HPV-16 viral load in oropharyngeal squamous cell carcinoma using digital PCR

Conclusions: We did not identify any strong associations between HPV-16 viral load and any of the clinical or lifestyle factors.

Objective: The epidemiology of oropharyngeal SCC is changing, with an increasing proportion of HPV-positive cases seen in the last decade. It is known that a high viral load is linked to the development of cervical cancer, the relation between viral load and oropharyngeal SCC is less clear. We sought to determine HPV-16 viral load in HPV-positive oropharyngeal SCCs using highly sensitive digital PCR and to identify clinical and lifestyle factors associated with viral load.

Subjects and methods: We analysed 45 HPV-16 positive oropharyngeal SCCs diagnosed between 2013 and 2015. All patients completed a lifestyle questionnaire and clinical data were extracted from medical charts. Viral load was determined using digital PCR assays for HPV-L1 and RNAseP.

Results: We found large variations in HPV-16 viral load from 1 to 930 copies per cell (median 34 copies per cell).     

Detection of human papillomavirus (HPV) type 16 DNA relative sequence in the tissue of laryngeal carcinoma]

By using alpha 32-P-dCTP labeled Chinese Human papillomavirus type 16 DNA (CHPV 16 DNA) probe, we have adopted Southern blot method and detected HPV subtype 16 DNA (1/6) and HPV 16 DNA relative sequence (3/6) in 6 biopsy specimens of laryngeal carcinoma. The result showed a severe infection of HPV in laryngeal carcinoma. This indicates that HPV infection may play a pathogenetic role in the cancer.     

喉癌中p16基因突变和甲基化的研究

目的 探讨ｐ１６基因在喉癌发生发展中的作用和在喉癌中的失活机制。方法 应用聚合酶链反应－单链构象多态性分析（ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ－ｓｉｎｇｌｅ ｓｔｒａｎｄ ｃｏｎｆｏｒｍａｔｉｏｎ ｐｏｌｙｍｏｒｐｈｉｓｍａｎａｌｙ－ｓｉｓ,ＰＣＲ－ＳＳＣＰ）银染方法和ＰＣＲ甲基化敏感内匹酶方法检测３２例喉癌和相应癌旁组织的ｐ１６基因第１,第２外显子突变和甲基化热点区域部分内切酶     

喉鳞状细胞癌P16和增殖细胞核抗原表达

目的探讨p16基因及增殖细胞核抗原(proliferatingcellnuclearantigen,PCNA)在喉鳞状细胞癌中的表达及其临床意义。方法应用免疫组织化学技术SP法检测抑癌基因p16蛋白及PCNA在40例喉鳞状细胞癌中的表达,并与癌旁组织和正常喉组织对比,结合喉鳞状细胞癌的临床病理特征进行分析。结果在喉鳞状细胞癌中p16阳性率(50.0%)明显低于癌旁组织(76.0%)和正常组织(100.0%,P<0.05);Ⅰ、Ⅱ期喉癌中p16阳性率明显高于Ⅲ、Ⅳ期。喉鳞状细胞癌中PCNA阳性率为55.0%,明显高于癌旁组织(32.0%)和正常组织(10.0%,P<0.05);Ⅰ、Ⅱ期喉鳞状细胞癌中PCNA表达明显低于Ⅲ、Ⅳ期(P<0.05)。p16阳性与阴性患者生存期≥5年的比率分别为66.7%和33.3%,P<0.05;PCNA阳性与阴性患者生存期≥5年的比率分别为37.5%和62.5%,P<0.05。结论p16和PCNA分别是喉鳞状细胞癌进展及预后判断的独立指标;p16和PCNA联合检测对喉鳞状细胞癌的预后评估可能更有价值。     

Intratumoral DNA content heterogeneity in laryngeal squamous cell carcinoma.

Multiple tissue samples obtained from sections cut by Michaels and Gregor's method obtained from 21 consecutive total laryngectomies for squamous cell carcinoma were studied for intratumoral DNA content heterogeneity or homogeneity. Concordant DNA ploidy was manifested in all samples of five (23.8%) carcinomas (two diploid and three aneuploid), while 16 carcinomas (76.2%) demonstrated a variable DNA ploidy (diploid and aneuploid). Analysis of cellular proliferative activity demonstrated remarkable intratumoral stability in both concordant and discordant carcinomas. These data indicate there is a considerable heterogeneity of DNA ploidy but the proliferative rate is relatively stable within the carcinomas. Clinical implications of our findings are also presented.     

线粒体基因组单倍型类群与喉鳞状细胞癌的相关性研究

目的检测常见线粒体基因组（mtDNA）单倍型A、B、C、D、F、G、M在正常人群及喉癌患者中的分布频率,探讨mtDNA单倍型与喉鳞状细胞癌（laryngeal squamous cell carcinoma, LSCC）发病的相关性。方法选取2013年12月~2015年12月80例LSCC患者作为实验组,同期选取健康体检者74例作为对照组（normal control, NC）,提取两组病例的全血DNA,经PCR-扩增、基因测序,划分单倍型及其子单倍型,计算mtDNA单倍型及其子单倍型在不同组的分布频率,运用SPSS 20.0统计学软件进行统计分析,P<0.05为有统计学意义。结果mtDNA单倍型B及D4在LSCC组的分布频率高于NC组(P﹤0.05),且与临床分期无关（P>0.05）。结论mtDNA单倍型B和D4可能是LSCC的风险因素,可以作为LSCC发病的基因筛查参考指标。     

喉鳞状细胞癌蛋白酪氨酸磷酸酶基因第5和第8外显子的突变分析

目的 探讨蛋白酪氨酸磷酸酶(phosphatase and tensin homology deleted on chromosome 10／mutated in multiple advanced cancer1／TGF-β regulated and epithelial cell-enriched phosphatase,PTEN／MMAC1／TEP1,以下简称PTEN)基因突变与喉鳞状细胞癌(简称鳞癌)的关系。方法 应用聚合酶链反应一单链构象多态性(polymerase chain reaction-single-strand conformation polymorphism,PCR-SSCP)分析银染系统和PCR产物直接测序技术,检测喉鳞癌新鲜或冰冻组织中PTEN基因第5、第8外显子的突变情况,对有电泳迁移率改变的进行PCR产物测序。结果 第5、第8外显子的突变均发生在声门上型喉癌,声门型喉癌未发现突变。其中,第5外显子有6例发生突变,突变率15%(6／40)。直接测序发现2例在85密码子第2、3碱基子之间发生了插入A突变,即TT→TAT,2例86密码子第3碱基缺失一个A,从而导致移码突变;1例同时存在以上两种突变,导致错义突变;1例85密码子第2、3碱基之间插入A,同时86密码子第2碱基缺失,即GCA→GA导致无义突变。这6例中有低分化3例,中分化3例,无高分化;淋巴结转移5例占83．3％(5／6),无淋巴结转移1例,占16．7%(1／6)。第8外显子仅有1例发生突变,突变率为2．5％(1／40),直接测序发现276密码子第2碱基A缺失,即AAT→AT．336密码子与337密码子之间插入1个C,该病例虽无淋巴结转移,但病理为低分化。结论 PTEN基因第5外显子中85、86密码子在人喉鳞状细胞癌中突变率较高,该基因突变可能与喉鳞癌淋巴结转移及病理中、低分化有关。     

p16基因治疗喉鳞状细胞癌的实验研究

OBJECTIVE: To assess the anti-tumor effects of p16INK4A gene transfer in laryngeal squamous cell carcinoma. METHODS: A Complete p16INK4A gene was inserted into a replication-defective recombinant adenovirus (Ad-p16) and the tumor cells were infected with Ad-p16. Confirmation of p16INK4A protein expression after Ad-p16 infection was performed by Western Blotting. The therapeutic effects were evaluated both in in vitro and in vivo study. RESULTS: The replication-defective recombinant adenovirus can direct a high level of p16INK4A protein expression in laryngeal squamous cell carcinoma. Studies both in in vitro and in vivo experiments demonstrated that Ad-p16 treatment significantly inhibits the cell growth and the established tumors in nude mice. The mean value of tumor volumes among the Ad-p16, Ad-Lacz and phosphatic buffered saline(PBS) groups was (91.00 +/- 6.32) mm3, (137.00 +/- 9.62) mm3 and (144.00 +/- 13.87) mm3 respectively. Statistic analysis showed that there was a significant difference between the Ad-p16 group and the control groups (P < 0.05). No significant difference was found between Ad-lacZ and PBS groups, which indicated that the antitumor effects was not influenced by the adenovirus. CONCLUSION: These results demonstrate a significant antitumor effect of Ad-p16 against human laryngeal squamous cell carcinoma. This data also further support the potential application of Ad-p16 to treat the human head and neck cancer.     

p16与C—myc在喉鳞状细胞癌中的表达意义

目的：研究p16和C－myc在喉鳞状细胞癌中的表达意义。方法：应从p16和C－myc单克隆抗体，通过免疫组织化学法检测6例正常喉粘膜和47例喉鳞癌中p16和C－myc的表达。结果：6例正常喉粘膜组织中未发现p16和C－myc蛋白阳性表达，47例喉鳞癌中p16和C－myc阳性表达率分别为48．9％（23／47）和57.4％（27／47），与性别、年龄和肿瘤发生的部位无相关性（P＞0．05），与临床分期和病理分级相关（P＜0．05）；C－myc还与颈淋巴结转移相关（P＜0．05）。结论：p16和C-myc蛋白阳性表达在喉鳞癌的发生和发展中起重要作用，可作为评价喉鳞癌预后的新指标。     

基质金属蛋白酶14基因在人喉鳞状细胞癌中表达差异的分析

OBJECTIVE: To investigate the role of expression of matrix mettallo-proteinase 14 (MMP14) in laryngeal carcinomas and the relationship between MMP14 expression and the laryngeal biological behavior. METHODS: The gene expression differences of MMP14 between fresh laryngeal cancer tissues and their surrounding normal mucosa was analyzed by RT-PCR. The expression of MMP14 protein in paraffin-embedded tissues was determined immunohistochemically. All statistical analyses were performed by SPSS version 10.0. RESULTS: In the 33 cases of matched specimens, MMP14 gene expression was much higher in tumor tissues than that in surrounding ing normal tissues in 26 cases and lower in 2 cases, whereas in other 5 cases, no significant difference was observed between the cancer tissue and the surrounding normal tissue. MMP14 gene expression was not different in different stages in laryngeal glottic cancers, but correlated to the differentiation and lymph node metastasis (P < 0.05). There was correlation between MMP14 gene expression and the stage, differentiation and lymph node metastasis in the laryngeal supraglottic cancers (P < .05). MMP14 protein was localized predominantly in the carcinoma cell cytoplasm and in the stromal fibroblasct cytoplasm, and weakly or not expressed in surrounding normal tissue. MMP14 protein expression was much higher in tumor tissue than that in surrounding normal tissue in most of the cases. In general, MMP14 protein expression was the same as MMP14 gene expression and related to the stage, differentiation and lymph node metastasis in the laryngeal cancers. There was no survival difference at 3, 5 and 7-year between the group with higher MMP14 protein expression in tumor tissues than surrounding normal tissues and the group with no difference of MMP14 protein expression (Log Rank, P=0.5535). CONCLUSIONS: The protein MMP14 may play role in laryngeal cancer invasion in a certain extent, but important role in lymph node metastasis of laryngeal cancer. The over-expression of MMP14 protein may be a marker for lymph node metastasis of laryngeal cancer.     

Proteomic signatures in laryngeal squamous cell carcinoma

Laryngeal cancer remains a worldwide health problem. The identification of biomarkers unique to laryngeal cancer may provide new insights into its pathogenesis, as well as provide potential targets for novel therapies and early detection. In order to identify potential biomarkers, we performed a proteomic analysis of laryngeal cancer specimens. Using two-dimensional differential in-gel electrophoresis and mass spectroscopy, protein expression profiles from two laryngeal carcinoma specimens and corresponding adjacent normal tissue were analyzed. The results of our analysis showed that the expression of a number of proteins was significantly altered in the tumor specimens when compared to matched normal controls. The differentially expressed proteins were identified, and they included stratifin, S100 calcium-binding protein A9, p21-ARC, stathmin, and enolase. With these findings, we have identified potential biomarkers which may contribute to the pathogenesis of laryngeal carcinoma, and which may be suitable as targets for novel therapeutic and/or diagnostic modalities.     

端粒酶与喉癌相关性研究进展

端粒酶激活与恶性肿瘤发生发展之间存在着密切的关系。本文就端粒酶结构、功能及其与喉癌发生发展关系的最新研究进展作简要综述。     

喉鳞癌组织两种P糖蛋白单克隆抗体的检测

为了解喉鳞癌的多药耐药性，以期提高综合治疗中化疗的效果，采用免疫组化技术对未经任何治疗的喉鳞癌患者在手术中取出的新鲜肿瘤组织进行了Ｐ糖蛋白（ＰＧＰ）单克隆抗体ＪＳＢ１及其单克期抗体Ｃ２１９的检测。对比了肿瘤组织分化程度、肿瘤大小及有无淋巴结转移与ＰＧＰ表达阳性之间的关系。结果发现，单抗ＰＧＰ ＪＳＢ１的阳性率为６５．２％（ｎ＝２３），而单抗Ｃ２１９检测的６例ＰＧＰ均为阴性，与分化无关，与肿瘤大小、     

p16蛋白表达与喉癌临床病理关系的研究

目的探讨p16蛋白表达与喉鳞状细胞癌的临床病理关系. 方法应用免疫组化SP法检测60例喉鳞癌组织中p16的蛋白表达情况. 结果 p16蛋白在喉鳞癌组织中的阳性表达率明显低于正常喉黏膜组织(P<0.05).随着临床分期的进展及组织学分化程度的降低,p16蛋白表达进一步降低.有颈淋巴结转移的喉癌组织p16蛋白阳性表达低于无转移的喉癌组织. 结论检测p16蛋白表达状况对喉癌的早期诊断和预后评估有临床参考价值.     

喉癌中p16基因突变和甲基化的研究

目的　探讨p16基因在喉癌发生发展中的作用和在喉癌中的失活机制。方法　应用聚合酶链反应 单链构象多态性分析 (polymerasechainreaction singlestrandconformationpolymorphismanalysis ,PCR SSCP)银染方法和PCR甲基化敏感内切酶方法检测 32例喉癌和相应癌旁组织的p16基因第 1,第 2外显子突变和甲基化热点区域部分内切酶位点的甲基化情况。结果　 32例喉癌中p16第 1第 2外显子无一例发生突变 ;在第 1外显子CfoⅠ位点有 6例发生甲基化 ,SacⅡ位点有 5例发生甲基化 ,HpaⅡ位点有 4例发生甲基化 ,甲基化发生频率为 (6 /32 ) 18 8%。在第 2外显子HpaⅡ位点有 5例发生甲基化 ,CfoⅠ位点有 6例发生甲基化 ,甲基化发生频率为 (6 /32 ) 18 6 %。结论　喉癌中p16基因的甲基化是该基因失活的重要机制之一 ,p16基因的失活与喉癌的发展和演进密切相关。     

Nuclear DNA cytofluorometry of normal human laryngeal epithelia and squamous cell carcinoma

Normal human laryngeal epithelia and laryngeal squamous cell carcinoma were assayed by Feulgen DNA cytofluorometry using free cell nuclei isolated from carnoy-fixed, paraffin-embedded specimens. In all of the 12 normal specimens, the epithelium showed typical diploid cell clones with low proliferative activity. Polyploid cells were seen in only two specimens from subjects aged 61 and 69 years respectively, and the number of polyploid cells seen in these two specimens was only two. Fifteen cancer cases were divided into three groups: an untreated group (5 cases), a chemotherapy group (5 cases) and a group of cases with recurrence after radiation therapy (5 cases). Among these three groups the DNA ploidy patterns were compared. In the untreated group, all cases showed a two-peak diploid pattern and a high proliferative activity, and polyploid cells were present. In the chemotherapy group, a wide one-peak histogram extending from 2C to about 5C was noted in 4 cases, and an aneuploid pattern in one case. Thus, the DNA ploidy pattern in the chemotherapy group differed from that in the untreated group. Of the 5 cases with recurrence after radiation therapy, one had a tetraploid pattern, but the remaining 4, a two-peak diploid pattern similar to that seen in the untreated group. Polyploid cells were observed in all these cancer cases. However, because they were also seen in some normal subjects, the finding of polyploid cells is not considered to be conclusive of cancer diagnosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

喉癌线粒体DNA拷贝数的临床意义

目的探讨喉鳞状细胞癌（laryngeal squamous cell carcinoma,LSCC）线粒体DNA（mitochondrialDNA,mtDNA）拷贝数异常与TNM分期和分化程度的关系.方法选取32例喉癌、相应癌周组织石蜡标本,显微切割分离癌及癌周组织,分别提取总DNA.以ND1和β-actin为目的基因,进行SYBR Green Ⅰ荧光定量PCR扩增,并对不同TNM分期和分化程度LSCC的mtDNA拷贝数进行比较分析.结果LSCC Ⅰ和Ⅱ期癌周、癌组织平均（β-actin/ND1 DNA）比分别为0.269和0.274,Ⅲ和Ⅳ期癌周、癌组织平均（β-actin/ND1 DNA）比为0.168和0.157.癌周组织低、中、高分化的平均（β-actin/ND1 DNA）比分别为0.184、0.237、0.239;癌组织分别为0.175、0.201、0.226.结论LSCC癌组织Ⅲ和Ⅳ期的mtDNA拷贝数显著高于Ⅰ和Ⅱ期,mtDNA拷贝数与肿瘤分化程度成负相关,mtDNA拷贝数的变化在肿瘤的发病机制中可能是一个早期事件.