Salivary zinc finger protein 510 peptide as a novel biomarker for detection of oral squamous cell carcinoma in early stages
Introduction
Oral cancer composes 2–3% of all malignancies [1], with over 300,000 cases newly diagnosed yearly worldwide [2]. Over 90% of these cases are categorized as oral squamous cell carcinoma (OSCC) with a high degree of local invasiveness and high rate of metastasis, which leads to its high mortality [1], [2], [3]. Prevalence of oral cancer is markedly higher in Asia, compared with other industrialized nations [4], [5], [6], [7]. In Taiwan, it is the fourth most common cause of death from cancer in males, after liver, lung and colorectal cancer (Department of Health, Executive Yuan, Taiwan, 2006) [8]. Common risk factors for oral cancer include tobacco, alcohol and chewing betel quid. The risks are synergistic and might result in large areas of mucosal change or stimulate carcinogenesis in the oral cavity [6].
Oral cancer progresses from premalignant lesions to serial histological and clinical changes [9]. Clinical examination of oral cancer includes a thorough head, neck and intraoral visual examination and palpation of the oral cavity. Although the oral cavity is amenable to direct examination, oral cancer is often not detected until a late stage [10]. Biopsy of suspicious lesions remains the standard method to determine their nature. Besides discomfort associated with invasive biopsy, non-uniform appearance of (pre)cancerous lesions may hamper determination of site for biopsy, crucial in histopathological verification of oral cancer. Even with significant advances in treatment modalities (surgery, radiotherapy, chemotherapy or combination thereof), overall 5-y survival rate for oral cancer hovers around 50% [11], [12], [13]. New screening tumor markers are vital to improving identification of early malignant oral lesions, especially in at-risk populations, yet there is no effective means of accurate and feasible mass screening. Since it is heterogeneous, there apparently exist multiple cellular pathways in progress of tumorigenesis. We attempted to identify useful biomarkers for rapid and accurate diagnosis of saliva samples that also can differentiate stages of oral cancer progression.
In case of oral cancer, saliva is a good candidate for analysis to identify biomarkers [14]. Whole saliva consists of secretions from major and minor salivary glands and gingival crevicular fluid [15]. Compared with blood sampling or biopsy, use of saliva for oral cancer screening holds advantages: e.g., easier, less invasive, and better tolerated by patients. Significant increase of salivary soluble CD44 (solCD44) levels has been identified in head and neck squamous cell carcinoma (HNSCC) patients versus normal controls [16]. Comprehensive analysis of salivary parameters shows that secretory immunoglobulin A, 8-oxoguanine DNA glycosylase, phosphorylated-Src and mammary serine protease inhibitor (Maspin) are lower, while insulin growth factor I, metalloproteinases MMP-9, carbonyls and Cyclin D1 (CycD1) are higher in OSCC patients [17], [18]. Recent proteomic analysis of saliva samples from OSCC patients and matched healthy subjects, using capillary reversed-phase liquid chromatography with quadruple time-of-flight (LC-Q-TOF) mass spectrometry, indicated five candidate OSCC biomarkers (M2BP, MRP14, CD59, profilin, and catalase), successfully validated by immunoassays on an independent set of OSCC patients and matched healthy subjects [19]. Still, these candidate OSCC biomarkers have no significant correlation with tumor size, stage and recurrence.
C8-magnetic bead and mass spectrum (C8/MS) have been applied to this approach in human diseases [20]. We thus intended to identify salivary biomarkers of early stages of OSCC, using C8/MS and ClinProTools software. Potential salivary peptides as OSCC biomarkers were then subjected to validation by competitive binding inhibition and direct binding ELISA analysis of all samples from OSCC patients and control individuals. We identified significant association of linear increase in salivary ZNF510 peptide levels with tumor progression of OSCC.
Section snippets
Human subjects
The 77 persons who enrolled in this study with or without OSCC were treated at China Medical University Hospital (Taichung, Taiwan) from February 2007 to March 2011. Ages ranged from 21 to 78 y (mean ± SD 53.3 ± 11.5 y); 62 subjects (87.3%) were male. Saliva collection protocol was approved by the Institutional Review Board of China Medical University Hospital (permission number DMR96-IRB-80). Subjects provided informed consent information prior to experimental protocol. Clinical staging for OSCC
Identification of biomarkers in OSCC saliva
To investigate potential biomarkers, we collected saliva samples from OSCC patients and OSCC-free control subjects. A total of 47 OSCC patients were recruited in this study, 45 (95.7%) males and 2 (4.3%) females. The age range of OSCC patients was 29 to 79 with an average age of 50.8 y (Supplemental Table 1). Of the 30 OSCC-free control subjects, 23 (76.6%) were male and 7 (23.4%) were female. The age range of OSCC-free control subjects was 29 to 66 with an average age of 44.9 y. Although no
Discussion
This study contrasted peptide profiles of saliva from OSCC patients with an OSCC-free control group, using C8/MALDI-TOF MS analysis and ClinProTools software. Our results indicate peptide mass of 2919 Da as significantly elevated in saliva of OSCC patients (Figs. 1 and 2, Supplemental Table 2), which was sequenced and matched with the residues 176–199 of ZNF510 (Fig. 3). Subsequently, we specifically focused on 24-mer ZNF510 peptide as a potential saliva biomarker for OSCC progression.
Acknowledgments
This work was supported by the National Science Council of Taiwan (NSC99-2628-B-039-006-MY3) and China Medical University (CMU99-NSC-08 and CMU99-S-23).
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