Identification of a truncated cystatin SA-I as a saliva biomarker for oral squamous cell carcinoma using the SELDI ProteinChip platform

New and more consistent biomarkers of oral squamous cell carcinoma (OSCC) are needed to improve early detection of the disease and to monitor patient management. The aim of this study was to detect new OSCC tumor markers in saliva. Unstimulated saliva, collected from patients with primary stage I OSCC as matched pre-and post-treatment samples, was used in the analysis. A surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF) ProteinChip system was used to screen for differentially expressed proteins in the saliva samples. This analysis revealed 26 proteins with significantly different expression levels in the pre-and post-treatment samples (P < 0.05). A 14 kDa protein detected in pre-treatment saliva from the OSCC patients was identified as a truncated cystatin SA-I, with deletion of three amino acids from the N-terminus. The authors propose that ProteinChip analysis may provide a reliable screening test for early diagnosis of OSCC and that truncated cystatin SA-I might be a useful tumor biomarker for OSCC.     

Recent trends of saliva omics biomarkers for the diagnosis and treatment of oral cancer

BackgroundRecent advances in surgery, radiotherapy, and chemotherapy have no significant effect on oral cancer survival rates due to late diagnosis, poor tumor response to chemotherapy and radiotherapy, as well as a lack of effective biomarkers for early diagnosis.HighlightsTherefore, an investigative study aimed at identifying genomics, proteomics, metagenomics, and, metabolomics derived biomarkers for early diagnosis may improve the survival rate of oral cancer patients. Identification and application of saliva-based ‘‘omics’’ biomarkers may overcome painful invasive procedures currently being used for the diagnosis of oral cancer. One single biomarker may not be able to differentiate between oral squamous cell carcinoma (OSCC) and controls. Thus, multiple sensitive and specific biomarkers may be needed for screening high-risk patients and following them up for early signs of OSCC occurrence. Validation of these biomarkers in large patient cohorts is, however, required before they can be used in clinical practice.ConclusionIn this review, we summarize the potential of omics derived salivary biomarkers as diagnostic and prognostic tools in oral cancer detection and the future clinical benefits associated with these markers.     

口腔鳞癌相关血清多肽的筛选和鉴定

目的:探讨口腔鳞癌不同发展阶段的差异血清多肽谱。方法:采用基质辅助激光解析飞行时间质谱结合弱阳离子纳米磁珠,检测120例健康正常人、37例早期口腔鳞癌患者、129例晚期口腔鳞癌患者血清多肽指纹图谱,应用ClinProtToolsTM 2.1软件对检测结果进行分析,建立差异多肽的鉴别诊断模型,对模型中的差异多肽进行质谱分析,以IPI数据库检索确定相应蛋白质名称。结果:在3组间的两两比较中,共筛选出385个差异多肽,初步建立口腔鳞癌与健康正常人及口腔鳞癌早、晚期的鉴别诊断模型。早期口腔鳞癌与健康正常人之间的鉴别诊断模型包括5个差异多肽峰,分别是FGA、ITIH4、PKM2、SCARA5和SDPR,诊断敏感度和特异度为82.4%和98.5%;晚期口腔鳞癌与早期口腔鳞癌之间的鉴别诊断模型包括4个差异多肽峰,诊断敏感度和特异度为98.5%和88.2%,分别是FGA、PKM2、ITIH4和ALB;晚期口腔鳞癌与健康正常人之间的鉴别诊断模型包括5个差异多肽峰,其中3个为SERPINA1、TTR和SDPR,诊断敏感度和特异度为89.4%和85.1%。结论:采用多肽组学方法,鉴定了部分口腔鳞癌不同发展阶段的差异多肽,为进一步开展血清多肽验证和差异蛋白质的机制研究奠定了基础。     

口腔鳞癌组织与口腔正常黏膜组织蛋白质差异表达的初步研究

目的:筛选口腔鳞癌及正常口腔黏膜组织的差异表达蛋白质,为研究口腔鳞癌发生机制提供实验依据.方法: 收集10 例口腔鳞癌组织及正常口腔黏膜组织,进行二维电泳,选择在表达差异量较大的29 个点进行质谱和生物信息学分析,确定所分析的蛋白质类型. 结果: 口腔鳞癌及相应正常口腔黏膜组织凝胶的平均蛋白质点数分别为2 325±390和2 487±281.双向凝胶电泳图显示,口腔鳞癌及正常口腔黏膜组织的差异表达蛋白质点数为29 个,这29 个点在癌组织中均为低表达,对其进行了质谱(PMF)和生物信息学分析,鉴定了其中的3 个点,它们是:β纤维蛋白(fibrin beta)、磷酸丙糖异构酶(triosephosphate isomerase TIM)、unknown蛋白.结论: β纤维蛋白、磷酸丙糖异构酶、unknown蛋白在口腔鳞癌发生发展过程中发生了改变,其机制尚待进一步阐明.     

比较蛋白组学在口腔鳞癌患者及健康人血清的初步研究

目的:筛选口腔鳞癌及健康人血清差异表达的蛋白质。方法:收集口腔鳞癌患者外周血清17例,健康人外周血清17例,通过固相pH梯度双向凝胶电泳以及质谱鉴定和生物信息学分析,寻找在口腔鳞癌患者血清中特异表达的蛋白质。结果:先后对口腔鳞癌和健康人血清表达差异明显的11个蛋白质点进行质谱鉴定和生物信息学分析,鉴定了其中的2个点为触珠蛋白(haptoglobin,Hp),在口腔鳞癌血清中均为高表达;1个点为载脂蛋白A(apolipoprotein A)在口腔鳞癌血清中为低表达。结论:触珠蛋白和载脂蛋白A在口腔鳞癌外周血清和健康人外周血清的表达发生了改变,但其确切的调控机制有待进一步研究。     

表面增强激光解吸-电离质谱法筛选唾液中口腔鳞状细胞癌蛋白标志物

目的 在唾液中筛选口腔癌前病变、鳞状细胞癌、转移癌并与健康人鉴别的肿瘤蛋白标志物.方法 在CM-10蛋白质芯片上采用表面增强激光解吸-电离(Surface enhanced laser desorption/ionization(SELDI)质谱法技术对口腔白斑(6例)、鳞状细胞癌(17例)、转移癌(7例)和健康者(15人)的非刺激性全唾液中的蛋白标志物进行检测,支持向量机法建立指纹图谱诊断模式.结果 口腔鳞状细胞癌和健康人唾液蛋白鉴别模式:蛋白质荷比峰(简称:质荷比)5797、2902、3883和4951组合,敏感性为88.24%、特异性为93.33%;口腔鳞状细胞癌和白斑鉴别模式:质荷比为5818、4617和3884的组合,敏感性为100.00%、特异性为100.00%;口腔鳞状细胞癌和局部转移癌的鉴别模式:质荷比为55 809和5383的组合,敏感性为94.12%、特异性为85.71%.结论 通过SELDI质谱法技术筛选出的肿瘤标志物可以辅助口腔鳞状细胞癌的早期诊断,预测白斑向鳞状细胞癌转化及癌局部转移的潜能.     

循环肿瘤细胞与口腔鳞状细胞癌相关性的研究进展

循环肿瘤细胞(CTCs)是从原发灶部位脱落、释放并转移到血液循环的肿瘤细胞,目前广泛应用于肺癌、乳腺癌等恶性肿瘤的早期诊断、治疗、疗效评价和预后判断,但是在口腔鳞状细胞癌(OSCC)中运用较少且缺乏相关的临床证据。有研究表明,CTCs是OSCC相对独立的预后指标,而且是复发或转移的重要原因之一。本文为深入阐述OSCC中CTCs的形成机制及其检测技术,探索OSCC相关CTCs生物标志物的临床意义,了解CTCs与OSCC进展及预后的关系,现就CTCs与OSCC的相关性研究作一综述。     

The role of cyclin‐dependent kinases in oral potentially malignant disorders and oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a major global health problem with a relatively low‐moderate 5‐year survival rate. OSCC is often preceded by lesions and conditions known as oral potentially malignant disorders (OPMDs) that have an increased risk of malignant transformation. Despite advances in diagnostic technology and cancer research, the prognosis of OSCC remains poor as it is frequently detected a late stage. Understanding the molecular pathways involved in oral carcinogenesis provides a platform to identify biomarkers that may allow the early detection of OSCC and accurate prediction of the malignant potential of OPMDs. In addition, specific molecular inhibitors can be developed to target these important pathways and allow advanced therapeutic management to improve the prognosis of this malignancy. A common feature across a number of different cancers is the dysfunction of cell cycle moderator proteins known as cyclin‐dependent kinases. This review summarises the current literature regarding the role of cyclin‐dependent kinases in oral carcinogenesis with a particular focus on cyclin‐dependent kinases 4 (CDK4) and 6 (CDK6). This is of particular relevance as CDK4 and CDK6 inhibitors have shown some promising results in other cancer types and are interesting potential treatments for OSCC.     

CIITA rs4774 and rs6498122 polymorphisms are associated with oral lichen planus in Chinese people: a case–control study

Oral lichen planus (OLP) is a common autoimmune inflammatory disorder that is difficult to cure, and its pathogenesis is still largely unknown. The major histocompatibility complex (MHC) class II transactivator (CIITA) gene has been reported to be an important candidate in some classical autoimmune diseases, and certain single nucleotide polymorphisms (SNPs) in CIITA have been confirmed to be associated with susceptibility to some autoimmune diseases. We conducted this research to investigate the existence of any correlation between OLP and SNPs in CIITA. A case–control study was performed to genotype 15 SNPs in the CIITA gene from 42 patients with OLP and from 86 controls; this was carried out by the PCR and then by a locus‐specific single‐base extension reaction. Allele detection was performed using matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) mass spectrometry. The SNP rs4774 variant in exon 11 (+1614G/C, Gly500Ala) of CIITA is significantly associated with OLP in healthy persons, both in genotype frequency and in allele frequency. Another intronic SNP, rs6498122, showed significant differences only in allele frequency. In conclusion, our data show that the two SNPs rs4774 and rs6498122 are associated with OLP and could also indicate the autoimmune characteristics of OLP.     

Survey of risk factors contributed to lymphatic metastasis in patients with oral tongue cancer by immunohistochemistry

J Oral Pathol Med (2011) 40 : 127–134 Objectives: The aim of this study was to define the biomarkers of lymphatic spread which facilitate the appropriate therapy for oral tongue squamous cell carcinoma (OTSCC) patients at early stage. Here, we investigated the expression levels of seven biomarkers in oral squamous cell carcinoma (OSCC) tissues as well as their associations with the clinicopathologic features of OTSCC patients. Methods: The OTSCC samples were obtained from 138 patients undergoing tumor resection. Immunohistochemical staining was performed by using ColIA, ColIVA, Fn1, MMP‐1, MMP‐2, uPA, and D2‐40 antibodies. Expression level of theses biomarkers in normal and tumor tissues were compared. Risk factors of lymphatic dissemination were evaluated by logistic regression equation. Results: LVD, MMP‐1, MMP‐2, and uPA in cancer tissues were significantly higher than those in normal tissue, and ColIA, ColIVA, and Fn1 in cancer tissue were significantly lower than those in normal tissue. Similar results were obtained from the comparison between metastatic tumor and non‐metastatic tumor. All biomarker expressions were closely related with lymph node status and clinical stage. Additionally, the regression equation demonstrated that LVD is the risk factor of lymphatic metastasis in OTSCC patients (OR = 1.732; 95% CIs: 1.167–2.057; P < 0.05). Conclusions: Down‐regulation of ColIA, ColIVA, and Fn1 and up‐regulation of LVD, MMP‐1, MMP‐2, and uPA might be important features of OSCC progression, which may exert their functions and favorably predict lymphatic dissemination for OSCC patients at relatively early stage. Among these biomarkers, increased LVD is an independent risk factor of lymphatic metastasis, which could better predict whether metastasis will occur or not.     

Endothelial cell Bcl-2 and lymph node metastasis in patients with oral squamous cell carcinoma

J Oral Pathol Med (2012) 41 : 124–130 Background: Loco‐regional spread of disease causes high morbidity and is associated with the poor prognosis of malignant oral tumors. Better understanding of mechanisms underlying the establishment of lymph node metastasis is necessary for the development of more effective therapies for patients with oral cancer. The aims of this work were to evaluate a possible correlation between endothelial cell Bcl‐2 and lymph node metastasis in patients with oral squamous cell carcinoma (OSCC), and to study signaling pathways that regulate Bcl‐2 expression in lymphatic endothelial cells. Methods: Endothelial cells were selectively retrieved from paraffin‐embedded tissue sections of primary human OSCC from patients with or without lymph node metastasis by laser capture microdissection. RT‐PCR was used to evaluate Bcl‐2 expression in tumor‐associated endothelial cells and in tumor cells. In vitro, mechanistic studies were performed to examine the effect of vascular endothelial growth factor (VEGF)‐C on the expression of Bcl‐2 in primary human lymphatic endothelial cells. Results:  We observed that Bcl‐2 expression is upregulated in the endothelial cells of human oral tumors with lymph node metastasis as compared to endothelial cells from stage‐matched tumors without metastasis. VEGF‐C induced Bcl‐2 expression in lymphatic endothelial cells via VEGFR‐3 and PI3k/Akt signaling. Notably, OSCC cells express VEGF‐C and induce Bcl‐2 in lymphatic endothelial cells. Conclusions: Collectively, this work unveiled a mechanism for the induction of Bcl‐2 in lymphatic endothelial cells and suggested that endothelial cell Bcl‐2 contributes to lymph node metastasis in patients with oral squamous cell carcinoma.     

转录抑制因子Snail在口腔鳞状细胞癌中的表达及其与临床病理特征及预后多因素的关系

目的 研究转录抑制因子Snail在口腔鳞状细胞癌(以下简称口腔鳞癌)中的表达及其与多种临床病理因素之间的关系,探讨其在口腔鳞癌治疗及预后评估中的意义。方法 采用免疫组织化学染色半定量的方法检测83例术前未接受放、化疗或其他特殊治疗的原发性口腔鳞癌患者标本中Snail的表达情况,并对其与口腔鳞癌患者的临床病理因素、转移及预后之间的关系进行统计学分析。结果 83例口腔鳞癌组织标本中,Snail的表达率为89.2%,其中,高表达率为49.4%,低表达率为39.8%。口腔鳞癌组织中Snail的表达水平与患者的性别、年龄、肿瘤分化程度、肿瘤大小、临床分期及淋巴结转移之间在统计学上无显著性差异（P>0.05）。Snail表达水平与口腔鳞癌患者的复发之间有显著性差异（P＝0.003）。结论 口腔鳞癌患者癌组织中Snail的高表达与肿瘤复发及预后之间有一定相关关系。因此,Snail有可能作为口腔鳞癌患者复发和预后的预测指标。     

Twist、Snail、Slug在口腔鳞状细胞癌中的表达及其与各临床病理因素的关系

目的:探讨Twist、Snail、Slug在口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)中的表达水平及其与各临床病理因素之间的关系.方法:采用免疫组织化学方法检测术前未接受放化疗的有颈部淋巴结转移或无颈部淋巴结转移的60例原发OSCC患者肿瘤切除石蜡标本中Twist、Snail、...     

FAS and ErbB2 expression in early local recurrent oral cancer

J Oral Pathol Med (2010) 39 176–181 Background: Local failure occurs in 13.9–62.6% and it is a well known indicator of poor prognosis in patients with oral squamous cell carcinoma (OSCC), despite aggressive treatments. The purpose of this study was to investigate the value of histopathology and molecular biomarkers in predicting the development of early local recurrence. Methods: This study included a total of 69 patients. There were 23 patients with early recurrent OSCC and 46 patients without local recurrence with the same clinical stage and tumor site, in a pair‐matched study design. Their charts were retrospectively analyzed. All surgical specimens of the primary tumors were evaluated according to the system proposed by Anneroth et al. and immunohistochemical for ErbB2 and FAS were performed. Results: A significant correlation of early local recurrence with grade of histological malignancy (more than 15 points) was observed (Fisher’s exact test, P = 0.03). Early local recurrence was also significantly associated with weak FAS expression and strong intracytoplasmic ErbB2 staining (Mantel–Haenszal chi‐square, P = 0.0038 and P = 0.0068, respectively). Histological grade of malignancy (more than 15 points) was also correlated with reduced survival (log‐rank, P = 0.06). Among the histopathological parameters, keratinization, pattern of invasion and inflammation were important for overall survival (log‐rank, P < 0.0001). Regarding the biomarkers, only FAS was significantly associated with overall survival (log‐rank, P = 0.0002). Moreover, a positive correlation of FAS and membrane ErbB2 expression with keratinization was noticed. Conclusion: Histopathological characteristics and the expression of FAS and ErbB2 carry prognosis importance in local recurrence and overall survival in OSCC.     

MMP-14在口腔癌患者预后及转移的临床病理学研究

目的 观察基质金属蛋白酶14（MMP-14）、波形蛋白\E-钙粘蛋白在口腔癌及癌旁组织中的表达,探讨MMP-14在口腔癌患者预后及转移的作用机制。方法 选取83例有随访资料的口腔癌患者标本和20例癌旁组织标本,应用免疫组织化学法检测MMP-14及EMT标记物Vimentin,E-cadherin的表达水平,结合随访患者临床参数,进行统计学分析。结果 MMP-14表达在口腔癌中较正常组织升高,有特别显著性差异(P＜0.01);在有淋巴结转移、中低分化及T3-T4期患者中高表达,有特别显著性差异（P<0.01）;且与Vimentin上调,E-cadherin下调具有显著相关性（P<0.001）。结论 MMP-14的高表达,提示口腔癌患者生存率低预后较差,易发生淋巴结转移。其机制可能通过对EMT的调控,从而促进口腔癌细胞的侵袭转移。     

Discovery of putative salivary biomarkers for Sjögren's syndrome using high resolution mass spectrometry and bioinformatics

The purpose of the current study was to determine if saliva contains biomarkers that can be used as diagnostic tools for Sj?gren's syndrome (SjS). Twenty seven SjS patients and 27 age-matched healthy controls were recruited for these studies. Unstimulated glandular saliva was collected from the Wharton's duct using a suction device. Two μl of salvia were processed for mass spectrometry analyses on a prOTOF 2000 matrix-assisted laser desorption/ionization orthogonal time of flight (MALDI O-TOF) mass spectrometer. Raw data were analyzed using bioinformatic tools to identify biomarkers. MALDI O-TOF MS analyses of saliva samples were highly reproducible and the mass spectra generated were very rich in peptides and peptide fragments in the 750-7,500 Da range. Data analysis using bioinformatic tools resulted in several classification models being built and several biomarkers identified. One model based on 7 putative biomarkers yielded a sensitivity of 97.5%, specificity of 97.8% and an accuracy of 97.6%. One biomarker was present only in SjS samples and was identified as a proteolytic peptide originating from human basic salivary proline-rich protein 3 precursor. We conclude that salivary biomarkers detected by high-resolution mass spectrometry coupled with powerful bioinformatic tools offer the potential to serve as diagnostic/prognostic tools for SjS.     

XIAP与口腔鳞癌局部淋巴结转移关系的研究

目的 探究XIAP的表达水平与口腔鳞癌局部淋巴结转移之间的关系。初步评价其表达水平是否可以作为预测口腔癌局部淋巴结转移新的靶基因。方法 收集40例原发口腔鳞癌标本及11例术后复发标本,运用免疫组化技术检测组织标本中XIAP蛋白的表达水平,评价其表达水平与淋巴结转移等临床参数之间的相关性。结果 40例原发口腔鳞癌标本中XIAP在细胞胞浆中均有不同程度的表达,其表达水平与淋巴结转移、病理分级及复发显著相关。结论 实验结果表明,细胞胞浆中XIAP的表达水平与口腔鳞状细胞癌局部淋巴结转移及术后复发密切相关,可能是口腔鳞癌淋巴转移新的促进基因。     

Evaluation of saliva and plasma cytokine biomarkers in patients with oral squamous cell carcinoma

The aim of this study was to investigate potential biomarkers in human saliva and plasma to aid in the early diagnosis of oral squamous cell carcinoma (OSCC). Saliva and plasma samples obtained from OSCC patients (n = 41) and non-oral cancer patients (n = 24) were analyzed by Luminex Bead-based Multiplex Assay. Data were analyzed using the non-parametric Mann–Whitney U-test, Kruskal–Wallis test, and receiver operating characteristics curve (ROC) to evaluate the predictive power of 14 biomarkers individually for OSCC diagnosis. The plasma level of IP-10 in early OSCC differed significantly from that in controls. Among the salivary biomarkers, IL-1β, IL-6, IL-8, MIP-1β, eotaxin and IFN-γ and TNF-α showed significant differences between OSCC patients and controls. With respect to carcinogenesis, significant differences in plasma levels of eotaxin, G-CSF, and IL-6 were found between OSCC stages III/IV and OSCC stages I/II. The area under the curve (AUC) for OSCC vs. control was greater than 0.7 for plasma IP-10 and saliva IL-1β, IL-6, IL-8, and TNF-α. The study findings indicate that salivary biomarkers may serve a useful role as a complementary adjunct for the early detection of oral OSCC. With regard to the evaluation of tumour progression, plasma eotaxin, G-CSF, and IL-6 may help in the detection of advanced OSCC. However, the correlation between saliva and plasma biomarkers in OSCC was weak.     

ANGPTL4 regulates the metastatic potential of oral squamous cell carcinoma

Lymph node metastasis is a major factor for poor prognosis in oral squamous cell carcinoma (OSCC). However, the molecular mechanisms of lymph node metastasis are unclear. We determined that angiopoietin‐like protein 4 (ANGPTL4) mRNA and protein expression were increased in OSCC cells established from the primary site in metastatic cases. In addition, ANGPTL4 expression in biopsy specimens was correlated with the presence of lymph node metastasis. Therefore, our initial findings suggest that OSCC cells expressing ANGPTL4 may possess metastatic ability. Furthermore, cell culture supernatants from OSCC cells that metastasized to the lymph node contain ANGPTL4 and promote invasive ability. These findings suggest that secreted ANGPTL4 may affect the invasive ability of OSCC. Moreover, the rates of positive ANGPTL4 expression at the primary site were significantly higher in the lymph node metastasis group. These results demonstrate that ANGPTL4 contributes to OSCC metastasis by stimulating cell invasion. Therefore, ANGPTL4 is a potential therapeutic target for preventing cancer metastasis.     

Lack of evidence for prognostic value of epidermal growth factor receptor intron‐1 CA repeats for oral carcinomas

Epidermal growth factor receptor (EGFR) expression is altered in several malignancies, including oral squamous cell carcinoma. A CA‐repeat polymorphism in intron‐1 (CA‐SSR‐1) of the EGFR gene is reported to influence EGFR expression and is associated with features of various solid tumors and outcomes of cancer patients. In the present study we evaluated the influence of length and zygosity of CA‐SSR‐1 on the survival of patients with oral squamous cell carcinoma. The length and zygosity of CA‐SSR‐1 was obtained through microsatellite analysis in 91 patients with oral cancer, who were treated in the Department of Oral and Maxillofacial Surgery of the University Medical Centre Hamburg Eppendorf, Germany, during the years 1998–2008. Follow up was conducted until 2016. Outcome measures were age, gender, tumor stage, occurrence of metastases, and date of recurrence or death. Statistical analysis was conducted using the chi‐square test and the log‐rank test. Neither length nor zygosity of the CA‐SSR‐1 in patients with oral squamous cell carcinoma was significantly correlated with sex, age, tumor size, tumor localization, lymph node involvement, metastasis status, disease‐free survival, or overall survival. Length and zygosity of the CA‐SSR‐1 polymorphism in EGFR is not able to serve as a prognostic biomarker in White European patients with oral squamous cell carcinoma.