DNA content in malignant salivary gland tumours

Objective:  Our aim was to evaluate the DNA content in malignant salivary gland tumours using image cytometry and its possible relationships with clinical and morphologic findings, disease course and prognosis.
Patients and methods:  The study sample comprised 31 patients diagnosed and treated for primary malignant salivary gland tumours. Formalin-fixed, paraffin-embedded surgical specimens of all patients were Feulgen-stained for DNA content analysis by image cytometry. Statistical analysis was used to investigate possible relationships between DNA content variables and clinical and histological findings, disease course and patient survival.
Results:  Seventeen (55%) cases of our sample were graded as DNA diploid, four (13%) as DNA aneuploid and 10 (32%) as DNA multiploid. In 15 (48%) cases, the 5c exceeding rate (5cER) was higher than 1.7%. DNA ploidy correlated with N stage and tumour size. DNA ploidy and 5cER had a statistically significant prognostic influence on overall and disease-free survival in univariate analysis. However, in multivariate analysis, stage classification was the only parameter with an independent prognosis value.
Conclusion:  Abnormal DNA content is a common finding in salivary gland cancers. Our results suggest an important role of DNA content analysis in the evaluation of these tumours.     

Nichtinvasive Bürstenbiopsie als innovative Methode in der Früherkennung des Mundhöhlenkarzinoms

Purpose

The aim of this prospective study was to investigate the diagnostic accuracy of DNA image cytometry in combination with non-invasive brush biopsies taken from suspicious oral lesions.

Material and methods

Cytological diagnoses obtained from 1328 exfoliative smears of 332 different lesions were compared with histology and/or clinical follow-ups of the respective patients. Additionally, nuclear DNA contents were measured after Feulgen restaining using a TV image analysis system. DNA aneuploidy was assumed if abnormal DNA stemlines or cells with DNA content greater than 9c were observed.

Results

The sensitivity of our cytological diagnosis in addition to DNA image cytometry on oral smears for the detection of cancer cells was 97.8%, specificity 100%, positive predictive value 100%, and negative predictive value 98.1%.

Conclusion

The application of DNA image cytometry with DNA aneuploidy as a marker for neoplastic transformation in oral smears secures cytologic diagnosis of carcinomas. Smears from brushings of all visible oral lesions are an easily practicable, cheap, noninvasive, painless, and safe screening method for detection of oral precancerous lesions and squamous cell carcinoma in all stages. We conclude that DNA image cytometry is a very sensitive and highly specific, objective, and reproducible adjuvant tool for identification of neoplastic cells in oral smears.     

口腔鳞状细胞癌DNA含量的测定及意义

目的：探讨细胞核DNA含量在判别口腔鳞状细胞癌（SCC）生物学行为中的意义。方法：应用图象细胞计（ICM）分析18例口腔鳞状细胞癌的DNA含量和细胞周期。结果：SCC中DNA指数（DI）和增殖指数（PI）显著高于正常上皮（P＜0．05）,随着SCC病理学分级的升高DI值和PI值明显升高（P＜0．05）。结论：用ICM检测细胞DNA含量,分析细胞周期,有助于了解鳞状细胞癌的增殖能力和恶性程度,对指导预后和评价治疗具有重要意义。     

The relationship of proliferating cell density at the invasive tumour front with prognostic and risk factors in human oral squamous cell carcinoma

BACKGROUND: We hypothesise that the density of proliferating cells at the invasive tumour front (ITF) has a positive relationship with prognostic and risk factors in human oral squamous cell carcinoma (SCC). METHODS: Tissues from 47 human oral SCC specimens were collected and stained with a monoclonal antibody directed against the Ki-67 antigen using a horseradish peroxidase based two-step immunostaining method. Counting was performed on two parallel sections at the ITF using an image analyser. The Ki-67 labelling index (LI) was determined by measuring the number of nuclei/mm(2) of epithelium. RESULTS: Our results show that the density of proliferating cells is related to clinical staging, with advanced stage of disease having a significantly higher Ki-67 LI compared with early stage of disease (2111 +/- 905 vs. 1908 +/- 913; P = 0.03). Importantly, this study shows that tumours that have metastasised have a significantly higher Ki-67 LI than tumours where distant metastasis was not detected (3257 +/- 650 vs. 1966 +/- 881; P < 0.0001). CONCLUSIONS: Cell proliferation, as measured by the Ki-67 LI at the ITF, has a positive relationship with clinical staging, tumour thickness, smoking status of the patient and alcohol consumption. Further, we suggest that a multicenter study with a large cohort of patients is indicated to fully elucidate whether cell proliferation at the ITF is directly related to patient survival.     

DNA-ploidy studies in a keratocyst undergoing subsequent malignant transformation

Using techniques that allow flow cytometry to be performed on tissue stored in paraffin wax, we have examined the DNA content of cells from an odontogenic keratocyst which underwent malignant transformation. On histological examination, the primary lesion was an odontogenic keratocyst showing dysplastic changes within the squamous epithelium. Flow cytometry revealed a prominent, abnormal DNA stemline, which was also present in the subsequent squamous cell carcinoma.     

DNA-ploidy studies in a keratocyst undergoing subsequent malignant transformation

Using techniques that allow flow cytometry to be performed on tissue stored in paraffin wax, we have examined the DNA content of cells from an odontogenic keratocyst which underwent malignant transformation. On histological examination, the primary lesion was an odontogenic keratocyst showing dysplastic changes within the squamous epithelium. Flow cytometry revealed a prominent, abnormal DNA stemline, which was also present in the subsequent squamous cell carcinoma.     

Cytologic and DNA-cytometric examination of oral lesions in lichen planus

BACKGROUND: The aim of the present study was to evaluate the diagnostic value of exfoliative cytology (EC) and DNA image cytometry applied to oral lesions of lichen planus (LP; n = 56), in order to detect or exclude malignant transformation. METHODS: Brush and excisional biopsies were obtained from 56 patients. In cases of oral LP in which brush biopsies were suspicious for tumor cells, nuclear DNA contents were measured, using a TV Image Analysis System. RESULTS: In 50 patients EC yielded tumor cell-negative, doubtful in four cases and suspicious results obtained in two cases. DNA image cytometry revealed DNA-aneuploidy only in the two suspicious cases. The comparison between cytologic/DNA-cytometric diagnosis and biopsy histology resulted in a total agreement (LP without dysplasia: 54 and squamous cell carcinoma in LP: two cases). CONCLUSIONS: In conclusion, cytology with DNA-cytometry is a highly sensitive, specific, and non-invasive method, which can be used for periodical follow up of oral LP lesions in order to early detect or exclude malignancy.     

Streptococcus anginosus infection in oral cancer and its infection route

OBJECTIVE: To elucidate a possible involvement of Streptococcus anginosus in oral cancer, we assessed the frequency of S. anginosus infection in oral cancer tissues, and investigated its infection route. MATERIALS AND METHOD: The tissue specimens were obtained from 46 oral cancer and three precancerous leukoplakia subjects. Frequency of S. anginosus infection was assessed by a species-specific polymerase chain reaction (PCR) assay. The genotype of the clinical isolates taken from cancer tissue and dental plaque samples was analyzed using pulsed-field gel electrophoresis (PFGE). RESULTS: S. anginosus DNA was frequently detected in squamous cell carcinoma (19/42), but not in other types of cancer (lymphoma and rhabdomyosarcoma) or leukoplakia samples. A subject-based analysis revealed that S. anginosus was solely detected in dental plaque and not in saliva from all 19 S. anginosus-positive squamous cell carcinoma cases. Further, the genotype of S. anginosus isolated from cancer tissue was identical to that from dental plaque of the same patients. CONCLUSION: Infection of S. anginosus could occur frequently in oral squamous cell carcinoma and that dental plaque could be a dominant reservoir of the S. anginosus.     

口腔鳞癌p16基因的表达及其病理意义

目的　探讨 p16基因在口腔鳞癌中的表达情况及其病理意义。方法　利用原位杂交和 Western blot-ting技术检测了 48例口腔鳞癌标本和 6例正常口腔粘膜标本 p16基因 m RNA的分布和强度及 p16蛋白的表达 ,并分析了 p16蛋白表达和患者临床病理参数的关系。结果　 6例正常口腔粘膜 p16 m RNA表达均阳性 ;48例口腔癌标本中有 2 1例 p16 m RNA表达阴性 (4 3.7% ) ,2 6例无 p16蛋白表达 (5 4.2 % ) ,其中 2 1例 m RNA表达阴性的病例均无 p16蛋白表达 ,另外 5例无 p16蛋白表达的鳞癌组织有 p16 m RNA杂交信号 ;p16蛋白表达与肿瘤的部位、癌细胞的分化程度及临床分期均无关 ,但与颈淋巴结是否转移密切相关 (P<0 .0 1)。结论　 p16基因产物丧失常发生于口腔鳞癌 ;p16基因可能通过某种机制抑制癌细胞的转移特性。     

淋巴显像技术在口腔鳞癌哨位淋巴结活检中的应用

目的:评价淋巴显像技术在口腔鳞癌哨位淋巴结活检中的价值。方法:应用颈淋巴显像技术结合蓝染法及SPECT/CT同机融合技术,对21例临床NO(cNO)口腔鳞癌患者的哨位淋巴结(sentinel lymph node,SLN)进行研究。结果:全组患者SLN检出率为100％,21例中有7例SLN活检阳性,颈清术后标本同样证实有颈淋巴结转移,无假阴性结果,SLN活检对全组病例颈淋巴结转移状况预测的准确性为100％。结论:颈淋巴显像技术结合蓝染法及SPECT/CT同机融合技术能有效地对口腔鳞癌SLN进行定位,从而准确预测颈淋巴结转移状况。     

三氧化二砷抑制口腔鳞癌细胞增殖的研究

目的:体外试验研究三氧化二砷对口腔鳞癌的可能治疗作用。方法:以人舌鳞状细胞癌细胞系Tca8113细胞为研究对象之一,采用台盼蓝拒染法计数活细胞,观察三氧化二砷作用于Tca8113细胞的量效关系、时效关系。采用平皿法姬姆萨染色观察三氧化二砷对Tca8113细胞的克隆形成抑制作用。以舌鳞状细胞癌临床新鲜标本组织块为研究对象之二,消化后细胞悬液与各浓度三氧化二砷、一定浓度的MTX、5Fu、PYM体外培养后MTT法测定临床舌癌细胞生长抑制率。结果:三氧化二砷对Tca8113细胞、临床舌癌细胞均有显著生长抑制作用。1μmolAs2O3台盼蓝染色活细胞计数Tca8113细胞生长抑制率28.57%,克隆形成抑制率31.01%;3μmolAs2O3浓度Tca8113细胞生长抑制率58.36%,克隆形成抑制率76.63%;5μmolAs2O3浓度Tca8113细胞生长抑制率86.03%,克隆形成抑制率91.30%。体外药敏测定:1μmol浓度As2O3的临床舌癌细胞的生长抑制率39.5%,3μmol浓度As2O3为47.2%,5μmol浓度As2O3为89%,MTX为36.5%,5Fu为41.3%,PYM为58.16%。临床药敏标准一般大于30%为敏感,PYM大于50%为敏感。结论:体外试验表明三氧化二砷可显著抑制人舌鳞状细胞癌细胞系Tca8113细胞的生长,对临床癌细胞也表现了良好的抗增殖作用,三氧化二砷可能成为治疗口腔鳞癌的有效药物     

Comparative evaluation of genetic assays to identify oral pre‐cancerous fields

Background: Oral squamous cell carcinomas often develop in a pre‐cancerous field, defined as mucosal epithelium with cancer‐related genetic alterations, and which may appear as a clinically visible lesion. The test characteristics of three genetic assays that were developed to detect pre‐cancerous fields were investigated and compared to histology. Methods: In total, 10 pre‐cancerous fields that were not visible at clinical inspection and gave rise to malignant transformation based on an identical TP53 mutation in tumor and mucosal epithelium in the surgical margin, as well as 10 normal oral mucosa specimens were analyzed for numerical chromosomal changes with multiplex ligation‐dependent probe amplification (MLPA), for loss of heterozygosity (LOH), with microsatellite PCR and for DNA index alterations with DNA image analysis. Results: No alterations were detected in normal tissue by either of the assays. Both MLPA and LOH assays detected all pre‐cancerous fields. DNA cytometry identified aneuploidy in four of 10 pre‐cancerous fields, while the corresponding tumors that developed in these fields were shown to be aneuploid. Conclusions: Both the MLPA and LOH assay seem suitable for screening pre‐cancerous fields in subjects at high risk for oral cancer even in the absence of clinically abnormal appearing oral mucosa. Measurements of DNA index might be valuable to determine the time to progression.     

MHC-Ⅰ类链相关基因A修饰的口腔鳞癌细胞疫苗诱导抗肿瘤免疫应答的实验研究

目的:研究MHC-Ⅰ类链相关基因A(MHC classⅠchain-related gene A,MICA)修饰的口腔鳞癌疫苗诱导机体抗肿瘤免疫应答的有效性并探讨其作用机制。方法:灭活稳定转染MICA基因的口腔鳞癌细胞,制备成肿瘤疫苗,通过腹腔注射严重联合免疫缺陷(severe combined immunodeficiency,SCID)鼠,观察其对皮下移植瘤生长的抑制作用;应用流式细胞术和乳酸脱氢酶(lactate dehydrogenase,LDH)释放法检测免疫重建荷瘤SCID鼠外周血单个核细胞(peripheral blood mononuclear cell,PBMC)和脾细胞表面NKG2D的表达及对肿瘤靶细胞的杀伤活性。采用SPSS16.0软件包对数据进行统计学处理。结果:转染MICA基因的口腔鳞癌疫苗能显著抑制免疫重建荷瘤SCID鼠皮下移植瘤的生长,上调PBMC和脾细胞表面NKG2D的表达,并增强对肿瘤靶细胞的杀伤活性,与未转染组及转染空白载体组比,差异有统计学意义(P<0.05)。结论:过表达MICA蛋白的口腔鳞癌疫苗能显著增强机体的抗肿瘤免疫应答能力,MICA有望作为口腔鳞癌免疫基因治疗的潜在靶标。     

舌鳞状细胞癌浸润前沿细胞增殖的研究

目的　研究舌磷癌浸润前沿分级、检测增殖细胞核抗原 (PCNA)、Ki6 7、核仁组织区相关嗜银蛋白 (AgNOR)在浸润前沿中的表达。 方法　对 5 7例舌鳞癌行浸润前沿分级 ,采用免疫组化SP法及银染色法检测浸润前沿和中心PCNA、Ki6 7、NORs的表达。结果　浸润前沿PCNA、Ki6 7的表达和AgNOR均数均明显高于非前沿部分 ,差异有极显著性 (P <0 .0 0 1) ;浸润前沿PCNA标记指数 (P <0 .0 5 )、AgNOR均数 (P <0 .0 1)与浸润前沿分级 (IFG)总分呈正相关关系 ;单因素COX分析显示 ,IFG总分 (P <0 .0 1)、浸润前沿AgNOR均数 (P <0 .0 5 )对判断预后有意义 ;多因素COX分析显示 ,IFG总分 (P <0 .0 1)可作为预测总体生存率和累积生存率的有意义的因子。结论　舌鳞癌浸润前沿肿瘤细胞分化较差 ,增殖活性明显高于非前沿部分 ;IFG总分和浸润前沿AgNOR均数与舌癌预后有关     

Analysis of the Ki-67 antigen at the invasive tumour front of human oral squamous cell carcinoma

BACKGROUND: It is hypothesised that cell proliferation, as measured by the Ki-67 labelling index (LI) at the invasive tumour front (ITF) was directly related to the histological grade in human oral squamous cell carcinomas (SCCs). METHODS: Tissues from 42 human oral SCCs were collected and stained with an antibody directed against the Ki-67 antigen using an advanced polymer staining system. Quantitation of the immunopositive cells was performed on two parallel sections at the invasive tumour front (ITF), using an image analyser. The Ki-67 LI was expressed as the number of positive nuclei/mm2 of epithelium. The control tissue used was normal epithelium at the excision margin. RESULTS: The mean Ki-67 LI for oral SCCs at the ITF was significantly greater than that for the excision margin tissue (P < 0.0001). There was a positive association between increasing Ki-67 LI and increasing Broders' grade (P < 0.05), with a well-differentiated tumour having the lowest mean Ki-67 LI (1549 +/- 806) and a poorly differentiated tumour having the highest value (2232 +/- 771). A similar trend was observed between the mean Ki-67 LI and Bryne's multifactorial grading system. CONCLUSIONS: It was concluded from this study that cell proliferation (as measured by the Ki-67 antigen) at the ITF had a strong positive relationship with histological grading in human oral SCC.     

大鼠颊黏膜鳞状细胞癌单克隆细胞系Rca-B的建立及其生物学特性研究

目的：建立大鼠颊黏膜鳞癌单克隆细胞系，并观察其生物学特性，为口腔癌的研究提供大鼠来源的恶性肿瘤细胞系、移植瘤和转移动物模型。方法：将4-硝基喹啉-1-氧化物诱发的SD大鼠颊黏膜鳞癌组织标本进行体外传代培养，利用有限稀释法获得单克隆细胞。光学和电子显微镜观察该细胞的形态变化，细胞计数和四唑盐比色法观察细胞的增殖能力，染色体分析确定细胞核型特点，流式细胞仪检测其细胞周期；动物实验观察细胞裸鼠皮下接种成瘤率及实验性肺转移能力。结果：成功得到单克隆细胞系，细胞形态和生物学观察结果表明，该细胞系符合鳞癌细胞的基本特征，65代细胞群体倍增时间为25．44h，平板克隆形成率为56．30％。细胞周期分布S期占20．13％；染色体为四倍体核型。角质蛋白和波形蛋白免疫组织化学染色均为阳性。细胞系裸鼠皮下接种成瘤为16／16；细胞系行裸鼠尾静脉注射后，其实验性肺转移为10／10；该单克隆细胞系SD大鼠同种异体皮下接种不能成瘤。结论：成功建立SD大鼠颊黏膜鳞癌细胞系Rca—B，细胞系具有高转移鳞癌细胞的典型生物学特征；该细胞系是研究口腔鳞癌分子发生机制和防治策略的又一理想模型。     

维甲酸受体-β基因联合维甲酸对Tca8113细胞的凋亡诱导作用

目的　研究已证实维甲酸受体 -β( Retinoic acid receptor-beta,RARβ)对某些类型的恶性肿瘤细胞有诱导分化和凋亡作用。现观察转导 RARβ基因联合全反式维甲酸 ( all trans retinoic acid,ATRA)对 Tca8113细胞的凋亡作用。方法　先应用脂质体将 RARβ基因导入 Tca8113细胞中 ,然后采用活细胞计数、透射电镜、流式细胞仪等方法观察 ATRA对转基因细胞的凋亡影响和生长抑制作用。结果　转 RARβ基因 Tca8113细胞在 1μmol/LATRA作用 10天后 ,细胞生长抑制率达 90 .2 % ;光镜和透射电镜下观察都可见到典型形态变化的凋亡细胞 ;FCM测定结果表明 ,细胞凋亡率高达 80 .7%。而母本细胞 Tca8113凋亡率小于 2 0 %。结论　 RARβ基因可能是 ATRA诱导 Tca8113细胞产生显著凋亡作用的“靶分子”。 RARβ基因联合维甲酸治疗某些口腔鳞癌将是有效和可行的。     

Flow cytometric analysis of squamous cell carcinoma of the oral and maxillofacial region]

Fifty fresh tissue samples and fifty paraffin embedded specimens from squamous cell carcinoma of the oral and maxillofacial region were analyzed for nuclear DNA content and cell kinetics by flow cytometry (FCM). Mean DNA index (DI) was 1.154, and 59% of them showed aneuploidy patterns. Mean S phase cell population, which represents the "proliferative activity", was 22.62%. With the increase of surgical stage, aneuploid population rose from 51.7% to 82.8%, and with the increase of histology grade, it rose from 54.17% to 71.4%. S phase cell population also showed a tendency to increase with the increase of surgical stage or histology grade, and in those with cervical metastasis. The results indicate that nuclear DNA analysis by FCM is quite useful as a supplement to histology diagnosis and evaluation of malignant grade of squamous cell carcinomas of the oral and maxillofacial region.     

口腔扁平苔藓病变中增殖细胞核抗原和DNA的检测

目的：探讨ＰＣＮＡ表达在反映口腔扁平苔藓及癌变组织增殖的可行性及临床意义。方法：应用Ｓ－Ｐ免疫组化法检测ＰＣＮＡ在口腔扁平苔藓、扁平苔鲜上皮异常增生及鳞癌组织中的表达；应用图像分析仪检测上述组织中ＤＮＡ含量的变化；用统计学方法分析ＰＣＮＡ与ＤＮＡ的相关关系。结果：随着病变程度的增加，ＰＣＮＡ的增殖指数增加，ＤＮＡ倍体逐渐增多，各组间有显著性差异。统计学检验证实ＰＣＮＡ与ＤＮＡ倍体值间有相关关系，ｒ＝０．８１５。结论：ＰＣＮＡ的表达可以反应病变细胞的增殖活性，在口腔扁平苔藓病变中有一定的应用价值     

LncTUG1通过靶向miR-212-3p对口腔鳞状细胞癌细胞NK细胞杀伤敏感性的影响

目的 探讨长链非编码RNA（Lnc）牛磺酸调节基因1(taurine up-regulated gene 1,TUG1)对口腔鳞状细胞癌细胞自然杀伤细胞(nature killer cell, NK)杀伤敏感性的影响。方法 以口腔鳞状细胞癌细胞作为体外实验对象,转染TUG1 siRNA,利用qRT-PCR、MTT、流式细胞术、LDH方法,分别检测TUG1表达量、细胞增殖、细胞凋亡和NK细胞杀伤率。利用生物信息学软件预测TUG1与miR-212-3p靶向互补结合,构建荧光素酶报告载体,鉴定靶向关系。在口腔鳞状细胞癌细胞中共转染TUG1 siRNA和miR-212-3p 抑制剂,评估miR-212-3p 抑制剂对TUG1 siRNA影响口腔鳞状细胞癌细胞增殖凋亡和NK细胞杀伤敏感性的影响。采用SPSS 21.0软件包对实验数据进行统计学分析。结果 TUG1 siRNA可显著降低口腔鳞状细胞癌细胞中TUG1的表达水平（P=0.000）,降低细胞增殖能力（P=0.001）,促进细胞凋亡（P=0.000）,增加NK细胞杀伤率（P<0.01）。TUG1 siRNA靶向提高miR-212-3p表达量。miR-212-3p 抑制剂可逆转TUG1 siRNA对口腔鳞状细胞癌细胞增殖、凋亡和NK细胞杀伤率的影响。结论 下调TUG1可靶向负调控miR-212-3p,抑制口腔鳞状细胞癌细胞增殖并诱导凋亡,提高NK细胞杀伤敏感性。