris L.N.
      • Lu X.
      • Li X.
      • Hwang J.
      • Gentleman R.
      • Iglehart J.D.
      • Miron A.
      Declining plasma fibrinogen alpha fragment identifies HER2-positive breast cancer patients and reverts to normal levels after surgery.
      ). SERPINA1 possesses inhibitory activities for wide variety of proteases and then can protect cells or tissues from proteases secreted from neutrophils (
      • Gettins P.G.
      Serpin structure, mechanism, and function.
      ). SERPINA1 has been reported as a potential biomarker in colorectal (
      • Mazzoccoli G.
      • Pazienza V.
      • Panza A.
      • Valvano M.R.
      • Benegiamo G.
      • Vinciguerra M.
      • Andriulli A.
      • Piepoli A.
      ARNTL2 and SERPINE1: potential biomarkers for tumor aggressiveness in colorectal cancer.
      ) and lung (
      • Topic A.
      • Ljujic M.
      • Radojkovic D.
      Alpha-1-antitrypsin in pathogenesis of hepatocellular carcinoma.
      ) cancers. Moreover, Kawahara et al. have revealed that SERPINA1 could act as a salivary biomarker for oral cancer with a targeted proteomic strategy in a small saliva cohort (
      • Kawahara R.
      • Bollinger J.G.
      • Rivera C.
      • Ribeiro A.C.
      • Brandao T.B.
      • Paes Leme A.F.
      • MacCoss M.J.
      A targeted proteomic strategy for the measurement of oral cancer candidate biomarkers in human saliva.
      ). The results collectively suggest that CFH, FGA, and SERPINA1 could be warranted for further biomarker evaluation.
      In conclusion, to improve OSCC diagnosis, the saliva proteomes of healthy controls, OPMD individuals, and OSCC patients were quantitatively profiled with the untargeted iTRAQ-based MS. The 24 proteins with elevated salivary levels in OSCC group were selected for further evaluation with the targeted MRM-MS assay in an independent cohort. Then, the three proteins (CFH, FGA, and SERPINA1) were demonstrated to have the potentials as biomarker candidates for early detection and/or prognosis of OSCC. To the best of our knowledge, this work is one of the few investigations into the identification of OSCC biomarkers, in which iTRAQ and MRM analyses of salivary proteome are in conjugation with antibody-based assays to prioritize candidates that are worthy to be further evaluated.

      Data Availability

      The MS raw data of iTRAQ analysis were deposited on the ProteomeXchange Consortium website (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (
      • Vizcaino J.A.
      • Csordas A.
      • Del-Toro N.
      • Dianes J.A.
      • Griss J.
      • Lavidas I.
      • Mayer G.
      • Perez-Riverol Y.
      • Reisinger F.
      • Ternent T.
      • Xu Q.W.
      • Wang R.
      • Hermjakob H.
      2016 update of the PRIDE database and its related tools.
      ), data set identifier: PXD008655. The information of identified proteins and iTRAQ ratio are provided as Supporting Information. The raw files of MRM data for individual saliva samples have been deposited in a storage server (ftp.peptideatlas.org) provided by PeptideAtlas with username (Dataset Identifier): PASS01181. Official URL for this dataset is http://www.peptideatlas.org/PASS/PASS01181.

      Acknowledgments

      We thank Biosignature Research Grant CIRPD3B0013 for supporting bioinformatics and computing resources.

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