Distinct expression of perforin and granzyme B in lip and oral cavity squamous cell carcinoma

J Oral Pathol Med (2011) 40 : 380–384 Background: Perforin and granzyme B (GB) are the main constituents of cytotoxic T‐lymphocyte granules, and they have important roles in preventing the initiation and progression of cancer. Methods: The aim of this study was to compare the expression of CD8⁺/perforin⁺ double‐staining and GB⁺ cells, by immunohistochemistry, in primary oral cavity squamous cell carcinoma (OCSCC), lip squamous cell carcinoma (LSCC), non‐dysplastic leukoplakia (LK), dysplastic LK, actinic cheilitis (AC), oral lichen planus (LP) and normal oral mucosa. Results: Our results showed a higher expression of CD8⁺/perforin⁺ and GB⁺ cells in LSCC when compared with the samples of OCSCC, non‐dysplastic and dysplastic LK, AC, oral LP and normal oral mucosa. In addition, increased CD8⁺/perforin⁺ and GB⁺ cell numbers were observed in all pre‐malignant lesions (non‐dysplastic LK, dysplastic LK, AC) when compared with the control. Conclusions: Perforin and GB proteins may contribute to antitumoural immunity, leading to the direct killing of tumour cells; however, it seems to occur more effectively in LSCC than OCSCC.     

不同癌基因在口腔白斑和鳞癌中表达的研究

目的 研究正常口腔黏膜，口腔黏膜白斑及口腔鳞癌组织中不同癌基因的表达。方法 通过HE染色确定口腔黏膜组织类型，提取组织中RNA，采用RT－PCR的方法研究不同组织中c-myc、ras、cyclinD1及bcl-2基因的表达。结果 正常口腔黏膜中未见c-myc、ras、cyclinD1及bcl-2基因表达；轻、中、重度异常增生白斑和鳞癌组织中c-myc、ras、cyclinD1及bcl-2基因表达显著增加。结论 c-myc、ras、cyclinD1及bcl-2基因的表达增加与口腔黏膜白斑及鳞癌的发生相关。     

Differential infiltration of CD8+ and NK cells in lip and oral cavity squamous cell carcinoma

J Oral Pathol Med (2010) 39 162–167 Background: CD8⁺ and natural killer (NK) cells have been considered the most effective cells in the combat of cancer, contributing to better prognosis and longer survival. Methods: The aim of this study was to evaluate the population of CD8⁺ and NK cells, by immunohistochemistry, in samples of oral cavity squamous cell carcinoma (OCSCC) and lip squamous cell carcinoma (LSCC), leukoplakia, actinic cheilitis, and healthy oral mucosa (control). The relationship of CD8⁺ and NK cells with survival data, lymph node metastasis, tumor size, and proliferative index was also evaluated. Results: The number of peritumoral and intratumoral CD8⁺ and NK cells was significantly higher in LSCC, when compared with control, pre‐malignant lesions, and OCSCC. A higher proportion of peritumoral CD8⁺ cells demonstrated correlation with a lower neoplastic proliferative index. Moreover, patients with OCSCC with a high density of peritumoral CD8⁺ cells showed a tendency towards a longer survival time. Conclusions: The differential CD8⁺ and NK cells infiltration in oral SCC might reflect a distinctive tumor microenvironment with a favorable local cytotoxic immune response against neoplastic cells.     

Morphometric computer‐assisted image analysis of oral epithelial cells in normal epithelium and leukoplakia

J Oral Pathol Med (2010) 39 149–154 Background: There are very few studies documenting morphometric parameters of normal oral mucosa and leukoplakia. The present study was undertaken to establish the morphometric parameters of the parabasal and spinous cells of normal oral epithelium. Analysis of changes occurring in these cells in leukoplakia was also done. Methods: This study was conducted on tissue sections of clinically normal oral mucosa and leukoplakia. Morphometric analysis was done for parabasal and spinous cells. Statistical analysis was done using one way ANOVA and Mann–Whitney test. Results: Morphometric parameters were greater in the spinous cells than in parabasal cells in normal oral mucosa. Leukoplakia showed greater cellular and nuclear parameters than normal mucosa. Conclusion: Normal oral epithelium showed site‐wise difference in cell and nuclear measurements. Nuclear parameters showed a statistically significant change than cellular parameters in dysplasia. These changes were expressed in the earliest stage of transformation to dysplasia.     

口腔黏膜癌前病变及口腔鳞癌中bFGF的表达及意义

目的　探讨口腔黏膜癌前病变及口腔鳞癌的发生、发展过程中bFGF的表达及意义。方法　应用免疫组织化学方法对 10例正常口腔黏膜、2 7例口腔扁平苔藓、2 4例口腔白斑及 3 0例口腔鳞癌分别进行检测。结果　口腔鳞癌组织中bFGF高表达 ,明显高于正常口腔黏膜、口腔扁平苔藓和口腔白斑组织 (P <0 .0 5 ) ;口腔扁平苔藓和口腔白斑组织中bFGF表达高于正常口腔黏膜 (P <0 .0 5 )。结论　bFGF的过表达在口腔鳞癌的发生、发展过程中起着十分重要的作用 ,可以将其作为预测口腔黏膜恶变潜能的重要标志物     

Expression of apoptotic signaling proteins in leukoplakia and oral lichen planus: quantitative and topographical studies

Although the pathogenesis of leukoplakia has been unclear, carcinogenic transformation is postulated to result from alterations of apoptotic signal transduction proteins in epithelial cells. The pathogenesis of oral lichen planus (OLP) has also been unclear, but apoptotic changes of the epithelial cells in OLP have been reported. In the present study, we used a histochemical approach to describe human keratinocyte-expression of several apoptotic signaling proteins in leukoplakia, in OLP, and in normal oral mucosa as a control. Mucosal biopsies from patients with leukoplakia (n=13), OLP (n=10), and normal oral mucosa (n=9) were frozen, sectioned and immunostained with monoclonal antibodies to wild-type (wt) tumor suppressive protein p53, cyclin-dependent kinase inhibitor p21WAF1/CIP1 and the oncoproteins MDM2, and Bcl-2. Apoptosis was assessed in all cases by the TUNEL method. MDM2 and Bcl-2 expression in keratinocytes were quantitatively greater in leukoplakia than in OLP. Wt-p53 and p21WAF1/CIP1 expression was quantitatively greater in keratinocytes in OLP than in leukoplakia. Keratinocyte maturation appeared histologically normal in OLP, even though wt-p53 and p21WAF1/CIP1 were expressed in these cells. Altered keratinocyte maturation was seen in leukoplakia lesions expressing MDM2 and Bcl-2. No significant difference for the number of apoptotic epithelial cells was observed between leukoplakia and OLP, in spite of the divergent outcomes of the apoptotic signaling proteins.     

E‐cadherin downregulation and Twist overexpression since early stages of oral carcinogenesis

There is some evidence of Twist participation in oral carcinogenesis; however, little is known about its interaction with E‐cadherin in oral squamous cell carcinoma (OSCC) development. This experimental study included an immunohistochemical analysis of Twist and E‐cadherin proteins in paraffin‐embedded specimens of oral leukoplakia (OL), OSCC, and normal oral mucosa. In addition, it was also performed a Western blot and double‐immunofluorescence analysis of Twist and E‐cadherin expression in OSCC cell lines. Significant differences in Twist and E‐cadherin immunoexpression were observed between normal oral mucosa and OL, with an inverse relation since the earliest stages of oral dysplasia (r = ?0,512; P < 0.001). Western blot and double‐immunofluorescence analysis showed differences in Twist and E‐cadherin expression among human oral keratinocytes and OSCC cell lines suggesting that downregulation of E‐cadherin occurs in a dependent manner of Twist in OSCC. Our results showed a possible value of Twist and E‐cadherin in the prediction of risk of oral epithelium malignant transformation.     

Short telomeres in an oral precancerous lesion: Q-FISH analysis of leukoplakia

J Oral Pathol Med (2012) 41 : 372–378 Objectives: A precancerous condition is a lesion that, if left untreated, leads to cancer or can be induced to become malignant. In the oral region, leukoplakia is a lesion that has been regarded as precancerous. In cases of oral carcinoma, we have frequently noticed that a type of leukoplakia histologically demonstrating hyper‐orthokeratosis and mild atypia (ortho‐keratotic dysplasia; OKD) is often associated with carcinoma, either synchronously or metachronously. Therefore, we consider OKD‐type leukoplakia to be a true precancerous lesion. Materials and Methods: In an attempt to clarify the relationship between OKD as a precancerous condition in the oral mucosa and telomere length, we estimated telomere lengths in this type of leukoplakia using quantitative fluorescence in situ hybridization, and also quantified the frequency of anaphase–telophase bridges (ATBs) in comparison with squamous cell carcinoma in situ (CIS) and the background tissues of CIS and OKD. Results: Ortho‐keratotic dysplasia was frequently associated with squamous cell carcinoma (45.0%) and showed significantly shorter telomeres than normal control epithelium, CIS, or the background of CIS or OKD. The frequency of ATBs was much higher in OKD than in control epithelium or CIS. Conclusion: Ortho‐keratotic dysplasia appears to be frequently associated with carcinoma, chromosomal instability, and excessively shortened telomeres, not only in the lesion itself but also in the surrounding background. Therefore, when this type of leukoplakia is recognized in the oral region, strict follow‐up for oral squamous cell carcinoma is necessary, focusing not only on the areas of leukoplakia, but also the surrounding background.     

ALDH1在OLP和LK组织中的表达及其意义

目的：通过检测干细胞标记物ALDH1在口腔扁平苔藓（OLP）和白斑（LK）中的表达水平,评价其与癌变的关系。方法：采用免疫组织化学SP法检测ALDH1在10例正常口腔黏膜,30例OLP,60例LK,10例口腔鳞癌（OS－CC）中表达水平;再检测ALDH1在30例癌变与30例未癌变LK中的表达差异。结果：ALDH1在正常口腔黏膜中不表达,在OLP、LK和OSCC中的表达率分别为26．7％,63．3％和90．0％（P＜0．05）;ALDH1在未癌变和癌变LK中的表达率分别是43．3％和83．3％（P＜0．01）。结论：ALDH1与口腔黏膜恶性潜能程度相关,可能是预测癌变的分子标记物。     

VEGFR—3表达与口腔癌淋巴道转移的关系

目的　了解血管内皮生长因子 (VEGF C)及血管内皮生长因子受体 3(VEGFR 3)在正常口腔黏膜、白斑及鳞癌等组织中的表达规律。方法　采用免疫组化染色及图像分析方法 ,确定VEGFR 3的表达及定量。结果　VEGFR 3主要表达定位在脉管结构 ,在正常及病变上皮组织中也有少量表达。口腔癌VEGF C表达阳性之VEGFR 3染色脉管数目比阴性增多 ,淋巴结转移比无转移增多 (P <0 .0 1) ;而鳞癌比正常黏膜、白斑增多 (P <0 .0 5 ) ;随临床分期或病理分级增高 ,VEGFR 3染色脉管数目有增多趋势 ,但无统计学差异 (P >0 .0 5 )。结论　VEGF C表达的增高 ,通过其受体VEGFR 3导致了淋巴管的增生 ,这种增生在肿瘤情况下很可能导致了淋巴道转移的增加     

Immunohistochemical study of oral keratoses including lichen planus

Biopsies of non-ulcerated oral mucosa from 13 patients with oral lichen planus and 12 patients with leukoplakia were immunohistochemically stained using monoclonal antibodies to pan T, pan B, T helper and T suppressor/cytotoxic cells and the stained lymphocytes enumerated using an image analyser. The results show the preponderance of T cells infiltrating both oral lichen planus and leukoplakia. The T helper: T suppressor/cytotoxic cell ratio was the same (1:2) for both oral lichen planus and leukoplakia. A similar proportion of T suppressor/cytotoxic cells was found infiltrating the epithelium. These data indicate that T cell subset analysis is of no value in distinguishing oral lichen planus from other oral keratoses.     

Lack of modulatory function of coding nucleotide polymorphism S100A2_185G>A in oral squamous cell carcinoma

Oral Diseases (2011) 17 , 283–290 Objective: S100A2, a Ca²⁺ ‐binding protein with two EF‐hands, is a tumor suppressor in oral cancer. Helix III flanking the C‐terminal EF‐hand is implicated to participate in the interaction of S100A2 and its target(s). The aim of this study was to examine if the coding sequence polymorphism S100A2_185G>A, leading to the peptide 62 substitution of asparagine (AAC, A allele) for serine (AGC, G allele) in helix III, had modulation effects on S100A‐mediated tumor suppression. Subjects and Methods: We sequenced the coding sequence of S100A2 gene in normal oral keratinocytes (NOKs), dysplastic oral keratinocytes (DOKs), eight oral cancer lines, and 54 pairwise oral cancer specimens. We also compared the in vitro anti‐tumor effect of wildtype (G allele) and variant (A allele) S100A2 expression using cell proliferation, migration, invasion, and colony formation assays. Results: With the exception of CAL27 and SCC‐15 cancer lines being heterozygotes of A and G alleles, the remaining oral cells were homozygotic in G alleles. No alterations of anti‐growth, anti‐migration, anti‐invasion, and anti‐colony formation were observed between variant and wildtype cells. Moreover, no minor S100A2_185A allele was detected in 54‐pairwise clinical specimens. Conclusion: The coding sequence polymorphism S100A2_185G>A had no regulatory role in S100A2‐mediated tumor suppression in oral cancer.     

CC趋化因子7和趋化因子受体7在口腔白斑及口腔鳞癌组织中的表达及意义

目的 :探讨CC趋化因子7在口腔白斑组织中的表达及意义。方法 :选取2011-03—2014-03期间,在我院口腔科治疗的口腔白斑患者43例,口腔鳞癌患者41例,以及正常口腔黏膜组织46份,利用免疫组化法对不同组织中CCL7和CCR7蛋白表达情况进行检测。结果 :CCL7蛋白在口腔白斑组织中的表达低于口腔鳞癌组织,而高于正常黏膜组织,差异有统计学意义(P<0.05);CCR7蛋白在口腔白斑组织中表达低于口腔鳞癌组织,而高于和正常黏膜组织,差异有统计学意义(P<0.05);Spearman相关分析显示,口腔白斑组织中CCL7蛋白和CCR7蛋白表达呈正相关(r=0.594,P<0.05)。结论:CCL7和CCR7蛋白表达均表现出从口腔正常黏膜组织到口腔白斑到口腔鳞癌逐渐升高的趋势,且二者呈正相关,可能参与了口腔白斑癌变过程。     

Myeloid-derived suppressor cells contribute to oral cancer progression in 4NQO-treated mice

Oral Diseases (2011) 18 , 67–73 Objective: Abnormal myelopoiesis especially the expansion of myeloid‐derived suppressor cells (MDSCs) is increasingly recognized as an important reason for the escape of tumor from immune surveillance. This study aims to investigate the role of this specific population of cells in oral cancer progression. Materials and Methods: 4‐Nitroquinoline 1‐oxide (4NQO) was used to induce oral cancer in C57BL/6 mice. The tongue mucosa was examined by hematoxylin and eosin staining. The distribution of MDSCs in the spleen and peripheral blood and T cell subsets in the spleen was analyzed by flow cytometry. The expression of MDSCs in the tongue tissues was investigated by immunohistochemical staining, and the expression of arginase‐1 (ARG‐1) and NOS‐2 in the tongue tissues was detected by real‐time PCR. Results: We found that during tumor progression, significantly increased frequency of MDSCs was observed in the spleens and peripheral blood of 4NQO‐treated mice, and the frequency of MDSCs in the spleens was positively correlated with systemic CD3⁺CD8⁺ T cells. Moreover, 4NQO‐treated mice showed significantly higher MDSCs infiltration and ARG‐1 mRNA level in the tumor site. Conclusions: Myeloid‐derived suppressor cells contribute to oral tumor progression and represent a potential target for immunotherapy of oral cancer.     

口腔鳞癌组织中Ki-67和p53蛋白的表达

目的 探讨口腔鳞癌组织中Ki-67和p53蛋白的表达及其与临床病理特征的关系。方法采用免疫组织化学S-P法对10例正常口腔黏膜组织、16例口腔白斑（OLK）组织、48例口腔鳞癌（OSCC）组织中的Ki-67和p53蛋白表达进行检测,结合患者临床病理资料进行分析,使用SPSS17.0 软件包对数据进行统计学处理。结果Ki-67蛋白在正常口腔黏膜组织、口腔白斑和口腔鳞癌组织中的阳性表达率分别为30.0%、56.3%和79.2%;p53的阳性表达率分别为0.0%、43.8%和70.8%,Ki-67和p53在正常黏膜组与口腔白斑和口腔鳞癌组差异均具有显著性（P<0.05）;Ki67蛋白在口腔鳞癌组织中的表达与肿瘤的临床分期、分化程度、有无淋巴结转移有关（P<0.05）,p53蛋白的表达与肿瘤的分化程度有关(P<0.05);Ki-67和p53蛋白在口腔鳞癌组织中的表达呈正相关（P<0.05）。结论Ki-67和p53蛋白在口腔鳞癌组织中高表达,可能在口腔鳞癌的发生、发展过程中起着重要作用。     

Immunohistochemical analysis of P53 and bcl-2 in benign and malignant salivary glands tumors

J Oral Pathol Med (2010) 39 : 48–55 Background: Salivary glands tumors are relatively uncommon neoplasm with widely variable histopathologic and biologic characteristics. Alteration in some proto‐oncogenes and tumor suppressor genes may lead to the development and progression of these tumors. Aims: The purpose of this study was to analyze the immunohistochemical expression of P₅₃ and bcl‐2 in some salivary gland tumors in relation to tumor size, histologic grade, and extent of invasion. Setting and design: The sample consisted of 22 formalin‐fixed paraffin embedded blocks of benign and malignant salivary glands tumors. Material and method: P₅₃ and bcl‐2 immunoreactivity was semi‐quantitatively evaluated in at least 1000 cells examined under the microscope at 40× magnification and recorded as percentage of P₅₃ or bcl‐2 positive tumor cells over the total number of cells examined in the same area. Percentage scores were subsequently categorized using the 5% cut‐off point for positive staining. Results and conclusion: Pleomorphic adenoma (PA) showed negative expression of P₅₃ and bcl‐2 in 70% of cases, whereas all malignant salivary glands tumors were positive for P₅₃ and bcl‐2. P₅₃ positive/bcl‐2 positive immunostaining reaction was week in small size PA and was totally absent in larger lesions. However, all malignant tumors expressed P₅₃, with the highest record in low‐grade adenocarcinoma (76%) and the lowest score was observed in both low‐grade carcinoma in PA and adenocystic carcinoma. bcl‐2 immunostaining was also assessed, the highest recorded score was in high‐grade adeno cystic carcinoma (90%) and the lowest in both low‐grade carcinoma ex‐PA and low‐grade cystic mucoepidermoid carcinoma (3%). P₅₃/bcl‐2 immunostaining reactivity could be helpful in demonstrating salivary glands tumor behavior in terms of progression and extent of invasion.     

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目的研究鸟氨酸脱羧酶（ODC）与口腔白斑的发生、发展乃至癌变潜能的关系。方法选取2009—2010年大连医科大学附属第一医院口腔科收治且经病理确诊的口腔白斑患者22例,以其病变黏膜组织为研究对象,同时以11例正常口腔黏膜、22例口腔扁平苔藓组织和22例口腔鳞癌组织为对照。采用免疫组化法检测ODC在各种组织中的表达,并对阳性率进行比较分析。结果在口腔正常黏膜、扁平苔藓、白斑和鳞癌组织中ODC的阳性率依次增加;ODC在口腔扁平苔藓中的阳性率高于正常黏膜,但差异无统计学意义（P〉0.05）,ODC在口腔白斑中的阳性率与正常黏膜和扁平苔藓的差异均有统计学意义（P〈0.05）,口腔鳞癌组织中ODC的阳性率显著高于其余组（P〈0.05）。结论 ODC表达程度可用于判断口腔白斑的恶变倾向及口腔鳞癌的恶性程度。     

口腔粘膜白斑组织中上皮细胞凋亡的实验研究

目的　观察和比较口腔粘膜白斑(LK)病变和正常对照的局部活检粘膜组织中上皮细胞凋亡和增殖的情况。方法　应用原位末端转移酶标记技术和ABC免疫组化技术,通过组织学定位、相对染色强度和阳性细胞计数来评估上皮细胞凋亡和增殖细胞核抗原的变化程度及主要部位。结果　与对照相比,LK病变中凋亡阳性信号在棘细胞层较强,而增殖细胞核抗原则在基底细胞层及其周围呈现强阳性反应,病变组织固有层中浸润的部分炎细胞和血管内皮细胞也表达出较强的阳性凋亡信号。病变组和对照组阳性细胞计数量有显著性差异(P<0·05)。结论　白斑病变时上皮层棘细胞凋亡增强,而基底细胞的增殖能力活跃,该处局部组织的免疫防御能力可能有所下降。     

N-乙酰基转移酶10蛋白和朊蛋白在口腔鳞状细胞癌组织中的表达及相关性研究

目的:检测N-乙酰基转移酶10蛋白(Naa10p)和朊蛋白(PrPc)在口腔鳞状细胞癌(OSCC) 、白斑、口腔正常黏膜中的表达及临床意义。方法:应用免疫组化EnvisionTM法检测OSCC(112例)、白斑(42例)及正常黏膜组织(11例)中Naa10p和PrPc的表达情况,并分析其与临床病理特征相关性。结果:Naa10p和PrPc在OSCC表达最高,白斑次之,正常黏膜组织中表达最低。Naa10p在3种不同组织中的表达两两比较均有显著统计学差异(P<0.05),PrPc分别在白斑和OSCC组织,正常口腔黏膜与OSCC组织中的表达有统计学差异(P<0.05)。Naa10p的表达水平与OSCC的TNM分期、淋巴结转移、组织学分化程度密切相关(P<0.05),与性别、年龄无关。PrPc表达仅与组织学分化程度密切相关(P<0.05)。PrPc在OSCC中的表达强度随组织学分化程度下降而明显升高(P<0.05),而Naa10p的表达强度随组织学分化程度下降而明显下降(P<0.05)。结论:Naa10p和PrPc与OSCC的发生和转移相关。     

Orofacial diseases in solid organ and hematopoietic stem cell transplant recipients

Oral Diseases (2012) 19 , 18–36 Objective: Solid organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) recipients are at risk of several diseases, principally attributable to immunosuppression. This global overview of SOT/HSCT‐associated orofacial diseases is aimed at providing a practical instrument for the oral healthcare management of SOT/HSCT recipients. Methods: Literature search was made through MEDLINE. The associations between orofacial diseases and SOT/HSCT were assessed using observational studies and case series and were classified into ‘association’, ‘no association’, and ‘unclear association’. Results: Lip/oral cancers, drug‐induced gingival overgrowth (DIGO), infections, including hairy leukoplakia and, less frequently, post‐transplantation lymphoproliferative disorders (PTLDs) and oral lichenoid lesions of graft‐versus‐host disease (GVHD), were associated with SOT. Lip/oral cancers, GVHD, mucositis, DIGO, infections and, less frequently, PTLDs were associated with HSCT. Associations of orofacial granulomatosis‐like lesions and oral mucosa‐associated lymphoid tissue‐type lymphoma with SOT, and of pyogenic granuloma and hairy leukoplakia with HSCT were unclear. Periodontal disease and dental caries were not associated with SOT/HSCT. For none of the local treatments was there a strong evidence of effectiveness. Conclusions: Solid organ transplant/HSCT recipients are at risk of orofacial diseases. Adequate management of these patients alleviates local symptoms responsible for impaired eating, helps prevent systemic and lethal complications, and helps where dental healthcare has been neglected.